Gram-negative alkaliphilic microorganisms

The present invention is in the field of microbiology and more particularly in the field of alkaliphilic microorganisms.
BACKGROUND OF THE INVENTION
Alkaliphiles are defined as organisms which exhibit optimum growth in an alkaline pH environment, particularly in excess of pH 8, and generally in the range between pH 9 and 10. Alkaliphiles may also be found living in environments having a pH as high as 12. Obligate alkaliphiles are incapable of growth at neutral pH.
Alkaliphiles may be found in such everyday environments as garden soil, presumably due to transient alkaline conditions caused by biological activity such as ammonification, sulphate reduction or photosynthesis. A much richer source of a greater variety of alkaliphilic organisms may be found in naturally occurring, stable alkaline environments such as soda lakes.
A more detailed study of soda lakes and alkaliphilic organisms in general is provided in Grant, W. D., Mwatha, W. E. and Jones, B. E. ((1990) FEMS Microbiology Reviews, 75, 255-270), the text of which is hereby incorporated by reference. Lists of alkaline soda lakes may be found in the publications of Grant, W. D. and Tindall, B. J. in Microbes in Extreme Environments, (eds. R. A. Herbert and G. A. Codd); Academic Press, London, (1986), pp. 22-54; and Tindall, B. J. in Halophilic Bacteria, Volume 1, (ed. F. Rodriguez-Valera); CRC Press-Inc., Boca Raton, Fla., (1988), pp. 31-70, both texts are also hereby incorporated by reference.
Alkaliphiles, the majority of which are Bacillus species, have been isolated from non-saline environments and are discussed by Horikoshi, K. and Akiba, T. in Alkalophilic Microorganisms (Springer-Verlag, Berlin, Heidelberg, N.Y., (1982)). However, alkaliphilic organisms from saline and alkaline environments such as lakes are not discussed therein. Strictly anaerobic bacteria from alkaline, hypersaline, environments have been recently described by Shiba, H. in Superbugs (eds. K. Horikoshi and W. D. Grant); Japan Scientific Societies Press, Tokyo and Springer-Verlag, Berlin, Heidelberg, N.Y., (1991), pp. 191-211; and by Nakatsugawa. N., ibid, pp. 212-220.
Soda lakes, which may be found in various locations around the world, are caused by a combination of geological, geographical and climatic conditions. They are characterized by the presence of large amounts of sodium carbonate (or complexes thereof) formed by evaporative concentration, as well as by the corresponding lack of Ca.sup.2+ and Mg.sup.2+ which would remove carbonate ions as insoluble salts. Other salts such as NaCl may also concentrate resulting in environments which are both alkaline and saline.
Despite this apparently harsh environment, soda lakes are nevertheless home to a large population of prokaryotes, a few types of which may dominate as permanent or seasonal blooms. The organisms range from alkaliphilic cyanobacteria to haloalkaliphilic archaeobacteria. Moreover, it is not unusual to find common types of alkaliphilic organisms inhabiting soda lakes in various widely dispersed locations throughout the world such as in the East African Rift Valley, in the western U.S., Tibet, China and Hungary. For example, natronobacteria have been isolated and identified in soda lakes located in China (Wang, D. and Tang, Q., "Natronobacterium from Soda Lakes of China" in Recent Advances in Microbial Ecology (Proceedings of the 5th International Symposium on Microbial Ecology, eds. T. Hattori et al.); Japan Scientific Societies Press, Tokyo, (1989), pp. 68-72) and in the western U.S. (Morth, S. and Tindall, B. J. (1985) System. Appl. Microbiol., 6, 247-250). Natronobacteria have also been found in soda lakes located in Tibet (W. D. Grant, unpublished observations) and India (Upasani, V. and Desai, S. (1990) Arch. Microbiol., 154, pp. 589-593).
Alkaliphiles have already made an impact in the application of biotechnology for the manufacture of consumer products. Alkali-tolerant enzymes produced by alkaliphilic microorganisms have already found use in industrial processes and have considerable economic potential. For example, these enzymes are currently used in detergent compositions and in leather tanning, and are foreseen to find applications in the food, waste treatment and textile industries. Additionally, alkaliphiles and their enzymes are potentially useful for biotransformations, especially in the synthesis of pure enantiomers.
SUMMARY OF THE INVENTION
The present invention provides pure cultures of novel aerobic, Gram-negative alkaliphilic bacteria. These bacteria have been isolated from samples of soil, water, sediment and a number of other sources, all of which were obtained from in and around alkaline soda lakes. These alkaliphiles have been analyzed according to the principles of numerical taxonomy with respect to each other and also to a variety of known bacteria in order to confirm their novelty. In addition, these bacterial taxa are further circumscribed by an analysis of various chemotaxonomic characteristics.
The present invention also provides data as to the composition of the environments from which the samples containing the microorganisms were obtained, as well as the media required for their efficient isolation and culturing such that one of ordinary skill may easily locate such an environment and be able to isolate the organisms of the present invention by following the procedures described herein.
It is also an object of the present invention to provide microorganisms which produce useful alkali-tolerant enzymes, as well as methods for obtaining substantially pure preparations of these enzymes. These enzymes are capable of performing their functions at high pH which makes them uniquely suited for applications requiring such extreme conditions. For example, alkali-tolerant enzymes may be employed in detergent compositions, in leather tanning and in the food, waste treatment and textile industries, as well as for biotransformations such as the production of pure enantiomers.
The genes encoding these alkali-tolerant enzymes may be isolated, cloned and brought to expression in compatible expression hosts to provide a source of larger volumes of enzyme products which may be, if desired, more easily purified and used in various industrial applications, should the wild-type strain fail to produce sufficient amounts of the desired enzyme, or does not ferment well.

BRIEF DESCRIPTION OF THE FIGURES
FIGS. 1A and 1B. Simplified dendrogram showing clusters (phenons) obtained with the S.sub.G coefficient and Unweighted Average Linkage procedure.
FIGS. 2A and 2B. Simplified dendrogram showing clusters (phenons) obtained with the S.sub.J coefficient and Unweighted Average Linkage procedure.
FIG. 3. Simplified dendrogram obtained with the S.sub.G coefficient and Unweighted Average Linkage procedure using the derived minimum discriminatory tests.
FIG. 4. A chart showing the relationship of the newly described species of bacteria with known species.

DETAILED DESCRIPTION OF THE INVENTION
Sampling
Several hundreds of strains of bacteria have been isolated from samples of soil, water, sediment and a number of other sources in and around alkaline lakes. These samples were obtained as part of an investigation over a period of three years. The isolated bacteria are non-phototrophic eubacteria. Up until now, such bacteria have not been well characterized.
The samples were collected in sterile plastic bags. Sampling was conducted at lakes Elmenteita, Nakuru, Bogoria, Crater (Sonachi), Little Naivasha (Oloidien), Magadi, and Little Magadi (Nasikie Engida), all of which are located in Kenya, East Africa. Alkaline soda lakes having similar environments may also be found in Tibet, China, Hungary and the western U.S. At each sampling site, physical parameters such as pH, conductivity and temperature were measured as well as the physical appearance of the site and the sample. Some of the samples were treated locally within 36 hours of collection of the sample but the majority were examined off-site, several weeks after collection.
Table 1 lists various strains which have been isolated. The strains are listed according to the location from which the sample was taken, the physical appearance of the sample itself and a reference to Table 2 which provides the chemical analysis of the lake water samples.
Table 3 provides a list of the isolated strains arranged according to the results of the numerical taxonomic analysis. Furthermore, Table 3 provides physical properties of the sample, in particular the temperature, conductivity and alkaline pH, as well as the numerous isolation media required for obtaining pure cultures of the new bacteria. These media are letter coded with reference to Appendix A.
Tables 1, 2 and 3 provide data from which the environment of the sampling locations may be characterized. The chemical and physical analysis of the samples confirm the presence of alkaline pH, as well as the presence of unusually high levels of Na.sub.2 CO.sub.3, coupled with low levels of Ca.sup.2+ and Mg.sup.2+.
No chemical analysis is available for mud samples. Furthermore, no chemical analysis is available for a few samples (see Table 1). However, other samples taken at the same location have been analyzed and are described in Tables 1-3. It is known that the basic environments of soda lakes are stable with respect to their pH and ionic composition. Moreover, the microbial populations found at these sites remain largely stable. Thus, it is to be expected that despite the lack of a chemical analysis of certain samples, the environment from which the bacteria were obtained may nonetheless be determined from the data presented in Tables 1-3.
The fresh soda-lake water samples were plated out on an alkaline nutrient medium (Medium A) soon after collection. Microscopic inspection showed an unexpectedly high diversity of bacterial types. Considering the extremely alkaline nature of the environment, viable counts showed unexpectedly high numbers of organotrophic bacteria, in the range of 10.sup.5 -10.sup.6 colony forming units per ml. The samples were stored either cooled or at ambient temperatures. After a few weeks' storage, the total numbers of bacteria in the sample rose, whereas the diversity of types decreased.
TABLE 1______________________________________Alkaliphilic Strains Arranged According to Their Place of Origin SAMPLE SAMPLE ANALYSISSTRAINS LOCATION APPEARANCE (Table 2)______________________________________1E.1, 2E.1, Lake Elmemteita Mud from dried up N.R.4E.1, 5E.1, (east bay) lake bed35E.2, 36E.2,37E.2, 38E.2wE.5, wEl1, Lake Elmenteita Sediment and water, N.T.wE12 (east bay) littoral zone.39E.3, 40E.3, Lake Elmemteita Mud and water, 141E.3, 42E.3, (east bay) littoral zone.44E.3, 53E.4, Spirulina scum.56E.445E.3, 47E.3, Lake Elmenteita Brown water 257E.4 swamp, south- and sediment east arm48E.3, 58E.4 Lake Elmenteita Grey mud 2 swamp, south- east arm59E.4 Lake Elmenteita, Water and sandy 3 (north-west bay) sediment, littoral zone16N.1, 17N.1, Lake Nakuru, Mud and water, N.T.18N.1, 19N.1, north beach littoral zone.20N.1, 26N.1, between Hippo28N.1, wN1, Point and NjorowN2, 2Nk1, Point.wNk2.49N.3, 50N.3, Lake Nakuru, Water column, 461N.4 north beach littoral zone. between Hippo Point and Njoro Point.51N.3, 52N.3, Lake Nakuru, Lake sediment, 4 north beach littoral zone. between Hippo Point and Njoro Point.63N.4 Lake Nakuru; Mud and water N.T. water hole, SW salt flats.6B.1, 7B.1, Lake Bogoria, Mud and water, N.T.8B.1, 9B.1, northern mud littoral zone.10B.1, 24B.1. flats.25B.1, wB1,wB2, wB4,wB5wBn464B.4 Lake Bogoria, Dried crust of N.R. northern mud soda mud. flats.65B.4 Lake Bogoria, Mud at water 5 norther mud line flats.wBs4 Lake Bogoria, Mud and water, N.T. south-west littoral zone. shore.11C.1, 12C.1, Crater Lake, Mud and water, N.T.29C.1, North point littoral zone.73aC.4, Crater Lake, Mud and water, 673bC.4, North point littoral zone.74C.475C.4 Crater Lake, Soad-mud, shore N.R. North point line.77LN.4, Little Lake Water column 778LN.4 Naivasha, and sediment south shore.21M.1, 22M.1, Lake Magadi, Mud and water N.T.27M.1 causeway upper western arm.92LM.4, Little Lake Spring water and 892LM.4 Magadi, north- sediment west springs.______________________________________ N.T. = not tested N.R. = not relevant
TABLE 2__________________________________________________________________________Chemical Analysis of Kenyan Soda Lake Waters Na.sup.+ K.sup.+ Ca.sup.2+ Mg.sup.2+ SiO.sub.2 PO.sub.4.sup.3- Cl.sup.- SO.sub.4.sup.2- CO.sub.3.sup.2- TON*ANALYSIS (mM) (mM) (mM) (mM) (mM) (mM) (mM) (mM) (mM) (mM) TA.sctn.__________________________________________________________________________1 196 3.58 0.07 b.l.d. 2.91 0.03 65.1 2.0 68.0 0.8 1192 140 3.32 0.48 0.13 1.85 0.02 46.8 1.7 32.0 1.2 863 167 3.32 0.06 b.l.d. 3.10 0.03 51.8 1.7 64.0 2.2 1034 326 5.63 0.15 b.l.d. 3.25 0.15 57.5 0.5 198.3 1.9 2595 735 5.50 0.21 0.01 2.23 0.09 100.9 1.0 476.7 0.9 6126 140 8.95 0.06 0.01 2.13 0.04 12.4 0.8 90.0 1.1 1337 8.7 1.79 0.28 0.65 1.02 0.003 4.8 0.5 <10.0 <0.07 188 483 4.35 0.03 0.03 0.64 0.08 157.8 1.7 166.0 1.2 105__________________________________________________________________________ b.l.d. = below the limits of detection *TON = Total Organic Nitrogen .sctn.TA = Total Alkalinity in milliequivalents/liter
TABLE 3__________________________________________________________________________Origin of the Strains Arranged by Cluster* SAMPLE Temp. Conductivity ISOLATIONCLUSTER STRAIN LOCATION pH .degree.C. mS/cm MEDIUM__________________________________________________________________________1 1E.1.sup.CT Elmenteita 9.5 35 n.t. A1 2E.1 Elmenteita 9.5 35 n.t. A1 wB2 Bogoria n.t. n.t. n.t. A1 wB5 Bogoria n.t. n.t. n.t. A1 wBs4 Bogoria 10.5 n.t. 19 A1 10B.1 Bogoria 10.5 36 45 A1 20N.1 Nakuru 10.5 36 30-40 A1 27M.1 Magadi 11.0 36 100 A1 Comamonas terrigena.sup.T (NCIMB 8193) --1 wNk2 Nakuru 10.5 n.t. 19 A1 Pseudomonas putida.sup.T (NCIMB 9494) --2 39E.3 Elmenteita 10-10.5 23 13.9 M2 41E.3 Elmenteita 10-10.5 23 11.3 N2 54E.3.sup.CT Elmenteita 10 27 11.3 P2 47E.3 Elmenteita 10 27 11.3 O2 51N.3 Nakuru 10-10.5 29 40.1 P2 52N.3 Nakuru 10-10.5 29 40.1 P2 42E.3 Elmenteita 10-10.5 23 13.9 N2 50N.3 Nakuru 10-10.5 29 40.1 N2 Pseudomonas stutzeri.sup.T (NCIMB 11358) ---- wN2 Nakuru n.t. n.t. n.t. A-- Pseudomonas beijerinckii.sup.T (NCIMB 9041) ---- 4E.1 Elmenteita 9.5 35 n.t. A-- 5E.1 Elmenteita 9.5 35 n.t. A3 6B.1 Bogoria 10.5 36 45 A3 7B.1 Bogoria 10.5 36 45 A3 8B.1 Bogoria 10.5 36 45 A3 38E.2 Elmenteita n.t. n.t. n.t. B3 56E.4 Elmenteita 10-10.5 23 13.9 C3 25B.1 Bogoria 10.5 36 45 A3 26N.1 Nakuru 10.5 36 30-45 A3 11C.1 Crater 9.0 30 10 A3 wB1 Bogoria n.t. n.t. n.t. A3 12C.1 Crater 9.0 30 10 A3 28N.1.sup.CT Nakuru 10.5 36 30-40 A3 61N.4 Nakuru 10-10.5 29 40.1 E3 36E.3 Elmenteita n.t. n.t. n.t. K3 40E.3 Elmenteita 10-10.5 23 13.9 M3 65B.4 Bogoria n.t. n.t. 41.9 C3 94LM.4 Little Magadi 9-9.5 81 35.0 L3 19N.1 Nakuru 10.5 36 30-40 A3 24B.1 Bogoria 10.5 36 45 A3 21M.1 Magadi 11.0 36 100 A3 29C.1 Crater 9.0 30 10 A3 35E.2 Elmenteita n.t. n.t. n.t. I3 37E.2 Elmenteita n.t. n.t. n.t. J3 48E.3 Elmenteita 10.0 27 11.3 P3 78LN.4 Little 8.5-9 30 1.2 G Naivasha3 73aC.4 Crater n.t. n.t. 10.2 D3 75C.4 Crater n.t. n.t. n.t. H3 73bC.4 Crater n.t. n.t. 10.2 D3 74C.4 Crater n.t. n.t. 10.2 H3 77LN.4 Little 8.5-9 30 1.2 F Naivasha3 wN1 Nakuru n.t. n.t. n.t. A3 49N.3 Nakuru 10-10.5 29 40.1 Q3 44E.3 Elmenteita 10.0 27 13.9 O3 58E.4 Elmenteita 10.0 27 11.3 G3 57E.4 Elmenteita 10.0 27 11.3 C4 wE5 Elmenteita n.t. n.t. n.t. A4 wB4.sup.CT Bogoria n.t. n.t. n.t. A4 wNk1 Nakuru 10.5 n.t. 19 A4 wEl1 Elmenteita 10.4 n.t. 13 A4 wEl2 Elmenteita 10.4 n.t. 13 A5 9B.1 Bogoria 10.5 36 45 A5 16N.1 Nakuru 10.5 36 30-40 A5 17N.1.sup.CT Nakuru 10.5 36 30-40 A5 22M.1 Magadi 11.0 36 100 A6 18N.1 Nakuru 10.5 36 30-40 A6 59E.4 Elmenteita 10.0 31-33 12.7 G6 64B.4.sup.CT Bogoria n.t. n.t. n.t. E6 63N.4 Nakuru 9.0 n.t. n.t. C6 53E.4 Elmenteita 10-10.5 23 13.9 G-- 92LM.4 Little Magadi 9-9.5 81 35.0 L-- wBn5 Bogoria 10.5 n.t. 19 A__________________________________________________________________________ *Clusters of microorganisms are obtained by analysis according to the principles of numerical taxonomy using the S.sub.G / UPGMA method (see discussion below and FIG. 1). n.t. = not tested The letter codes given for the Isolation Media refer to Appendix A.
Treatment of the Samples: Enrichment and Isolation of Alkaliphilic Bacteria
A wide diversity of enrichment and isolation methods were applied. Some of the methods were specifically designed for the enrichment and isolation of alkaliphilic bacteria which exhibit specific types of enzyme activity at an alkaline pH. Other techniques of a more general nature were applied for the isolation of diverse sorts of alkaliphilic bacteria. In some cases, the specific conditions prevailing in the lakes (Table 2) were taken into account when experiments were performed for the isolation of bacteria.
The different nutrient media employed for the isolation of the new alkaliphilic bacteria are designated Medium A--Medium Q. The composition of the various media employed is shown in Appendix A.
For the isolation of non-specific alkaliphilic organotrophic bacteria, soda-lake water samples or dilutions thereof were streaked out on an alkaline nutrient agar, pH 10-pH 10.5 (Medium A). Samples of a more solid consistency, mud, sediment, etc. were first suspended in an alkaline nutrient broth (Medium A) before spreading on an alkaline nutrient agar (Medium A). The bacteria were cultivated in a heated incubator, preferably at 37.degree. C. In some cases, the samples were suspended in an alkaline nutrient broth (Medium A) and the bacteria cultivated by shaking, preferably at 37.degree. C. for 2-3 days before spreading the broth onto an alkaline nutrient agar (Medium A) for the isolation of bacterial colonies.
For the isolation of alkaliphilic bacteria exhibiting specific types of enzyme activity, samples were spread onto alkaline nutrient agar containing specific substrates such as lactalbumin or casein or olive oil. In some instances, the bacteria in the sample may be enriched for 1 day or several weeks in a non-specific alkaline nutrient broth such as Medium A before spreading the broth onto an alkaline nutrient agar specific for the detection of bacteria exhibiting enzyme activity such as lipolytic or proteolytic activity.
Taxonomic Analysis
Seventy strains of bacteria isolated from in and around alkaline lakes were assigned to the type of bacteria known as Gram-negative bacteria on the basis of (1) the Dussault modification of the Gram's staining reaction (Dussault, H. P., (1955), J. Bacteriol., 70, 484-485); (2) the KOH sensitivity test (Gregersen, T., (1978), Eur. J. Appl. Microbiol. and Biotech. 5, 123-127; Halebian, S. et al., (1981), J. Clin. Microbiol., 13, 444-448); (3) the aminopeptidase reaction (Cerny, G., (1976), Eur. J. Appl. Microbiol., 3, 223-225; ibid, (1978), 5, 113-122); and in many cases, confirmation also on the basis of (4) a quinone analysis (Collins, M. D. & Jones, D., (1981), Microbiol. Rev., 45, 316-354) using the method described by Collins, M. D. in Chemical Methods in Bacterial Systematics (eds. M. Goodfellow & D. Minnikin) pp. 267-288, Academic Press, London, 1985.
The seventy strains were tested for 104 characters. The results were analyzed using the principles of numerical taxonomy (Sneath, P. H. A. and Sokal, R. R., in Numerical Taxonomy, W.H. Freeman & Co., San Francisco, 1973). The characters tested and how they were tested are compiled in Appendix B. In addition, Appendix C records how each character was coded for taxonomic analysis.
Since there are no well-documented strict or obligate non-phototrophic, alkaliphilic Gram-negative eubacteria known to the inventors, a diverse collection of 20 known Gram-negative bacteria were subjected as controls to the same analysis, using modified pH conditions. These 20 known reference bacteria are recorded in Table 4 from which it will be seen that in most cases the "Type Strain" of the known species has been used.
TABLE 4______________________________________Gram-Negative Non-Alkaliphilic Reference Strains______________________________________(C.t.)* Comamonas terrigena.sup.T NCIMB 8193(P.p.) Pseudomonas putida.sup.T NCIMB 9494(P.s.) Pseudomonas stutzeri.sup.T NCIMB 11358(A.t.) "Alcaligenes tolerans" Leicester University strain(V.c.) Vibrio costicola.sup.T NCIMB 701(P.a.) Providencia alcalifaciens.sup.T NCTC 10286(P.v.) Proteus vulgaris.sup.NT ATCC 13315(M.v.) Moellerella wisconsensis.sup.T NCTC 12132(E.t.) Edwardsiella tarda.sup.T NCTC 10396(A.h.) Aeromonas hydrophilia.sup.T NCTC 8049(A.s.) Aeromonas sp S5 Leicester University strain(F.a.) Flavobacterium aquatile.sup.T NCIMB 8694(E.c.) Escherichia coli.sup.T NCTC 9001(E.a.) Enterobacter aerogenes.sup.T NCTC 10006(K.a.) Klebsiella pneumonia ATCC 15380 ("K. aerogenes")(H.a.) Hafnia alvei.sup.T ATCC 13337(C.f.) Citrobacter freundii.sup.T NCTC 9750(S.m.) Serratia marcescens.sup.T NCTC 10211(P.b.) Pseudomonas beijerinckii.sup.T NCIMB 9041(H.e.) Halomonas elongata.sup.T ATCC 33173______________________________________ *abbreviation used in FIGS. 1A and 1B and FIGS. 2A and 2B .sup.T denotes "Type Strain"- .sup.NT denotes "Neotype Strain"-
Analysis of Test Data
The Estimation of Taxonomic Resemblance
The phenetic data, consisting of 104 unit characters were scored as indicated in Appendix C, and set out in the form of an "n.times.t" matrix, whose t columns represent the t bacterial strains to be grouped on the basis of resemblances, and whose n rows are the unit characters. Taxonomic resemblance of the bacterial strains was estimated by means of a similarity coefficient (Sneath, P. H. A. and Sokal, R. R., Numerical Taxonomy, supra, pp. 114-187). Although many different coefficients have been used for biological classification, only a few have found regular use in bacteriology. We have chosen to apply two association coefficients (Sneath, P. H. A. and Sokal, R. R., ibid, p. 129 et seq.), namely, the Gower and Jaccard coefficients. These have been frequently applied to the analysis of bacteriological data and have a wide acceptance by those skilled in the art since they have been shown to result in robust classifications.
The coded data were analyzed using the TAXPAK program package (Sackin, M. J., "Programmes for classification and identification". In Methods in Microbiology, Volume 19 (eds. R. R. Colwell and R. Grigorova), pp. 459-494, Academic Press, London, (1987)) run on a DEC VAX computer at the University of Leicester, U.K.
A similarity matrix was constructed for all pairs of strains using the Gower Coefficient (S.sub.G) with the option of permitting negative matches (Sneath, P. H. A. and Sokal, R. R., supra, pp. 135-136) using the RTBNSIM program in TAXPAK. As the primary instrument of analysis and the one upon which most of the arguments presented herein are based, the Gower Coefficient was chosen over other coefficients for generating similarity matrices because it is applicable to all types of characters or data, namely, two-state, multistate (ordered and qualitative), and quantitative.
Cluster analysis of the similarity matrix was accomplished using the Unweighted Pair Group Method with Arithmetic Averages (UPGMA) algorithm, also known as the Unweighted Average Linkage procedure, by running the SMATCLST sub-routine in TAXPAK.
The result of the cluster analysis is a dendrogram, a simplified version of which is provided in FIG. 1. The dendrogram illustrates the levels of similarity between the bacterial strains. The dendrogram is obtained by using the DENDGR program in TAXPAK.
The phenetic data, omitting multistate characters (characters 1-5, 11 and 12; Appendix C) and thus consisting of 193 unit characters, and scored in binary notation (positive=1, negative=0) were re-analyzed using the Jaccard Coefficient (S.sub.J) (Sneath, P. H. A. and Sokal, R. R., ibid, p. 131) by running the RTBNSIM program in TAXPAK. A further dendrogram was obtained by using the SMATCLST with UPGMA option and DENDGR sub-routines in TAXPAK. A simplified version of this dendrogram is illustrated in FIG. 2. Appendix E gives the percentage positive states of characters in each cluster.
Results of the Cluster Analysis
S.sub.G /UPGMA Method
FIG. 1 shows the results of cluster analysis, based on the Gower Coefficient and the UPGMA method, of 70 new, Gram-negative, alkaliphilic bacteria isolated from in and around alkaline lakes, together with 20 known Gram-negative bacteria.
Six natural clusters or phenons of alkaliphilic bacteria which include 65 of the 70 alkaliphilic strains are generated at the 73% similarity level. Although the choice of 73% for the level of delineation may seem arbitrary, it is in keeping with current practices in numerical taxonomy (Austin, B. and Priest, F., in Modern Bacterial Taxonomy, p. 37; Van Nostrand Reinhold; Wokingham, U.K., (1986)). Placing the delineation at a lower percentage would combine groups of clearly unrelated organisms while a higher percentage would produce a multitude of less well-defined clusters. At the 73% level, the individual clusters may represent separate bacterial genera. Furthermore, the significance of clustering at this level is supported by chemotaxonomic data (see below) and the pattern of clusters obtained using the Jaccard Coefficient (FIGS. 2A and 2B).
The significance of the clustering at the 73% level is supported by the results of the TESTDEN program. This program tests the significance of all dichotomous pairs of clusters (comprising 4 or more strains) in a UPGMA generated dendrogram with squared Euclidean distances, or their complement, as a measurement. The program assumes that the clusters are hyperspherical. The critical overlap was set at 0.25%. As can be seen from Table 5, the separation of the clusters is highly significant.
TABLE 5______________________________________Significance of the Clusters Generated by the S.sub.G /UPGMAMethod Provided by TESTDENCLUSTER separates from CLUSTER at Significance Level______________________________________1 2 p = 0.991 + 2 3 + 4 p = 0.991 + 2 + 3 + 4 5 + 6 p < 0.901 + 2 + 3 + 4 + 5 + 6 controls p = 0.995 6 p = 0.99______________________________________
A further measure of cluster separation can be estimated from the probability of cluster overlap. This was achieved using the OVERMAT program in TAXPAK with the critical overlap set out at 2.5%. As can be seen from Table 6, there is a greater than 95% probability of less than 2.5% overlap between the clusters. For many of the cluster combinations the overlap is effectively nil. Only Clusters 3 and 4 have a lower probability of <2.5% overlap, but these clusters may be clearly distinguished from one another on the basis of chemotaxonomic data (see below).
TABLE 6______________________________________Percentage Probability that Cluster Overlap is < 2.5%CLUSTER 1 2 3 4 5 6______________________________________2 953 99 954 95 95 905 99 >99 >99 >996 >99 >99 99 >99 >99______________________________________
The controls show that, as expected, the cluster analysis groups the Enterobacteriaceae separately. Additionally, the Aeromonas and Pseudomonas species, included as controls, also group separately. This is entirely consistent with the current taxonomy of these organisms (Bergey's Manual Of Systematic Bacteriology, Volume 1, Williams and Wilkins, Baltimore/London, 1984).
Five of the alkaliphilic strains fall outside the major clusters. Two strains, 4E.1 and 5E.1 form a separate but related pair and are obviously associated with the major groups of alkaliphilic bacteria. Strain wN2 is also unclustered but is apparently related to a Pseudomonas species and the major phenons of alkaliphilic bacteria. Strains 92LM.4 and wBn5 do not associate with the major alkaliphilic phenons and probably represent distinct groups of new alkaliphilic bacteria.
Clusters 1 and 2 are the only phenons which show an association with known organisms, i.e. Pseudomonas and Comamonas species. The separation of Pseudomonas putida and Pseudomonas stutzeri into separate taxa is entirely in keeping with the current taxonomic status of these organisms (Palleroni, N. J. et al, (1973), Int. J. Systematic Bacteriol., 23, 333-339; Gavini, F. et al., (1989), ibid, 39, 135-144; Bergey's Manual of Systematic Bacteriology, supra).
It was clear from the original dendrogram that Pseudomonas stutzeri is an outlier to Cluster 2 and is not closely related to the other members of the cluster. This is seen when the Euclidean distances of the strains from the centroid of the cluster are computed and used to calculate the cluster radius (Sneath, P. H. A. and Sokal, R. R., supra, pp. 194 et seq). The cluster radius is 3.91 (99% confidence level) and the mean distance of the strains from the centroid is 2.84 (standard deviation 0.46). Pseudomonas stutzeri at a distance from the centroid of 3.91 is clearly at the very boundary of phenetic hyperspace which defines Cluster 2.
A clear discrimination between Clusters 1 and 2 is possible using the concept of the minimum discriminatory tests (see below).
Each of the alkaliphilic strains in Cluster 2 have been examined by two independent laboratories expert in the identification of bacteria, namely, the German Culture Collection (DSM, Braunschweig, FRG) and the Laboratory for Microbiology at Delft University of Technology, The Netherlands. Neither of these laboratories was able to make a positive identification of the strains, although both agreed there was a resemblance with Pseudomonas either placing them in RNA homology group I (Palleroni, N. J. et al, supra) or more specifically in the Comamonas testosteroni/Pseudomonas alcaligenes or Pseudomonas pseudoalcaligenes groups (Gavini, F. et al, supra). However, no Pseudomonas species are known which are able to grow under the same highly alkaline conditions (pH 10) as the new strains described herein. An attempt was made to cultivate Pseudomonas pseudoalcaligenes.sup.T DSM 50188 and Pseudomonas alcaligenes.sup.T DSM 50342 in an alkaline broth medium (Medium A, Appendix A), but without success.
The results of these experts together with the discoveries described here, clearly indicate that these alkaliphilic strains in Clusters 1 and 2 represent new species of bacteria.
Clusters 3, 4, 5 and 6 are discrete phenons distinguished from each other on the basis of the minimum discriminatory tests (see below) and chemotaxonomic markers (see below). These phenons show no significant similarity with known groups of bacteria, and thus represent new genera or species.
Whole cell protein patterns generated by PAGE-electrophoresis indicate that a number of strains are likely to be identical. Examples include: 1E.1.sup.CT and 2E.1; 6B.1, 7B.1 and 8B.1; 45E.3.sup.CT and 47E.3. The dendrogram reveals that these strains are related at an average S.sub.G value of 92.3%, indicating a probable test error of 3.8% (Sneath, P. H. A. and Sokal, R. R., supra). Strains 73bC.4 and 74C.4, which appear to be closely related (90% S.sub.G), have similar but not identical gel patterns.
S.sub.J /UPGMA Method
The Jaccard coefficient is a useful adjunct to the Gower coefficient as it can be used to detect phenons in the latter generated by negative matches or distortions owing to undue weight being put on potentially subjective qualitative data. Consequently, the Jaccard coefficient is useful for confirming the validity of clusters defined initially by the use of the Gower coefficient. The Jaccard coefficient is particularly useful in comparing biochemically unreactive organisms (Austin, B., and Priest, F. G., supra, p. 37).
In the main, all of the clusters generated by the S.sub.G /UPGMA method are recovered in the dendrogram produced by the S.sub.J /UPGMA method (FIG. 2). Although the composition of the clusters is virtually identical in both dendrograms, a few strains have changed position. Non-clustering strains 4E.1 and 5E.1 move into Cluster 1/5, strains 42E.3 and 50N.3 move from Cluster 2 to Cluster 3/4. The strains wNk2, Pseudomonas stutzeri, Pseudomonas putida, wE5 become non-clustering.
Not surprisingly, the S.sub.J transformation combines (S.sub.G) Clusters 1 and 5. Both of these clusters are characterized as consisting of biochemically fairly unreactive strains. However, Clusters 1 and 5 are clearly distinct. Cluster 1 consists of strains producing cream/beige, circular colonies while the strains of Cluster 5 exclusively produce bright yellow, irregular colonies.
Furthermore, the S.sub.J transformation groups most of the strains of Cluster 4 with the strains of Cluster 3. However, it is evident from the chemotaxonomic data (see below), which shows that the strains of Cluster 4 contain Q9 and the strains of Cluster 3 contain mainly Q6, that these clusters should not be combined since they contain distinctly different strains. For these reasons, it is considered that the clustering produced by the S.sub.G /UPGMA method is the better representation of the actual taxonomic status of these strains. However, the S.sub.J /UPGMA serves to re-emphasize that with the single exception of a Comamonas species none of the known strains, not even the Pseudomonas control strains, bear significant resemblance to the clusters of the new alkaliphilic bacteria.
Chemotaxonomic Definition of the Clusters
Chemotaxonomy is the study of the chemical compositions of organisms in relation to their systematics. The analysis of chromosomal DNA, ribosomal RNA, proteins, cell walls and membranes, for example, can give valuable insights into taxonomic relationships and may be used as a further tool to construct or to verify the taxonomies of microorganisms (Goodfellow, M. and Minnikin, D. E. in Chemical Methods in Bacterial Systematics, (eds. Goodfellow, M. and Minnikin, D. E.), Academic Press, London and Orlando, Fla., (1985), pp. 1-15). However, it is not always possible to decide a priori which type of chemical information will be most diagnostic for a given classification. The amphipathic polar lipids, the major respiratory quinones, fatty acids located in the bacterial membranes and analysis of chromosomal DNA all have taxonomic significance for the classification of various bacteria (Lechevalier, H. and Lechevalier, M. P., in Microbial Lipids, volume 1 (eds. Ratledge, C. and Wilkinson, S. G.) Academic Press, London and San Diego, Calif., (1988), pp. 869-902).
Polar Lipids
The extraction of polar lipids from bacteria and their analysis by two dimensional thin layer chromatography (2D-TLC) may yield patterns of diagnostic value. Stationary phase cells were extracted in 1:1 (v/v) CHCl.sub.3 :CH.sub.3 OH and examined by 2D-TLC as described by Ross, H. N. H., Grant, W. D. and Harris, J. E., in Chemical Methods in Bacterial Systematics, (eds. Goodfellow, M. and Minnikin, D. E.), Academic Press, London and Orlando, Fla. (1985), pp. 289-300. The types of lipids present on the chromatograms were visualized using a variety of differential stains (Ross, H. N. M., et al., supra, p. 291; and Trincone, A., et al., J. Gen. Microbiol., (1990), 136, pp. 2327-2331). The identity of components were confirmed by co-chromatography with known lipids.
The results of this analysis for representative strains of Gram-negative alkaliphiles are set out in Table 7. These show no clear polar lipid pattern which is distinct for any one cluster. All strains contain phosphatidylglycerol, diphosphatidylglycerol, phosphatidylglycerol phosphate and phosphatidylethanolamine. In addition, certain strains, particularly in Cluster 3, contain phosphatidylglycerol sulphate (PGS). The distribution of PGS within Cluster 3 coincides broadly with the suspected sub-group structure of the cluster evident from the phenetic and other chemotaxonamic data. PGS is therefore a non-exclusive marker for Cluster 3.
We were surprised to find that a majority of the bacteria contained a glycolipid which on the basis of numerous co-chromatographic analyses appeared common to Gram-negative bacteria of the present invention. Glycolipids have not previously been demonstrated to be present in alkaliphilic bacteria (Krulwich, T. A., et al, CRC Critical Reviews in Microbiology, (1988), 16, 15-36). Furthermore, as judged by co-chromatography of lipids obtained from several strains, the glycolipid is also found in Gram-positive alkaliphiles isolated from soda lakes. It is possible therefore, that the chemical structure of the glycolipid may be a chemotaxonomic marker for the obligate alkaliphiles.
TABLE 7______________________________________Polar Lipid Componemts of Gram-Negative Alkaliphilic BacteriaCLUS-TER STRAIN PG DPG PGP PE PGS GL AL UPL______________________________________1 1E.1.sup.CT + + + + 2E.1 + + + + + 10B.1 + + + + 3+ 20N.1 + + + + + 27M.1 + + + + wNk.2 + + + + +2 39E.3 + + + + + 41E.3 + + + + + 45E.3.sup.CT + + + + + 51N.3 + + + + + 52N.3 + + + + + 42E.3 + + + + + 50N.3 + + + + + P.s. + + +-- 5E.1 + + + + + + +3 6B.1 + + + + + 7B.1 + + + + + 8B.1 + + + + + 25B.1 + + + + + 26N.1 + + + + + 12C.1 + + + + + + 28N.1.sup.CT + + + + + 36E.2 + + + + + + 40E.3 + + + + + + 94LM.4 + + + + + + 19N.1 + + + + + 24B.1 + + + + + 21M.1 + + + + + 29C.1 + + + + + 35E.2 + + + + + 37E.2 + + + + + + 48E.3 + + + + + + 73aC.4 + + + + + + 74C.4 + + + + + + 49E.3 + + + + + + 44E.3 + + + + + + 58E.4 + + + + +4 wE5 + + + + + wB4.sup.CT + + + + wNk1 + + + + + + wEl1 + + + + + + wEl2 + + + + +5 9B.1 + + + + 16N.1 + + + + 17N.1.sup.CT + + + + + 22M.1 + + + +6 18N.1 + + + + + 59E.4 + + + + 64B.4.sup.CT + + + + 63N.4 + + + + + 53E.4 + + + + +______________________________________ (PG) phosphatidylglycerol; (DPG) diphosphatidylglycerol; (PGP) phosphatidylglycerol phosphate; (PE) phosphatidylethanolamine; (PGS) phosphatidylglycerol sulphate; (GL) unidentified glycolipid(s), .alpha.-naphthol positive (the number in the column gives the number of positive spots on the TLC plate); (AL) unidentified aminolipid (ninhydrin positive); (UPL) unidentified phospholipid(s).
Isoprenoid Quinones
The isoprenoid or respiratory quinones are characteristic components of the plasma membrane of aerobic bacteria. There are two types; menaquinones and ubiquinones. The value of isoprenoid quinones as taxonomic criteria lies in the variation in the length of the polyprenyl side-chain and the degree of saturation (Collins, M. D. and Jones, D. (1981), supra).
Freeze dried stationary phase bacterial cells were extracted, using a modified procedure of Collins, M. D. (in Chemical Methods in Bacterial Systematics, supra, pp. 267-284), in 1:1 (v/v) CHCl.sub.3 :CH.sub.3 OH at 50.degree. C., for 16 hours. The quinones were examined by reverse phase thin layer chromatography as described by Collins, M. D. (supra).
The results of quinone analyses of nearly all the strains of Gram-negative alkaliphiles are illustrated in Table 8. All of the strains tested contained exclusively ubiquinones which confirms their status as Gram-negative bacteria (Collins, M. D. and Jones, D., supra). Table 8 shows quite clearly that the major ubiquinones are Q6 and Q9. It is also evident that the strains containing Q6 are exclusive to Cluster 3 and that this distinguishes Cluster 3 from all the other clusters since they contain strains possessing Q9 as the major ubiquinone.
TABLE 8______________________________________Major Respiratory Quinones of the StrainsArranged per Cluster______________________________________CLUSTER 1 CLUSTER 2 CLUSTER 3STRAIN Q STRAIN Q STRAIN Q______________________________________1E.1.sup.CT Q9 39E.3 Q9 6B.1 Q62E.1 Q9 41E.3 Q9 7B.1 Q6, Q9wB2 Q9 45E.3.sup.CT Q9 8B.1 Q6, Q9wB5 Q9 47E.3 Q9, Q10 38E.2 Q6wBs4 Q9 52N.3 Q9 56E.4 Q610B.1 Q9 42E.3 Q9 25B.1 Q6, Q920N.1 Q9 50N.3 Q9 26N.1 Q627M.1 Q9 12C.1 Q6C.t.* �Q8 + Q9(t)! 28N.1.sup.CT Q6wNk2 Q9 61N.4 Q6P.p.* �Q9! 36E.2 Q6 40E.3 Q6, Q9 65B.4 Q6 94LM.4 Q6, Q9 19N.1 Q6, Q9 24B.1 Q6, Q9 21M.1 Q6 29C.1 Q6 35E.2 Q6 37E.2 Q6 48E.3 Q6 78LN.4 Q6 73aC.4 Q6 75C.4 Q6 73bC.4 Q6 74C.4 Q6, Q9 77LN.4 Q6 49E.3 Q6 58E.4 Q6 57E.4 Q6______________________________________CLUSTER 4 CLUSTER 5 CLUSTER 6 NON-CLUSTERSTRAIN Q STRAIN Q STRAIN Q STRAIN Q______________________________________wE5 Q9 9B.1 Q9 18N.1 Q9 wN.2 Q9wB4.sup.CT Q9 16N.1 Q9 59E.4 Q9 5E.1 Q8wNk1 Q9 17N.1.sup.CT Q9, 64B.4.sup.CT Q9 92LM.4 Q9 Q10wE11 Q9, 22M.1 Q9 63N.4 Q9 wBn5 Q8, Q10 Q9(t) (t)wE12 Q9 22M.1 Q9 53E.4 Q9______________________________________ Q = Ubiquinone, the number indicates the number of sidechain isoprene units. (t) = trace *C.t. = Comomonas terrigena.sup.T NCIMB 8193, the quinone result is obtained from J. Tamaoka et al, International Journal of Systematic Bacteriology, 37, 52-59, (1987). P.p. = Pseudomonas putida.sup.T NCIMB 9494, the quinone result is obtaine from M. D. Collins and D. Jones, Microbiological Reviews, 45, 316-354, (1981).
Fatty Acids
The analysis of fatty acid profiles has had a significant impact on bacterial classification especially in the circumscription of genera and species among Gram-positive bacteria and actinomycetes (Kroppenstedt, R. M., in Chemical Methods in Bacterial Systematics (eds. M. Goodfellow and D. E. Minnikin), Academic Press; London and Orlando, Fla., (1985), pp. 173-199); Lechevalier, H. and Lechevalier, M. P., supra.
Freeze dried stationary phase cells (200-300 mg) were extracted for 16 hours at 75.degree. C. in toluene:methanol:conc. sulphuric acid (2.5 ml:2.5 ml:0.2 ml) and after cooling, the lipids were partitioned into hexane (twice times 1 ml). Residual acid was removed using NH.sub.4 HCO.sub.3. Lipid extracts were concentrated under O.sub.2 -free N.sub.2, dissolved in 300 .mu.l hexane and applied to preparative silica gel plates (Merck F254, Type T). The plates were developed in hexane: diethyl ether 85:15 (v/v) and the fatty acid methyl esters scraped off, extracted with hexane and concentrated under a stream of O.sub.2 -free N.sub.2.
The fatty acid methyl esters were dissolved in heptane and analyzed by gas chromatography using a Packard model 439 chromatograph equipped with flame ionization detectors. The samples were divided by a sample splitter and analyzed simultaneously over two columns, namely, CP-SIL-88 (Chrompack) (length 50 meter, internal diameter 0.22 mm) and Ultra-2 (Hewlett/Packard) (length 50 m, internal diameter 0.20 mm). The carrier gas was nitrogen; the injection temperature 120.degree. C.; temperature gradient 2.5.degree. C. per minute to 240.degree. C. and isothermal at 240.degree. C. for 30 minutes. Fatty acid methyl esters were assigned by reference to known standard mixtures. The identity of some peaks was confirmed by means of gas chromatography-mass spectrometry using a Carlo Erba HRGC 5160 Mega series gas chromatograph equipped with a CP-SIL-88 column (length 50 meter, internal diameter 0.22 mm) with helium as carrier gas and direct injection into the source of a AMD 403 mass spectrometer.
The fatty acid compositions of representative individual Gram-negative bacteria are set out in Table 9. Table 10 shows the unique fatty acid profiles of each of the clusters. Clusters 1, 2, 3 and 4 are fairly typical of the majority of Gram-negative bacteria where the major saturated fatty acid is C16:0 with lesser amounts of C14:0 and C18:0. The major unsaturated fatty acids in these alkaliphilic bacteria are C16:0 and C18:1 (11-cis), which is also typical, as is the lack of odd-numbered fatty acids (Wilkinson, S. G., in Microbial Lipids, volume 1 (eds. Ratledge, C. and Wilkinson, S. G.), Academic Press, London and San Diego, Calif., (1988), pp. 299-488). Minor amounts of C17:0 and C19:0 cyclopropane acids are found in some strains of Gram-negative bacteria. The strains of Cluster 3 exhibit fairly simple fatty acid profiles with C16:0 and C18:1 contributing 67-88% of the total acids, and C16:1 plus C18:0 up to 20% of the remainder. Even so, the fatty acid patterns support the notion that Cluster 3 contains several sub-groups, a conclusion that is also inferred from phenetic (numerical taxonomy) and polar lipid analyses (Table 7).
The strains of Cluster 1 can be distinguished from those of Cluster 2 on the relative abundance of straight chain saturated and unsaturated fatty acids, as well as the percentage amounts of C18:1(11-cis). The alkaliphilic bacteria of Clusters 1 and 2 have more complex fatty acid profiles than those of Cluster 3, with many more minor components. From the numerical taxonomy evidence, the alkaliphilic strains of Clusters 1 and 2 exhibit some resemblance to Pseudomonas species. However, the total lack of any hydroxy-fatty acids which are typical of most Pseudomonas species, further indicating that a close relationship is doubtful.
The strains of Clusters 5 and 6 are remarkable in that besides containing major amounts of C16:0, the other major fatty acids are odd-numbered branched chain acids (40-85%). Also, these strains lack significant amounts of C18:1 or any other unsaturated acids which are present in appreciable amounts in the alkaliphilic strains of Clusters 1, 2, 3 and 4. The presence of large amounts of C15:0 and C17:0 iso and anteiso acids is characteristic of only a very few classes of Gram-negative bacteria, notably species from exotic environments such as Thermus, or poorly defined taxa such as Flavobacterium (Wilkinson, S. G., supra). This result further emphasizes the novelty of the alkaliphilic strains of the present invention. The strains of Clusters 5 and 6 can be distinguished from each other by the proportion of branched-chain fatty acids they contain and more especially by the relative proportions of even- and odd-numbered fatty acids.
TABLE 9__________________________________________________________________________Fatty Acid Composition.sup.+ of Gram-Negative Alkaliphiles__________________________________________________________________________CLUSTER .fwdarw. 1 2 3FATTY ACID 1E.1.sup.CT 2E.1 45E.3.sup.CT 50E.3 25B.1 28N.1.sup.CT 36E.2 24B.1 37E.2 48E.3__________________________________________________________________________10:0 0.412:0 t t 3.6 4.7 0.5 0.5 0.3 t12:1 0.2 0.213:0 <0.114:0 0.7 0.7 3.8 3.9 4.5 4.4 3.4 2.6 2.9 2.614:0 iso14:1 2.6 0.9 0.115:0 0.3 0.6 0.9 0.7 0.6 0.9 0.315:0 iso 0.1 0.215:0 anteiso <0.1 0.315:0 cyclo16:0 29.0 32.0 28.5 34.4 37.3 24.1 42.0 36.7 33.3 26.016:0 iso 0.3 0.216:1 7.4 9.6 4.1 4.3 11.6 15.0 10.0 5.8 3.4 8.017:0 0.5 2.3 0.9 1.1 0.2 1.2 0.317:0 iso 0.2 0.617:0 anteiso17:0 cyclo 0.4.sup.a 1.8 1.8 0.317:1 0.3 1.6 0.617:1 br 1.718:0 12.0 4.7 17.9 10.8 8.9 0.2 0.9 2.0 0.6 1.118:0 unknown 0.2 0.418:1 9-cis 0.2 t 0.5 0.3 t t t 0.6 t18:1 9-trans 0.5 0.6 5.9 2.9 2.4 0.2 t 1.0 0.6 0.618:1 11-cis 42.0 44.0 23.9 24.1 27.5 54.2 43.0 50.6 41.6 57.718:1 unknown 0.2 0.318:2 0.5 1.9 2.7 0.5 t t19:0 0.119:0 cyclo 1.3 0.4 12.9 2.019:1 br20:0 0.4 5.3 2.7 2.420:1 3.6 1.222:0 3.3 1.5 1.424:0 0.2__________________________________________________________________________CLUSTER .fwdarw. 4 5 6 non-clusteringFATTY ACID WB4.sup.CT WEl1 9B.1 16N.1 17N.1.sup.CT 59E.4 64B.4.sup.CT 5E.1 92LM.4__________________________________________________________________________10:0 0.512:0 0.9 0.212:1 0.813:014:0 3.8 1.3 3.2 1.9 1.2 3.9 6.4 3.8 1.514:0 iso 0.7 t t 0.7 2.014:1 0.115:0 0.7 0.2 t 3.8 t15:0 iso 8.7 9.3 8.2 3.9 12.6 44.015:0 anteiso 32.3 27.1 35.3 24.8 18.1 9.915:0 cyclo <0.116:0 26.8 26.9 22.5 17.4 12.5 26.3 40.5 74.7 18.716:0 iso 4.7 5.8 6.7 2.4 3.7 2.116:1 7.9 2.4 2.917:0 0.4 0.3 t 2.417:0 iso 0.3 3.2 6.0 5.1 0.6 1.7 15.717:0 anteiso 21.9 24.4 29.8 6.1 3.7 6.817:0 cyclo 0.217:1 <0.117:1 br18:0 5.2 3.5 2.3 5.3 1.2 16.2 4.8 2.6 1.518:0 unknown 5.918:1 9-cis t 2.518:1 9-trans 1.7 0.9 *0.5 *1.6 *1.0 *5.7 *0.5 *10.018:1 11-cis 47.4 48.118:1 unknown18:2 1.0 0.3 2.719:019:0 cyclo 1.019:1 br 11.220:0 1.3 0.4 4.620:1 0.322:0 0.8 0.2 2.624:0__________________________________________________________________________ *includes all C18:1 isomers .sup.+ % total fatty acids t = trace br = branched .sup.a C17:0 cyclo or C18:0 unknown
TABLE 10__________________________________________________________________________Fatty Acid Profiles of the Clusters of Gram-Negative Alkaliphiles Cluster 1 2 3A/B 3C 3D 4 5 6__________________________________________________________________________Predominant Fatty C16:0 C16:0 C16:0 C16:0 C16:0 C16:0 C15:0 anteiso C15:0 brAcids (>10%) C18:1 11-cis C18:0 C16:1 C18:1 11-cis C18:1 11-cis C18:1 11-cis C16:0 C16:0 C18:1 11-cis C18:1 11-cis C17:0 anteison-saturated .about.40% 60-65% 30-55% .about.40% 30-40% 30-40% 15-30% 55-60%n-unsaturated .about.60% .apprxeq.33% 45-70% .about.60% 50-65% 50-60% <2% <10%iso <1% <1% 0% 0% 0% <1% .about.20% 10-20%anteiso 0% <1% 0% 0% 0% 0% 50-65% 20-30%total branched <1% <3% 0% 0% 0% 1-12% >70% .about.40%cyclo 0% <5% 0% 2-15% <2% 0% 0%even carbon no. >95% >90% >99% >99% >80% >85% 20-35% 55-65%odd carbon no. <5% <10% <1% <1% <20% <15% 65-80% 35-45%additional markers C17:0 cyclo C17:0 cyclo C19:0 cyclo C19:0 cyclo C17:1 br__________________________________________________________________________ br = branched
Nucleic Acids
An essential component of any taxonomic study is analysis of the genetic material--the nucleic acids. The composition of chromosomal DNA is unaffected by the growth conditions of the organism and an appropriate analysis may confirm or refute the taxonomic position of the organism. Chromosomal DNA may be analyzed by the determination of the base composition (G+C mol %) of individual strains, and the base sequence homologies between pairs of strains by DNA-DNA reassociation (hybridization) (Owen, R. J. and Pitcher, D., in Chemical Methods in Bacterial Systematics (eds. M. Goodfellow and D. E. Minnikin), Academic Press, London and Orlando, Fla. (1985), pp. 67-93).
DNA Base Composition
The guanine plus cytosine (G+C mol %) composition is constant for the chromosomal DNA from any given organism. Closely related organisms have similar G+C compositions. However, G+C results must be interpreted within the context of independent taxonomic data since similar G+C mol % of DNA samples from different organisms does not in itself imply biological relatedness.
DNA was extracted from cells grown to exponential phase in Medium A by the chloroform:phenol method and was precipitated with ethanol. Base composition was determined by the thermal denaturation method (Marmur, J. and Doty, P. (1962), J. Mol. Biol., 3, 585-594) on a Phillips model Pv8764 spectrophotometer with temperature programming. A second method involved HPLC analysis on a Beckman system gold using a Beckman ultrasphere ODS column and 0.04M potassium dihydrogen phosphate plus acetonitrile (9+1, v/v) as eluent at a flow rate of 1.5 ml/min, after treatment of the DNA with nuclease P1 and alkaline phosphatase.
The results of these analyses are set out in Table 11. The G+C mol % values for the alkaliphilic bacteria cover a range of 30 mol % (37.6-67.1 mol %). However, within the clusters the variation is only 3-7 mol %, which further confirms that the strains within a cluster are closely related to each other.
TABLE 11______________________________________DNA Base Composition of Gram-NegativeAlkaliphilic Bacteria G + C mol %Cluster Strain HPLC T.sub.M______________________________________1 2E.1 55.2 wBs4 51.2 20N.1 51.1 wNk2 53.02 42E.2 62.73 wB1 63.0 28N.1.sup.CT 64.1 37E.2 67.1 wN1 64.84 wE5 58.5 wB4.sup.CT 65.3 wNk1 61.0 wEl1 59.7 wEl2 58.15 17N.1.sup.CT 50.0 22M.1 43.86 64B.4.sup.CT 41.0 53E.4 37.6non wN2 64.1 wBn5 54.6______________________________________
DNA-DNA Molecular Hybridization
The method used was essentially that of Crosa, J. H. et al. (Int. J. Systematic Bacteriol., 29, 328-332, 1979). Tritium labelled DNA was prepared using a nick-translation kit (Amersham, N5000) according to the manufacturer's instructions. The reassociation mixtures were incubated at 65.degree. C. for 16 hours. The results are set out in Table 12 from which it can be seen that the DNA sequence homology is higher within the clusters that between the clusters.
TABLE 12______________________________________Inter-Cluster and Intra-Cluster DNA--DNA HomologyValues for Gram-Negative Alkaliphilic BacteriaCluster 1 2 3 4 5 6Strain 2E.1 45E.3.sup.CT 28N.1.sup.CT wEl2 17N.1.sup.CT 64B.4.sup.CT______________________________________1 2E.1 100 33 35 25 25 20N.1 562 45E.3.sup.CT 100 25 42E.3 76 303 28N.1.sup.CT 20 34 100 26 30 30 56E.4 51 21M.1 53 37E.2 37 44E.3 554 wEl2 100 wB4.sup.CT 41 435 17N.1.sup.CT 45 44 36 24 100 44 22M.1 656 64B.4.sup.CT 21 31 35 25 34 100 53E.4 60 SS 17 20 20______________________________________ The values give the percent hybridization between the strains (rows) and H.sup.3 -labelled strains (columns). SS = salmon sperm DNA.
Determination of Representative Strains
The centroid of each individual cluster generated by the S.sub.G /UPGMA method was computed using the RGROUPS program in TAXPAK. The centroid of a cluster of points representing real organisms projected into hyperspace represents a hypothetical average organism. The centroid rarely, if ever represents a real organism. Therefore, the Euclidean distances of each of the members of the cluster from the centroid of the cluster were calculated in order to establish which strain was closest to the hypothetical average organism. The strain closest to the centroid was designated the "centrotype" organism (indicated with the superscript "CT").
The centrotype organism can be thought of as the "Type Strain" which most closely represents the essential and discriminating features of each particular cluster. The centrotype strains are recorded in Table 13.
TABLE 13______________________________________Centrotype Strains Mean Euclidean Centrotype Distance of Euclidean Number of Strains DistanceCluster Strains in from Standard fromNumber Cluster Centroid Deviation Strain Centroid______________________________________1 11 3.67 0.30 1E.1 2.882 9 3.20 0.52 45E.3 2.303 34 3.52 0.32 28N.1 2.904 5 3.97 0.29 wB4 2.875 4 3.25 0.29 17N.1 2.136 5 3.11 0.41 64B.4 1.93______________________________________
A description of each of the centrotype organisms has been made so as to be able to distinguish these organisms from all other bacteria previously known and described. In addition, the minimum number of discriminating tests to define each cluster has been computed so that it may be clearly seen that the clusters containing these novel bacteria can be easily distinguished from each other and from all other known bacteria.
Description of Centrotype Strains
Strain 1E.1.sup.CT (Cluster 1)
An aerobic, motile, Gram-negative rod-shaped bacterium, 1.7-3.3 .mu.m.times.0.5-0.7 .mu.m.
Obligate alkaliphile, grows best between pH 9 and pH 10.
On alkaline-agar, (Medium A) forms smooth, cream colored colonies, initially translucent but becoming opaque after a few days. The colonies are circular, entire and convex, 2-3 mm in diameter.
In alkaline-broth, (Medium A) growth (37.degree. C.) is flocculent with the formation of a sediment and surface pellicle.
Grows well between 20.degree. C. and 40.degree. C. Grows slowly at 10.degree.-15.degree. C. No growth at 8.degree. C. or 45.degree. C.
______________________________________KOH test: positiveAminopeptidase: weak positiveOxidase: negativeCatalase: positiveNaCl tolerance: 0% to <8%. No growth at 8%Hydrolysis of Gelatin: positiveHydrolysis of Starch: positiveMajor polar lipid components: phosphatidylglycerol diphosphatidylglycerol phosphatidylglycerol phosphate phosphatidylethanolamineMajor ubiquinone: Q9Major fatty acids: C16:0, C18:0, 11-cis C18:1______________________________________
Chemoorganotroph. Grows on complex substrates such as yeast extract and peptones. Growth on simple sugars and organic acids very restricted (e.g., growth only observed on ribose, sucrose and pyruvate).
Strain 45E.3.sup.CT (Cluster 2)
An aerobic, Gram-negative, rod-shaped bacterium, 3-4.5 .mu.m.times.0.6 .mu.m. Motile by a single polar flagellum.
Obligate alkaliphile growing between pH 7.8 and pH 11.2. On alkaline-agar, (Medium A) forms smooth, opaque, cream colored colonies, 1-2 mm in diameter. The colonies are circular, convex and entire.
In alkaline-broth, (Medium A) growth (37.degree. C.) is slow, slight with an even turbidity, surface pellicle and no sediment.
Grows well between 20.degree. C. and 40.degree. C. Grows slowly at 10.degree. C. No growth at 8.degree. C. or 45.degree. C.
______________________________________KOH test: positiveAminopeptidase: positiveOxidase: positiveCatalase: positiveNaCl tolerance: 0% to 12%. Growth at 12% is slow No growth at 15%Hydrolysis of Gelatin: positiveHydrolysis of Starch: positive (weak)Major polar lipid components: phosphatidylglycerol diphosphatidylglycerol phosphatidylglycerol phosphate phosphatidylethanolamine glycolipid (.alpha.-naphthol positive)Major ubiquinone: Q9Major fatty acids: C16:0, C18:0, 11-cis C18:1______________________________________
Chemoorganotroph. Grows on complex substrates such as yeast extract and peptones. No growth on simple sugars. Grows on organic acids (e.g., fumarate, succinate, pyruvate, acetate, lactate) and some fatty acids (e.g., propionate, valerate) and amino acids (e.g., proline, alanine, phenylalanine).
Strain 28N.1.sup.CT (Cluster 3)
An aerobic, motile, Gram-negative, rod-shaped bacterium, 4.8-5.5 .mu.m.times.0.6-0.8 .mu.m. Obligate alkaliphile growing between pH 8.5 and pH 10.7.
On alkaline-agar, (Medium A) forms smooth, circular, opaque colonies with a stringy texture. The colonies have a convex elevation and entire margin. The colony color is initially cream/beige becoming pink after a few days.
In alkaline-broth, (Medium A) growth (37.degree. C.) is heavy, flocculent with a surface pellicle and a sediment.
Grows well between 20.degree. C. and 45.degree. C. Grows slowly at 10.degree. C. and 15.degree. C. No growth at 50.degree. C.
______________________________________KOH test: positiveAminopeptidase: positiveOxidase: positiveCatalase: positiveNaCl tolerance: 0% to 12%. No growth at 15%Hydrolysis of Gelatin: negativeHydrolysis of Starch: positiveMajor polar lipid components: phosphatidylglycerol diphosphatidylglycerol phosphatidylglycerol sulphate phosphatidylethanolamineMajor ubiquinone: Q6Major fatty acids: C16:0, C16:1, 11-cis C18:1G + C: 64.1 mol % (HPLC)______________________________________
Chemoorganotroph. Grows well on complex substrates such as yeast extract and peptones. Grows on simple sugars, organic acids, fatty acids and amino acids.
Strain wB4.sup.CT (cluster 4)
An aerobic, Gram-negative, rod-shaped bacterium, 3-4 .mu.m.times.0.6-0.8 .mu.m, frequently occurring as pairs of cells.
Alkaliphile, grows well between pH 7.5 and pH 10.9.
On alkaline-agar, (Medium A) forms smooth, beige to brown colonies. The colonies are somewhat variable: 1 to >5 mm in size, circular to irregular in form, low convex or raised in elevation with an undulate or entire margin.
In alkaline broth, (Medium A) growth (37.degree. C.) is flocculent, sediment forming with a surface pellicle.
Grows best between 15.degree. C. and 45.degree. C., no growth at 50.degree. C.
______________________________________KOH test: positiveAminopeptidase: positiveOxidase: very weakly positive, may be seen as negativeCatalase: positiveNaCl tolerance: 0% to .gtoreq.12%, no growth at 15%Hydrolysis of Gelatin: negativeHydrolysis of Starch: negativeMajor polar lipid components: phosphatidylglycerol diphosphatidylglycerol phosphatidylglycerol phosphate phosphatidylethanolamineMajor ubiquinone: Q9Major fatty acids: C16:0, 11-cis C18:1G + C: 65.3 mol % (T.sub.M)______________________________________
Chemoorganotroph. Grows well on complex substrates such as yeast extract. Growth on simple sugars is restricted. Grows on organic acids (e.g., lactate, acetate, fumarate), fatty acids (e.g., propionate, valerate, caprate) and amino acids (e.g., proline, serine, lysine).
Strain 17N.1.sup.CT (cluster 5)
An aerobic, Gram-negative, long, thin, rod-shaped, bacterium, 5.5-10.5 .mu.m.times.0.6 .mu.m, sometimes forming short chains of cells. With age pleomorphic, peculiar swollen forms predominate.
Obligate alkaliphile, grows best between pH 8 and pH 10.5.
On alkaline-agar, (Medium A) forms smooth, opaque, yellow, colonies, 2-3 mm in diameter. The colonies vary from circular to irregular in form, with a convex to umbonate elevation, and entire, undulate or lobate margin, depending upon age.
In alkaline-broth (Medium A), growth (37.degree. C.) is even and sediment forming with no surface pellicle.
Grows well between 15.degree. C. and 37.degree. C. Grows slowly at 10.degree. C. and not at all at 8.degree. C. No growth at 40.degree. C. or above.
______________________________________KOH test: positiveAminopeptidase: weakly positiveOxidase: negativeCatalase: positiveNaCl tolerance: 0% to <12%, grows best at 0% NaClHydrolysis of Gelatin: positiveHydrolysis of Starch: weakly positiveMajor polar lipid components: phosphatidylglycerol diphosphatidylglycerol phosphatidylglycerol phosphate phosphatidylethanolamine glycolipid (.alpha.-naphthol positive)Major ubiquinone: Q9, Q10Major fatty acids: C15:0 anteiso, C16:0, C17:0 anteisoG + C: 50.0 mol % (HPLC)______________________________________
Chemoorganotroph. Grows well on complex substrates such as yeast extract. Growth on simple sugars is restricted (e.g., growth observed only on fructose; no growth observed on glucose, ribose, lactose). Grows on organic acids (e.g., fumarate, succinate, pyruvate, 2-ketogluconate) and amino acids.
Strain 64B.4.sup.CT (cluster 6)
An aerobic, Gram-negative, rod-shaped bacterium 2.0-3.5 .mu.m.times.0.8-1.0 .mu.m.
Obligate alkaliphile, grows best between pH 8.2 and pH 10.9.
On alkaline-agar, (Medium A) forms smooth, opaque colonies, first creamy yellow in color, becoming beige with age. The colonies are about 4 mm in diameter, circular becoming irregular; flat or low convex in elevation becoming convex; with an entire margin becoming undulate.
In alkaline-broth (Medium A), growth (37.degree. C.) is sediment forming with no surface pellicle.
Grows well at 15.degree. C. to 45.degree. C., no growth at 10.degree. C. or 50.degree. C.
______________________________________KOH test: positiveAminopeptidase: negativeOxidase: positiveCatalase: positiveNaCl tolerance: 0% to .ltoreq.12%, no growth at 15%Hydrolysis of Gelatin: positiveHydrolysis of Starch: weakly positiveMajor polar lipid components: phosphatidylglycerol diphosphatidylglycerol phosphatidylglycerol phosphate phosphatidylethanolamine glycolipid (.alpha.-naphthol positive)Major ubiquinone: Q9Major fatty acids: C15:0 iso, C15:0 anteiso, C16:0G + C: 41.0 .+-. 0.9 mol % (HPLC)______________________________________
Chemoorganotroph. Grows well on complex substances such as yeast extract. Grows on some simple sugars (e.g., glucose, ribose, maltose and fructose), organic acids (e.g., acetate, lactate, citrate and fumarate), some fatty acids (e.g., propionate and caprate) and amino acids (e.g., proline, histidine and alanine).
Non-Clustering Strains
The strains which do not fall into the clusters defined here are also novel bacteria not previously known or described. These strains, coded wN2, 4E.1, 5E.1, 92LM.4 and wBn5, may represent rarer varieties of alkaliphilic bacteria and are probably members of clusters of bacteria representing new genera and species at present not described. A description of these "non-clustering" strains has been made so as to be able to distinguish these organisms from all other bacteria previously known and described.
Strain wN2
An aerobic, Gram-negative, motile, rod-shaped bacterium, frequently in pairs.
Obligate alkaliphile, grows best between pH 9 and pH 10.
On alkaline-agar, (Medium A) forms smooth, translucent, beige colored colonies, 1-2 mm in diameter. The colonies are circular, convex with an entire margin.
In alkaline-broth (Medium A), growth (37.degree. C.) is flocculent with a ring or surface pellicle and formation of a sediment.
Grows well at 20.degree. C. to 30.degree. C. No growth at 15.degree. C. or 40.degree. C.
______________________________________KOH test: positiveAminopeptidase: weak positiveOxidase: weak positiveCatalase: positiveNaCl tolerance: obligate halophile, growth at 4% NaCl no growth at 0% or 8% NaClHydrolysis of Gelatin: slow positiveHydrolysis of Starch: positiveMajor ubiquinone: Q9G + C: 64.1 (T.sub.M)______________________________________
Chemoorganotroph. Metabolically unreactive. No growth on simple sugars or organic acids. Grows on complex substrates such as yeast extract and peptones, and on some amino acids.
Strain 4E.1
An aerobic, Gram-negative, motile, rod-shaped bacterium, 1.7-5.2 .mu.m.times.0.75 .mu.m.
Obligate alkaliphile, grows best between pH 8.2 and pH 10.9.
On alkaline-agar, (Medium A) forms smooth, opaque, beige or brown colored colonies, 2-4 mm in diameter. The colonies are circular in form, convex in elevation, with an entire margin.
In alkaline-broth (Medium A), growth (37.degree. C.) is heavy and flocculent with a sediment and surface pellicle.
Grows well between 20.degree. C. and 37.degree. C. Grows very slowly at 10.degree. C. and not at all at 8.degree. C. No growth at 40.degree. C. or above.
______________________________________KOH test: positiveAminopeptidase: positiveOxidase: very weakly positive, can appear negativeCatalase: positiveNaCl tolerance: 0% to 12%, may grow weakly at 15% no growth at 20%Hydrolysis of Gelatin: negativeHydrolysis of Starch: negative______________________________________
Chemoorganotroph. Does not grow on simple sugars, except for ribose. Grows well on complex substrates such as yeast extract, and on organic acids (e.g., succinate, pyruvate, citrate, malonate, acetate and lactate), fatty acids (e.g., propionate, valerate and suberate), and amino acids (e.g., proline, serine, histidine and lysine).
Strain 5E.1
An aerobic, Gram-negative, rod-shaped bacterium, 3.0-5.3 m.times.1.3 .mu.m.
Obligate alkaliphile, grows best between pH 9 and pH 10.5.
On alkaline-agar, (Medium A) forms smooth, opaque, brown colored colonies, 3-4 mm in diameter. The colonies are fairly irregular in form, generally flat to slightly umbonate in elevation with a lobate margin.
In alkaline-broth (Medium A), growth (37.degree. C.) is moderate to heavy, becoming flocculent with a sediment and surface pellicle.
Grows well between 20.degree. C. and 40.degree. C. Grows slowly at 10.degree. C. No growth at 45.degree. C.
______________________________________KOH test: positiveAminopeptidase: positiveOxidase: negativeCatalase: positiveNaCl tolerance: 0% to 12%, may grow weakly at 15% no growth at 20%Hydrolysis of Gelatin: positiveHydrolysis of Starch: weakly positiveMajor polar lipid components: phosphatidylglycerol diphosphatidylglycerol phosphatidylglycerol phosphate phosphatidylglycerol sulphate phosphatidylethanolamine glycolipid (.alpha.-naphthol positive)Major ubiquinone: Q8Major fatty acids: C16:0, C18:1______________________________________
Chemoorganotroph. Does not grow on simple sugars. Grows well on complex substrates such as yeast extract, organic acids (e.g., pyruvate, citrate, acetate and lactate), fatty acids (e.g., propionate, caprate and valerate) and amino acids (e.g., proline, alanine and lysine).
Strain 92LM.4
An aerobic, Gram-negative, rod-shaped bacterium, 2.0-3.5 .mu.m.times.0.5-1.0 .mu.m.
Obligate alkaliphile, no growth below pH 7.5.
On alkaline-agar, (Medium A) forms smooth, cream colored colonies, initially translucent but becoming opaque. The colonies develop from circular, entire to irregular, lobate in form, with a convex elevation.
In alkaline-broth (Medium A), growth (37.degree. C.) is slow, slight, flocculent with a sediment but no surface pellicle.
Grows between 10.degree. C. and 40.degree. C., no growth at 8.degree. C. or 45.degree. C.
______________________________________KOH test: positiveAminopeptidase: negativeOxidase: positiveCatalase: positiveNaCl tolerance: 0% to 15%, growth at 15% is slow no growth at 20%Hydrolysis of Gelatin: positiveHydrolysis of Starch: weakly positiveMajor ubiquinone: Q9Major fatty acids: C15:0 iso, C15:0 anteiso, C16:0, C17:0 iso______________________________________
Chemoorganotroph. Grows on complex substrates such as yeast extract and peptones, and a variety of sugars, organic acids and amino acids.
Strain wBn5
An aerobic, Gram-negative, small, rod-shaped bacterium, frequently forming short chains of cells.
Obligate alkaliphile, no growth below pH 8.
On alkaline-agar, (Medium A) forms smooth, circular, convex colonies with an entire margin, about 1 mm in diameter. The colonies are initially cream/beige, transparent becoming opaque, brown.
In alkaline-broth (Medium A), growth (37.degree. C.) is initially evenly turbid with a sediment but no surface pellicle becoming after 4 days flocculent with formation of a pellicle.
Grows at 30.degree. C. and 37.degree. C. No growth at 40.degree. C.
______________________________________KOH test: positiveaminopeptidaes: positiveOxidase: positiveCatalase: positiveNaCl tolerance: obligate halophile. Growth at 4% NaCl no growth at 0% or 8%Hydrolysis of Gelatin: slow positiveHydrolysis of Starch: negativeMajor ubiquinone: Q8, Q9G + C: 54.6 mol % (T.sub.M)______________________________________
Chemoorganotroph. Grows on a range of complex substrates such as yeast extract and peptones, as well as sugars, organic acids, fatty acids and amino acids.
Cluster Definition by the Calculation of the Minimum Number of Discriminatory Tests, and the Construction of a Probability Matrix for the Identification of Gram-Negative Alkaliphiles
One of the purposes of a numerical classification study is to use the phenetic data, which defines the clusters at a selected similarity level, for the assignment or identification of unknown strains. The classification test data cam be used to determine the minimum set of tests which are required to define the clusters at the 73% (S.sub.G) similarity level, and to identify those characters which are most diagnostic (predictive) for the individual clusters. In other words, the minimum number of tests required to assign an unknown organism to a pre-determined cluster with a high degree of predictability.
From the minimum discriminatory tests, a probability matrix can be constructed for the identification of unknown strains. The analysis is achieved by using a combination of the CHARSEP and DIACHAR (TAXPAK) and MCHOICE (not on TAXPAK but available by Data-Mail from the University of Leicester, U.K.) programs. An evaluation of the identification matrix is provided by using the MOSTTYP and OVERMAT programs. Practical examples of the use of these programs for the probabilistic identification of bacteria have been published by Williams, S. T., et al., (1983), J. Gen. Microbiol., 129, pp. 1815-1830; and Priest, F. G. and Alexander, B., (1988), J. Gen. Microbiol., 134, pp. 3011-3018; ibid, (1990), 136, pp. 367-376.
A "n.times.t" table was constructed from the test data using characters 6 to 10 and 13 to 104 (Appendix C) scored in binary notation (positive=1, negative=0). This data matrix was supplemented with the following four extra character states:
�105! Bright yellow colonies (character number 1, Appendix C)
�106! Translucent colonies (grown on Medium A, Appendix A)
�107! Lipase (lipolytic activity on olive oil (Medium M))
�108! Oxidase positive within 10 secs. (test 9, Appendix B)
The data matrix is first examined using the CHARSEP program which calculates separation indices and thus the diagnostic value of the individual characters for discriminating between the clusters. Tests with a VSP index>25% (Sneath, P. H. A., (1979), Computers and Geosciences, 5, 349-357) are accepted, characters with a low diagnostic value (VSP<25%) were rejected. A preference is made for characters with the highest VSP indices, provided that the criteria in the DIACHAR and MCHOICE programs are also met. In this example, 38 tests have a VSP index >25%, and 9 of the 24 characters finally chosen have a VSP index >50% (Table 11).
The data matrix is next re-examined by means of the DIACHAR program, which determines the most diagnostic character states of each of the clusters. The number of character states was set at 10. This result allows the choice of mutually exclusive character states between the clusters. As many of these tests as possible are retained in the final identification matrix of minimum discriminatory tests; in this example between 6 and 9 diagnostic characters per cluster. The remaining, unused tests are also noted and may be applied as additional tests for the confirmation of identification (Table 12).
The MCHOICE program ranks the tests in groups which can be displayed in the form of a dendrogram using the MDEND sub-routine. The groups identify tests with similar discriminatory value, thus allowing the rejection of tests which fail to make a significant discrimination as well as allowing choices to be made between tests of equal or very similar diagnostic value.
Table 13 shows the set of 24 tests which is the minimum number required to define the clusters and which can be used for the assignment of unknown strains. In addition, Table 13 shows the identification matrix which consists of the percentage of positive characters which define the clusters on the basis of the 24 minimum discriminatory tests. This is computed by the IDMAT program.
TABLE 14______________________________________Separation Values of Characters used for theMinimum Discriminatory TestsCHARACTER VSP Index______________________________________ �23! N-acetylglucosamine 35.4 �26! Saccharose 44.8 �27! Maltose 41.4 �32! Lactate 51.6 �41! Propionate 60.9 �43! Valerate 63.4 �44! Citrate 45.1 �45! Histidine 38.0 �47! Glycogen 31.7 �51! 3-hydroxybutyrate 66.1 �52! 4-hydroxybenzoate 38.0 �58! Leucine arylamidase 36.6 �59! Valine arylamidase 50.5 �64! Phosphohydrolase 52.8galactosidase 33.9 �85! Ampicillin 36.8 �92! Fusidic Acid 68.7 �93! Methicillin 58.3 �99! Polymixin 62.8�102! Vancomycin 48.3______________________________________
TABLE 15______________________________________Discriminatory Tests for Each of the Six Clusters (S.sub.G)Positive Negative______________________________________Cluster 1: cream, circular, opaque, mucoid colonies. �58! Leucine arylamidase (91%) �23! N-acetylglucosamine (9%) �59! Valine arylamidase (91%) �27! Maltose (9%) �64! Phosphohydrolase (91%) �41! Propionate (9%) �99! Polymixin (89%) �42! Caprate �43! Valerate (9%) �44! Citrate (9%) �45! Histidine �47! Glycogen (9%) �52! 4-hydroxybenzoategalactosidase �65! .alpha.Cluster 2: small, cream, translucent colonies. �21! Starch �23! N-acetylglucosamine �31! Acetate �26! Saccharose �41! Propionate �45! Histidine �43! Valerate �50! 2-ketogluconate �53! Proline �52! 4-hydroxybenzoateglucosidasee �68! .alpha.�108! Oxidase (within 10 secs.)glucosidase �69! .beta. �92! Fusidic AcidCluster 3: cream, opaque colonies. �31! Acetate �64! Phosphohydrolase (3%)galactosidase (3%) �65! .alpha. �41! Propionate (94%) �92! Fusidic Acid (3%) �43! Valerate (97%) �96! Tetracycline (3%) �44! Citrate (94%) �102! Vancomycin �51! 3-hydroxybutyrate (94%) �104! Bacitracin �53! Proline �58! Leucine arylamidase (94%)Cluster 4: beige to brown, opaque colonies. �32! Lactate �45! Histidine �33! Alanine �85! Ampicillin �48! 3-hydroxybutyrate �86! Naladixic acid �59! Valine arylamidase �88! Trimethoprim �99! Polymixin �89! Penicillin G �93! MethicillinCluster 5: bright yellow �105!, opaque colonies. �64! Phosphohydrolase �23! N-acetylglucosaminegalactosidase �32! Lactategalactosidase �33! L-alanine �85! Ampicillin �34! Mannitol �92! Fusidic Acid �41! Propionate �93! Methicillin �42! Caprate �96! Tetracyclinee �43! Valerate�102! Vancomycin �45! Histidine�104! Bacitracin �48! 3-hydroxybenzoate �51! 3-hydroxybutyrate �52! 4-hydroxybenzoate �99! PolymixinCluster 6: cream, irregular, flat colonies. �21! Starch �17! Pyruvate �23! N-acetylglucosamine �52! 4-hydroxybenzoate �26! Saccharose �58! Leucine arylamidase �27! Maltose �59! Valine arylamidasegalactosidase �65! .alpha. �33! Alanine �99! Polymixin �44! Citrate �47! Glycogen �51! 3-hydroxybutyrate �89! Penicillin G �92! Fusidic Acid �93! Methicillin �96! Tetracyclinee�104! Bacitracin______________________________________ Note: The numbers in square brackets proceeding the character state refers to the character states and unit tests in Appendices B and C. The percentage in parenthesis refers to positive character states.
TABLE 16__________________________________________________________________________A Probability Matrix for the Identification of Alkaliphiles:Percentage Distribution of Positive Discriminatory CharactersWhich Define the Clusters of Gram-Negative Alkaliphilic Bacteriaat the 73% Level (S.sub.G) CLUSTERTEST 1 2 3 4 5 6__________________________________________________________________________ �23! N-acetylglucosamine 13 0 26 20 0 100 �26! Saccharose 25 0 74 20 25 100 �27! Maltose 25 0 68 60 50 100 �32! Lactose 38 50 100 100 0 40 �41! Propionate 0 100 91 60 0 80 �43! Valerate 13 100 97 80 0 40 �44! Citrate 13 50 94 20 50 100 �45! Histidine 0 0 71 0 0 80 �47! Glycogen 0 13 26 20 25 100 �51! 3-hydroxybutyrate 13 25 94 100 0 100 �52! 4-hydroxybenzoate 0 0 71 80 0 0 �58! Leucine arylamidase 88 63 94 60 50 0 �59! Valine arylamidase 88 25 65 100 25 0 �64! Phosphohydrolase 88 13 3 20 75 40galactosidase 0 0 3 20 75 0 �85! Ampicillin 50 63 56 0 100 80 �92! Fusidic Acid 25 0 3 20 100 100 �93! Methicillin 50 13 50 0 100 100 �99! Polymixin 88 50 81 100 0 0�102! Vancomycin 13 13 3 20 100 75�105! Yellow colony 0 0 0 0 100 0�106! Translucent colony 0 100 3 0 0 0�107! Lipase 0 100 21 0 0 0�108! Oxidase (10 secs) 25 88 6 0 0 0__________________________________________________________________________
Evaluation of the Discriminatory Tests and Assessment of the Reliability of Identification
The evaluation of the discriminatory tests has two aspects. Firstly, the validity of the tests can be analyzed using practical examples, which can be further evaluated using statistical theory, or the tests can be directly subjected to theoretical assessment using statistical methods.
Illustration 1
A Practical Evaluation of the Discriminatory Tests
Many workers assess the accuracy of the discriminatory tests only by redetermining the character states of selected cluster representatives. This approach has been used here for the centrotype strains (see below). A far more stringent approach which is seldom applied, is to examine all the strains which were used in the original numerical taxonomic analysis. When subjected to cluster analysis using only the data acquired from the derived set of minimum discriminatory tests, the reconstructed dendrogram can be compared with the original. Using only the 24 discriminatory tests previously described (Table 16), the data (two-state, binary form) for all 70 of the novel Gram-negative alkaliphilic bacteria were subjected to cluster analysis by the S.sub.G /UPGMA method. The reconstructed dendrogram is reproduced in FIG. 3. This reconstructed dendrogram compares very favorably with the original dendrogram (FIG. 1).
Although there has been some rearrangement of position of the clusters, their composition is largely unchanged and they are defined at approximately the same similarity level as the original. Cluster 4 however, has combined with Cluster 3, with a single strain moving to Cluster 1. This further serves to emphasize the difficulty of defining Cluster 4 on phenetic data alone. It has been stressed several times that supplementary chemotaxonomic data are required to make the proper distinction between Cluster 3 and Cluster 4.
In both the original dendrogram and the reconstruction (FIG. 3), Cluster 3 appears to comprise several sub-clusters above the 73% similarity level. The fine structure of cluster 3 is also supported by the chemotaxonomic data (see above).
Illustration 2
A Theoretical Evaluation of the Discriminatory Tests
An assessment of cluster overlap is achieved using the OVERMAT program. This program examines the matrix constructed from the percentage positive values for the selected character states against a critical overlap value by considering the clusters defined by the coordinates of the centroid and the cluster radius (twice root mean square of the distances of the strains from the centroid). If there is significant overlap between the clusters, unknown strains may not identify with sufficient confidence to any one of them (Sneath, P. H. A. and Sokal, R. R., supra, p. 394-400). At a chosen critical overlap value of 2.5% (which is a more stringent condition than is used by most workers: see Priest, F. G. and Alexander, B., (1988), supra; and Williams, S. T. et al., (1983), supra) there was no significant overlap between the clusters (95% confidence level) except between Cluster 3 and Cluster 4 where the actual overlap was calculated to be 4%. However, chemotaxonomic data (see above) was not taken into account when constructing the identification matrix. On the basis of quinone analyses, strains from Cluster 3 can be distinguished from the strains of Cluster 4.
Illustration 3
A Theoretical Assessment of the Reliability of Identification
The hypothetical median organism (HMO) is another estimate of the "average" organism in a cluster (Sneath, P. H. A. and Sokal, R. R., supra, pp. 194 et seq.). A HMO is not a real strain but a hypothetical organism possessing the most common state for each character. The MOSTTYP program calculates HMO's for each cluster in the identification matrix and then attempts to identify them. In other words, MOSTTYP is a program to evaluate an identification matrix by calculating identification scores of the most typical strains against the clusters. A good identification matrix should give a high probability of a HMO being reassigned to its own cluster. The results of this analysis were very satisfactory (Table 17), especially since MOSTTYP was programed to consider only the first 20 diagnostic tests of the identification matrix (Table 16), i.e. excluding tests 105-108. Each HMO was reassigned to its original cluster with Willcox probabilities of 0.998-1.000 (Willcox, W. R. et al., (1973) J. Gen. Microbiol., 77, 317-330). The Taxonomic Distances were all low and the standard errors of the Taxonomic Distance were all negative, indicating that the HMO's were all closer to the centroid of the cluster than the average for the cluster (Table 17).
TABLE 17______________________________________Identification Scores for the Hypothetical Median Organismof each cluster provided by the MOSTTYP ProgramIdentification Score Willcox Taxonomic Standard Error orCLUSTER Probability Distance Taxonomic Distance______________________________________1 0.999 0.194 -2.7422 0.999 0.236 -2.2143 1.000 0.231 -2.1154 0.998 0.195 -2.9985 1.000 0.217 -1.8396 1.000 0.182 -2.502______________________________________
Illustration 4
A Practical Evaluation of Identification Score
Identification of strains using the minimum set of discriminatory tests is achieved using the MATIDEN program in TAXPAX. The program compares presence-absence data for an unknown strain against each cluster in turn in an identification matrix of percentage positive characters. Identification coefficients are computed, namely Willcox probability, Taxonomic Distance and the Standard Error of the Taxonomic Distance. The results are displayed, showing the identification scores to the best cluster and to the two next best alternative clusters. Additionally, the atypical results ("characters against") are recorded. In an analysis using data from real strains, the centrotypes were reassigned to their original clusters with Willcox probabilities of 0.9996-1.000 (Table 18). The Taxonomic Distances were low. The Standard Errors of the Taxonomic Distance were all negative indicating that the centrotypes were closer to the centroid of the cluster than the average for the cluster.
TABLE 18______________________________________Identification Scores for the Centrotype Organisms ofEach Cluster Provided by the MATIDEN Program Identification ScoreClus- Assigned Willcox Taxonomic Standardter Strain to Cluster Probability Distance (D) Error of D______________________________________1 2E.1 1 1.000 0.309 -0.2832 45E.3.sup.CT 2 1.000 0.226 -1.7493 28N.1.sup.CT 3 1.000 0.305 -0.6224 wB4.sup.CT 4 0.9996 0.265 -1.0925 17N.1.sup.CT 5 0.9999 0.255 -0.4786 64N.4.sup.CT 6 1.000 0.211 -1.126______________________________________
Illustration 5
Identification of Unknown Isolates
The identification matrix was assessed for the ability to assign unknown Gram-negative alkaliphiles to the clusters defined herein. The criteria for a successful identification were:
(a) bacteria isolated from a habitat similar to, but geographically separate from, the East African soda lakes;
(b) a Willcox probability greater than 0.95 and low values for Taxonomic Distance and its standard error (<3);
(c) an identification score to the best cluster significantly better than those against the two next best alternatives;
(d) "characters against" the best cluster should be zero or few in number.
Unknown microorganisms may be examined using the minimum tests listed in Table 16. The character states are determined and identification scores obtained using the MATIDEN program. This program compares the character states of the unknown with the identification matrix determined for all of the predetermined clusters, computes the best match and assigns the unknown to the most appropriate cluster.
A Willcox probability is calculated to determine the acceptability of identification. Willcox probabilities of 0.85 and 0.95 have been accepted as criteria for a successful identification (Williams, S. T., et al. (1983), supra; Priest, F. G. and Alexander, B., (1988), supra). The Taxonomic Distance of the unknown from the cluster centroid is calculated and may be compared to the radius of the cluster. The Standard Error of the Taxonomic Distance should be less than the upper value of +3.0 suggested by Sneath, P. H. A. ((1979), pp. 195-213). Moreover, physical characteristics, additional biochemical data and chemotaxomomic markers may be used to further confirm the identity of the unknown in a particular cluster.
The results provided by these five illustrations, together with the statistical data provided by the numerical taxonomic analysis and the chemotaxonomic data, indicate a robust classification which identifies 6 major groups of new, Gram-negative, alkaliphilic bacteria.
Production and Application of Alkali-Tolerant Enzymes
The alkaliphilic microorganisms of the present invention produce a variety of alkali-tolerant enzymes. Examples of enzyme activities present in representative strains of the Gram-negative bacteria of the present invention may be found in Appendices D and E. These enzymes are capable of performing their functions at an extremely high pH, making them uniquely suited for their application in a variety of processes requiring such enzymatic activity in high pH environments or reaction conditions.
Examples of the various applications for alkali-tolerant enzymes are in detergent compositions, leather tanning, food treatment, waste treatment and in the textile industry. These enzymes may also be used for biotransformations, especially in the preparation of pure enantiomers.
The alkaliphilic bacteria of the present invention may easily be screened for the production of alkali-tolerant lipases, proteases and starch-degrading enzymes, inter alia, using the methods described herein.
The broth in which alkaliphilic bacteria are cultured typically contains one or more types of enzymatic activity. The broth containing the enzyme or enzymes may be used directly in the desired process after the removal of the bacteria therefrom by means of centrifugation or filtration, for example.
If desired, the culture filtrate may be concentrated by freeze drying, before or after dialysis, or by ultrafiltration. The enzymes may also be recovered by precipitation and filtration. Alternatively, the enzyme or enzymes contained in the broth may be isolated and purified by chromatographic means or by gel electrophoresis, for example, before being applied to the desired process. The exact methods used to treat the culture filtrate and/or to extract and/or purify the alkali-tolerant enzymes is not critical to the present invention, and may be determined by one skilled in the art.
The genes encoding alkali-tolerant enzymes of interest may be cloned and expressed in organisms capable of expressing the desired enzyme in a pure or easily recoverable form.
The following examples are provided to illustrate methods for the identification of Gram-negative alkaliphilic bacteria of the present invention, as well as methods of screening these alkaliphilic bacteria for the presence of various alkali-tolerant enzymes and methods for the subsequent production and application of these enzymes in industrial processes. These examples are not to be construed so as to limit the scope of the present invention.
EXAMPLE 1
Identification of Unknown Isolates
Six strains of Gram-negative, alkaliphilic bacteria were isolated from Mono Lake, a hypersaline, alkaline lake situated in California, U.S.A. (Javor, B., in Hypersaline Environments, Springer Verlag, Berlin and Heidelberg (1988), pp. 303-305). The strains were isolated from samples of partially submerged soda-encrusted wood, tufa and soda-soil collected from the environs of Mono Lake (Calif., U.S.A.) in May, 1990 by enrichment culture at 37.degree. C. in Medium A (Appendix A). The six strains are described in Table 19. The strains were examined using 21 of the 24 minimum tests listed in Table 16. The character states were determined and identification scores obtained using the MATIDEN program. The results are outlined in Table 20.
TABLE 19______________________________________Alkaliphilic Strains from Mono Lake Colony CellStrain Sample Color Form Elevation Margin Shape______________________________________ML005 tufa beige circular convex entire rodML104 wood pink/beige circular convex entire rodML203 wood pink/beige circular convex entire rodML206 wood yellow circular convex entire short rodML301 soil beige circular convex entire rod______________________________________
TABLE 20______________________________________Example of the Output from the MATIDEN Program to IdentifySix Unknown Strains against the Identification Matrix______________________________________A. Reference Number of unknown is ML005 Value in Percent in:Character unknown Best Taxon Next Best Taxon______________________________________�23! N-acetylglucos- + 26 20 amine�26! Saccharose + 74 20�27! Maltose + 68 60�32! Lactate + 99 99�41! Propionate + 91 60�43! Valerate + 97 80�44! Citrate + 94 20�45! Histidine + 71 1�47! Glycogen + 26 20�51! 3-Hydroxy- + 94 99 butyrate�52! 4-Hydroxyben- - 71 80 zoate�58! Leucine + 94 60 arylamidase�59! Valine + 65 99 arylamidase�64! Phosphohydrolase - 3 20�65! .alpha.-Galactosidase n.t. 3 20�85! Ampicillin - 56 1�92! Fusidic Acid - 3 20�93! Methicillin - 50 1�99! Polymix + 81 99�102! Vancomycin - 3 20�105! Yellow colony - 1 1�106! Translucent n.t. 3 1 colony�107! Lipase n.t. 21 1�108! Oxidase (10 sec.) + 6 1______________________________________Isolate ML005 best identification is Cluster 3. Scoresfor coefficients: 1 (Willcox probability), 2 (Taxonomic Distance),3 (Standard Error of Taxonomic Distance). 1 2 3______________________________________Cluster 3 0.9999 0.407 1.475Cluster 4 0.8266 .times. 10.sup.-4 0.526 4.590Cluster 2 0.4086 .times. 10.sup.-7 0.584 6.296______________________________________ Cluster 3Characters against % in Taxon Value in unknown______________________________________�108! Oxidase (10 sec.) 6 +______________________________________Additional characters that assist in separating Cluster 3 % from Cluster 4 %______________________________________�106! Translucent colony 3 99�107! Lipase 21 99______________________________________B. Reference Number of unknown is ML104 Value in Percent in:Character unknown Best Taxon Next Best Taxon______________________________________�23! N-acetylglucos- - 13 1 amine�26! Saccharose - 25 1�27! Maltose - 25 1�32! Lactate - 38 50�41! Propionate - 1 99�43! Valerate - 13 99�44! Citrate - 13 50�45! Histidine - 1 1�47! Glycogen - 1 13�51! 3-Hydroxy- - 13 25 butyrate�52! 4-Hydroxyben- - 1 1 zoate�58! Leucine + 88 63 arylamidase�59! Valine + 88 25 arylamidase�64! Phosphohydrolase + 88 13�65! .alpha.-Galactosidase n.t. 1 1�85! Ampicillin - 50 63�92! Fusidic Acid - 25 1�93! Methicillin - 50 13�99! Polymix + 88 50�102! Vancomycin - 13 13�105! Yellow colony - 1 1�106! Translucent n.t. 1 99 colony�107! Lipase n.t. 1 99�108! Oxidase (10 sec.) + 25 88______________________________________Isolate ML104 best identification is Cluster 1. Scoresfor coefficients: 1 (Willcox probability), 2 (Taxonomic Distance),3 (Standard Error of Taxonomic Distance). 1 2 3______________________________________Cluster 1 0.9999 0.271 -1.108Cluster 2 0.825 .times. 10.sup.-5 0.473 3.797Cluster 4 0.407 .times. 10.sup.-7 0.534 4.767______________________________________ Cluster 1Characters against % in Taxon Value in unknown______________________________________(none)______________________________________Additional characters that assist in separating Cluster 1 % from Cluster 2 %______________________________________�106! Translucent colony 1 99�107! Lipase 1 99______________________________________ Cluster 1 % from Cluster 4 %______________________________________(none)______________________________________C. Reference Number of unknown is ML201 Value in Percent in:Character unknown Best Taxon Next Best Taxon______________________________________�23! N-acetylglucos- - 1 13 amine�26! Saccharose - 25 25�27! Maltose - 50 25�32! Lactate - 1 38�41! Propionate - 1 1�43! Valerate - 1 13�44! Citrate - 50 13�45! Histidine - 1 1�47! Glycogen - 25 1�51! 3-Hydroxy- - 1 13 butyrate�52! 4-Hydroxyben- - 1 1 zoate�58! Leucine + 50 88 arylamidase�59! Valine + 25 88 arylamidase�64! Phosphohydrolase + 75 88�65! .alpha.-Galactosidase n.t. 75 1�85! Ampicillin + 99 50�92! Fusidic Acid + 99 25�93! Methicillin + 99 50�99! Polymix - 1 88�102! Vancomycin + 99 13�105! Yellow colony + 99 1�106! Translucent n.t. 1 1 colony�107! Lipase n.t. 1 1�108! Oxidase (10 sec.) - 1 25______________________________________Isolate ML201 best identification is Cluster 5. Scoresfor coefficients: 1 (Willcox probability), 2 (Taxonomic Distance),3 (Standard Error of Taxonomic Distance). 1 2 3______________________________________Cluster 5 0.9999 0.267 -0.176Cluster 1 0.835 .times. 10.sup.-4 0.437 2.435Cluster 2 .sup. 0.177 .times. 10.sup.-11 0.641 7.593______________________________________ Cluster 5Characters against % in Taxon Value in unknown______________________________________(none)______________________________________Additional characters that assist in separating Cluster 5 % from Cluster 1 %______________________________________Galactosidase 75 1______________________________________ Cluster 5 % from Cluster 2 %______________________________________Galactosidase 75 1�106! Translucent colony 1 99�107! Lipase 1 99______________________________________D. Reference Number of unknown is ML203 Value in Percent in:Character unknown Best Taxon Next Best Taxon______________________________________�23! N-acetylglucos- + 26 20 amine�26! Saccharose + 74 20�27! Maltose + 68 60�32! Lactate + 99 99�41! Propionate + 91 60�43! Valerate + 97 80�44! Citrate + 94 20�45! Histidine + 71 1�47! Glycogen + 26 20�51! 3-Hydroxy- + 94 99 butyrate�52! 4-Hydroxyben- + 71 80 zoate�58! Leucine + 94 60 arylamidase�59! Valine + 65 99 arylamidase�64! Phosphohydrolase - 3 20�65! .alpha.-Galactosidase n.t. 3 20�85! Ampicillin - 56 1�92! Fusidic Acid - 3 20�93! Methicillin - 50 1�99! Polymix + 81 99�102! Vancomycin + 3 20�105! Yellow colony - 1 1�106! Translucent n.t. 3 1 colony�107! Lipase n.t. 21 1�108! Oxidase (10 sec.) + 6 1______________________________________Isolate ML203 best identification is Cluster 3. Scoresfor coefficients: 1 (Willcox probability), 2 (Taxonomic Distance),3 (Standard Error of Taxonomic Distance). 1 2 3______________________________________Cluster 3 0.9989 0.437 2.076Cluster 4 0.1090 .times. 10.sup.-2 0.526 4.590Cluster 2 0.8137 .times. 10.sup.-9 0.650 7.793______________________________________ Cluster 3Characters against % in Taxon Value in unknown______________________________________�102! Vancomycin 3 +�108! Oxidase (10 sec.) 6 +______________________________________Additional characters that assist in separating Cluster 3 % from Cluster 4 %______________________________________(none)______________________________________E. Reference Number of unknown is ML206 Value in Percent in:Character unknown Best Taxon Next Best Taxon______________________________________�23! N-acetylglucos- - 1 13 amine�26! Saccharose + 25 25�27! Maltose - 50 25�32! Lactate - 1 38�41! Propionate - 1 1�43! Valerate - 1 13�44! Citrate - 50 13�45! Histidine - 1 1�47! Glycogen + 25 1�51! 3-Hydroxy- - 1 13 butyrate�52! 4-Hydroxyben- - 1 1 zoate�58! Leucine + 50 88 arylamidase�59! Valine + 25 88 arylamidase�64! Phosphohydrolase + 75 88�65! .alpha.-Galactosidase n.t. 75 1�85! Ampicillin + 99 50�92! Fusidic Acid + 99 25�93! Methicillin + 99 50�99! Polymix - 1 88�102! Vancomycin + 99 13�105! Yellow colony + 99 1�106! Translucent n.t. 1 1 colony�107! Lipase n.t. 1 1�108! Oxidase (10 sec.) - 1 25______________________________________Isolate ML206 best identification is Cluster 5. Scoresfor coefficients: 1 (Willcox probability), 2 (Taxonomic Dis-tance), 3 (Standard Error of Taxonomic Distance). 1 2 3______________________________________Cluster 5 0.9999 0.345 1.766Cluster 1 0.2530 .times. 10.sup.-5 0.512 4.013Cluster 6 .sup. 0.2531 .times. 10.sup.-13 0.652 10.883______________________________________ Cluster 5Characters against % in Taxon Value in unknown______________________________________(none)______________________________________Additional characters that assist in separating Cluster 5 % from Cluster 1 %______________________________________Galactosidase 75 1______________________________________F. Reference Number of unknown is ML301 Value in Percent in:Character unknown Best Taxon Next Best Taxon______________________________________�23! N-acetylglucos- - 26 1 amine�26! Saccharose + 74 1�27! Maltose + 68 1�32! Lactate + 99 50�41! Propionate + 91 99�43! Valerate + 97 99�44! Citrate + 94 50�45! Histidine + 71 1�47! Glycogen + 26 13�51! 3-Hydroxy- + 94 25 butyrate�52! 4-Hydroxyben- - 71 1 zoate�58! Leucine + 94 63 arylamidase�59! Valine + 65 25 arylamidase�64! Phosphohydrolase - 3 13�65! .alpha.-Galactosidase n.t. 3 1�85! Ampicillin + 56 63�92! Fusidic Acid - 3 1�93! Methicillin + 50 13�99! Polymix + 81 50�102! Vancomycin - 3 13�105! Yellow colony - 1 1�106! Translucent n.t. 3 99 colony�107! Lipase n.t. 21 99�108! Oxidase (10 sec.) + 6 88______________________________________Isolate ML301 best identification is Cluster 3. Scoresfor coefficients: 1 (Willcox probability), 2 (Taxonomic Dis-tance), 3 (Standard Error of Taxonomic Distance). 1 2 3______________________________________Cluster 3 1.000 0.429 1.918Cluster 2 0.2841 .times. 10.sup.-6 0.603 6.731Cluster 4 0.9313 .times. 10.sup.-8 0.623 6.704______________________________________ Cluster 3Characters against % in Taxon Value in unknown______________________________________�108! Oxidase (10 sec.) 6 +______________________________________Additional characters that assist in separating Cluster 3 % from Cluster 2 %______________________________________�106! Translucent colony 3 99�107! Lipase 21 99______________________________________ n.t. = not tested
EXAMPLE 2
Production of Proteolytic Enzymes
Two alkaliphilic strains (1E.1 and 9B.1) were tested for the production of proteolytic enzyme(s) in 7 different media poised at an alkaline pH. The experiments were carried out in 2 liter shake flasks with a baffle, each of the flasks contained 400 ml of the nutrient media R to X (Appendix A). The flasks were placed in an orbital incubator rotating at 280 revolutions per minute at a constant temperature of 37.degree. C. Samples of culture media were removed from the flasks at intervals of 1, 2, 3, 4, 5, 6 and 8 days for the determination of enzyme content which is expressed in Alkaline Delft Units (ADU--as described in British Patent Specification 1,353,317).
Table 21 presents the maximum enzyme yields and the pH of the cultivation medium at the moment at which the measurement of enzyme levels were made.
TABLE 21______________________________________Production of Proteolytic Enzymes STRAIN 1E.1.sup.CT STRAIN 9B.1 pH of pH ofMEDIUM ADU/ml MEDIUM ADU/ml MEDIUM______________________________________R 100 8.2 14 9.7S 140 8.5 49 9.1T 111 8.7 6 9.1U 6 9.7 4 9.7V 51 9.5 7 9.6W 94 9.2 7 9.3X 100 9.6 28 9.6______________________________________
The results of the test clearly indicate the presence of proteolytic enzymes, produced by the alkaliphilic bacteria of the present invention, in the culture broth.
EXAMPLE 3
Wash Performance Test Using Proteolytic Enzymes
Enzyme preparations from the alkaliphilic bacteria were tested in a specially developed mini-wash test using cotton swatches (2.5.times.2.5 cm) soiled with milk, blood and ink (obtained from EMPA, St. Gallen, Switzerland, and designated EMPA 116). Prior to the wash test the swatches were pre-treated with a solution containing an anionic surfactant, sodium perborate and a bleach activator (TAED) at ambient temperature for 15 minutes. After this treatment the test swatches were rinsed in running demineralized water for 10 minutes and air-dried. This treatment results in the fixation of the soil, making its removal more difficult.
The washing tests were performed in 100 ml Erlenmeyer flasks provided with a baffle and containing 30 ml of a defined detergent composition plus 300 ADU protease to be tested. In each flask were placed two pre-treated EMPA 116 test swatches. The flasks were placed in a reciprocal shaking water bath (2 cm stroke) and agitated at 320 revolutions per minute. The tests were carried out at 40.degree. C. for 30 minutes. After washing, the swatches were rinsed in running demineralized water for 10 minutes and air-dried. The reflectance of the test swatches was measured at 680 nm with a Photovolt photometer (Model 577) equipped with a green filter.
The wash performance of the supernatant fraction of cultures of various alkaliphilic bacteria in European powder detergents was determined according to the method specified above. The supernatant fractions were subjected to various treatments so as to produce enzyme-containing preparations.
100 ml Erlenmeyer flasks were charged with powder detergent IEC dissolved in standard tap water of 15.degree. German Hardness so as to give a final concentration of 4 g per liter.
The composition of the powder detergent IEC was as follows:
______________________________________Component wt %______________________________________Linear sodium alkyl benzene sulphonate 6.4(mean chain length of alkane chain (C11.5))Ethoxylated tallow alcohol (14EO) 2.3Sodium soap 2.8Sodium tripolyphosphate (STPP) 35.0Sodium silicate 6.0Magnesium silicate 1.5Carboxy methyl cellulose 1.0Sodium sulphate 16.8Sodium perborate tetrahydrate 18.5TAED 1.5Miscellaneous + water up to 100______________________________________
Standard tap water is composed of CaCl.sub.2.2H.sub.2 O, 0.291 g/l; MgCl.6H.sub.2 O, 0.140 g/l and NaHCO.sub.3, 0.210 g/l dissolved in demineralized water.
To each flask, two EMPA 116 swatches were added and sufficient enzyme-containing preparations to give a final activity of 300 ADU. The final volume of the sud was 30 ml. By way of comparison, one flask contained no enzyme preparation, which was replaced with water. The trial was repeated either two or three times. The results are shown in Table 22.
TABLE 22______________________________________Application Washing TrialsPerformance of Proteolytic Enzyme-Containing Preparationsin a Washing Formulation.Preparationfrom Average Remission of EMPA 116 Test SwatchesStrain Trial 1 Trial 2 Trial 3______________________________________Untreated Culture SupernatantNone 11.4 11.8 13.0(control)1E.1.sup.CT 29.2 22.2 24.79B.1 23.7 23.9 24.217N.1.sup.CT 11.8 18.424B.1 17.3 16.3Freeze Dried Supernatant FractionNone 10.4 11.8 13.0(control)1E.1.sup.CT 15.1 28.9 30.09B.1 21.7 14.9 17.417N.1.sup.CT 13.7 17.924B.1 17.8 17.3Dialyzed Supernatant FractionsNone 11.4 11.8 13.0(control)1E.1.sup.CT 26.4 22.7 26.39B.1 18.7 16.7 17.017N.1.sup.CT 12.0 12.624B.1 12.6 12.4Ultrafiltration Concentrate of Supernatant FractionsNone 10.4 11.4(control)1E.1.sup.CT 14.6 26.09B.1 15.5 16.1Acetone Precipitates of Supernatant FractionsNone 10.4 11.4(control)1E.1.sup.CT 13.4 23.49B.1 12.6 14.7______________________________________
The results of the trials demonstrate the efficacy of the proteolytic enzymes produced by the strains of the present invention, provided in various forms, in detergent formulations and the improved washing performance obtained.
EXAMPLE 4
Production of Starch Degrading Enzymes
Strain 1E.1.sup.CT was tested for the production of starch degrading enzymes on a starch containing medium poised at an alkaline pH.
500 ml Erlenmeyer flasks were charged with 100 ml of alkaline medium (Medium Y, Appendix A) containing 2% soluble starch. The flasks were inoculated (5%) with cells of strain 1E.1.sup.CT grown for 24 hours on Medium A (37.degree. C.). As controls, similar flasks of alkaline medium not containing starch were also inoculated.
The flasks were placed in an orbital shaking incubator rotating at 280 revolutions per minute, at a constant temperature of 37.degree. C. for 24 hours. The fluid containing the enzyme activity was separated from the cells by centrifugation for 10 minutes at 4000 r.p.m.
The enzyme activity of the supernatant was determined using the reducing sugar assay of Nelson and Somogyi (Methods in Microbiology, volume 5B, pp. 300-301; (eds. J. R. Norris and D. W. Ribbons), Academic Press, London, 1971).
Determination of Starch Degrading Enzyme Activity by the Reducing Sugar Assay Solutions
Reagent 1
144 g Na.sub.2 SO.sub.4 is dissolved by gentle warming in 500 ml demineralized water. 12 g potassium sodium tartrate tetrahydrate, 24 g Na.sub.2 CO.sub.3 and 16 g NaHCO.sub.3 are added to the solutions. The total volume of the solution is brought to 800 ml by the addition of demineralized water.
Reagent 2
36 g Na.sub.2 SO.sub.4 is dissolved by gentle warming in 100 ml demineralized water and 4 g CuSO.sub.4.5H.sub.2 O is added to the warmed solution. The total volume of the solution is brought to 200 ml by the addition of demineralized water.
Directly before use, Reagents 1 and 2 are mixed in the ratio of 4:1 (Reagent 1: Reagent 2).
Reagent 3
25 g ammonium molybdate tetrahydrate is dissolved in 450 ml demineralized water and 21 ml concentrated sulphuric acid is added with thorough mixing. 3 g Na.sub.2 HAsO.sub.4.7H.sub.2 O are dissolved in 25 ml demineralized water and this solution is added to the molybdate solution. The total solution is warmed for 48 hours at 37.degree. C. and any precipitate is filtered off.
Standard
100 mg glucose is dissolved in demineralized water and the total volume is brought to 100 ml. Before use, the solution is diluted 10 fold with demineralized water.
Substrate
0.25% soluble starch (Merck, product number 1257) dissolved in 0.1M Na.sub.2 CO.sub.3 -NaHCO.sub.3 buffer, pH 10.1.
Assay
0.9 ml starch substrate solution, pH 10.1 is placed in a test-tube. The test-tube is placed in a water bath at 25.degree. C. and allowed to equilibrate. The enzyme reaction is started by adding 0.1 ml of the enzyme-containing culture supernatant. The reaction is allowed to proceed for 30 minutes. The reaction is stopped by adding 1 ml of Reagent 1/2 and heating for 10 minutes at 100.degree. C. The mixture is cooled on ice for 5 minutes and then 0.5 ml of Reagent 3 is added and the blue color is allowed to develop during 30 minutes at room temperature. The mixture is diluted by adding 1.0 ml demineralized water and the extinction is measured at 500 nm in a spectrophotometer. The reducing sugars are measured as glucose equivalents from a standard curve.
One unit of starch degrading enzyme activity is defined as 1 .mu.g of reducing sugars measured as glucose released per milliliter per minute at pH 10.1 and 25.degree. C.
The number of starch degrading enzyme units formed is shown in Table 23.
TABLE 23______________________________________Production of Starch Degrading Enzymes by Strain 1E.1 OPTICAL DENSITY FINAL ENZYMEMEDIUM at 550 nm pH units per liter______________________________________plus starch 2.25 9.4 1150no starch 0.75 10.3 660______________________________________
The results of the test clearly indicate the presence of starch degrading enzymes, produced by the alkaliphilic bacterial strain of the present invention, in the culture broth.
EXAMPLE 5
Stability of Starch Degrading Enzymes in Detergent
The ability of the starch degrading enzymes from strain 1E.1.sup.CT to withstand detergents, which is essential for their application in laundry detergents or textile desizing, is demonstrated.
100 ml Erlenmeyer flasks provided with a baffle were each charged with 30 ml of 0.1M Na.sub.2 CO.sub.3 /NaHCO.sub.3 buffer, pH 10.1 containing 0.12 g of sodium dodecyl sulphate (equivalent to 4 g per liter). To one-half of the flasks 0.3 g potato starch (equivalent to 1%) was added.
Each flask was dosed with enzyme-containing supernatant from strain 1E.1.sup.CT by adding 0.5, 1.0 or 2.0 ml (see Table 23). As a control, the supernatant fluid was replaced with 1.0 ml water. Immediately after adding the enzyme, a 0.1 ml sample was removed (time=zero hours) for the measurement of enzyme activity.
The flasks were incubated with shaking at 25.degree. C. for 2.5 hours at which time a second 0.1 ml sample was removed for the measurement of enzyme activity.
As a comparison the experiment was repeated using a conventional .alpha.-amylase derived from Bacillus subtilis.
Enzyme activity was determined using the reducing sugars method previously described.
The results are recorded in Table 24.
TABLE 24______________________________________Stability of Starch Degrading Enzymes from Strain 1E.1.sup.CT inDetergentsENZYME-CONTAININGSUPERNATENT ENZYME UNITSADDED RECOVERED(ml) CONDITIONS pH 0 h. 2.5 h.______________________________________0* SDS 10.4 0 00.5 10.3 26 201.0 10.3 44 482.0 10.3 109 1130* SDS + 10.3 0 00.5 STARCH 10.2 12 171.0 10.1 36 482.0 10.2 79 120Standard .sctn. SDS 10.4 0 0Standard .sctn. SDS + STARCH 10.2 0 0______________________________________ *replaced with 1 ml water .sctn. 2.8 RAU Bacillus subtilis .alpha.-amylase One RAU (Reference Amylase Unit) is defined as the quantity of enzyme that will convert 1 mg of starch per minute at pH 6.6 and 30.degree. C. into a product which upo reaction with iodine has an equal absorbance at 620 nm. as a solution containing 25 g CoCl.sub.2.6H.sub.2 O, 3.84 g K.sub.2 Cr.sub.2 O.sub.7 an 1 ml 1 M HCl in 100 ml distilled water.
The results of this test clearly demonstrate the stability of the starch degrading enzymes, produced by the alkaliphilic bacterial strain of the present invention, in the presence of detergent.
EXAMPLE 6
Production of Lipolytic Enzymes
Eleven of the new strains which clearly exhibited lipase activity (Appendix D) were tested further for the production of lipolytic enzymes. The eleven strains are examples from Cluster 2 and Cluster 3 (FIGS. 1A and 1B).
The experiments were carried out in 100 ml conical flasks containing 30 ml sterile alkaline nutrient medium, pH 9.6, inoculated with the appropriate bacterial strain. Three different media were used, designated medium Z to BB (Appendix A). The flasks were placed in an orbital shaking incubator (300 rpm) at 30.degree. C. for 48 hours.
The cells were separated from the culture broth by centrifugation and the supernatant dialyzed against 50 volumes 0.1 mM Tris-HCl buffer pH 9, with 3 changes of buffer over 24 hours. The dialysate was freeze dried to give a lipase preparation (Table 25).
The lipase preparations obtained according to this example were used for the washing test described in Example 7, below.
TABLE 25______________________________________Production of Lipase PRODUCTION LIPASE LIPASESTRAIN MEDIUM TLU/ml* TLU/g______________________________________39E.3 BB 1.3 13440E.3 Z 1.2 11841E.3 Z 1.1 8242E.3 Z 1.2 7644E.3 Z 1.2 9945E.3.sup.CT AA 1.4 9848E.3 BB 2.0 15249N.3 BB 1.5 12350N.3 BB 2.0 10051N.3 BB 1.0 9852N.3 BB 1.2 128______________________________________ *TLU = True Lipase Unit as defined in U.S. Pat. No. 4,933287.
The results of this test clearly demonstrate the presence of lipolytic enzymes, produced by alkaliphilic bacteria of the present invention, in the culture broth and in a freeze-dried preparation of the dialyzed culture broth.
EXAMPLE 7
Lipase Washing Test
The lipase preparations from Example 6 were tested for performance under washing conditions in TIDE.TM. powder (1.5 g/l), a detergent product from Procter & Gamble.
The washing test (SLM-test) was carried out as described in U.S. Pat. No. 4,933,287, which is hereby incorporated by reference. As control, a lipase derived from Pseudomonas alcaligenes strain M1 (CB3 473.85) as described in U.S. Pat. No. 4,933,287 was used. The results are shown in Table 26.
TABLE 26______________________________________Lipase Washing TestDetergent: TIDE.sup.R (powder), 1.5 g/lLipase: 2 TLU/mlCa.sup.2+ : 10.sup.-5 M (sodium tripolyphosphate added)pH: 9.5 RECOVERY (%)STRAIN TRIGLYCERIDES TOTAL LIPID______________________________________39E.3 55.3 73.440E.3 82.0 89.941E.3 44.7 78.742E.3 55.5 74.844E.3 6.6 76.945E.3.sup.CT 81.6 92.948E.3 76.5 82.949E.3 72.4 82.050N.3 50.5 76.651N.3 80.4 87.652N.2 77.0 84.7M1 88.5 91.5control* 98.7 98.7______________________________________ *Standard tap water as defined in U.S. Pat. No. 4,933,287
The decrease in the percent recovery of triglycerides and total lipids, as compared to the control, clearly indicate the ability of the lipolytic enzymes, produced by the alkaliphilic bacteria of the present invention, to break down and remove triglycerides and their degradation products embedded on a fabric sample, as well as their improved performance as compared to a known lipase.
______________________________________Appendix AMedia Used in the Present Invention______________________________________MEDIUM AGlucose 10.0 gl.sup.-1Peptone (Difco: Detroit, MI, USA) 5.0 gl.sup.-1Yeast Extract (Difco) 5.0 gl.sup.-1K.sub.2 HPO.sub.4 1.0 gl.sup.-1MgSO.sub.4.7H.sub.2 O 0.2 gl.sup.-1NaCl 40.0 gl.sup.-1Na.sub.2 CO.sub.3 10.0 gl.sup.-1*Agar 20.0 gl.sup.-1MEDIUM BGlucose 10.0 gl.sup.-1Peptone (Difco) 5.0 gl.sup.-1Yeast Extract (Difco) 5.0 gl.sup.-1K.sub.2 HPO.sub.4 1.0 gl.sup.-1MgSO.sub.4.7H.sub.2 O 0.2 gl.sup.-1NaCl 40.0 gl.sup.-1Na.sub.2 CO.sub.3 10.0 gl.sup.-1Novobiocin 50.0 mgl.sup.-1Agar 20.0 gl.sup.-1MEDIUM CGlucose 10.0 gl.sup.-1Peptone (Difco) 5.0 gl.sup.-1Yeast Extract (Difco) 5.0 gl.sup.-1K.sub.2 HPO.sub.4 1.0 gl.sup.-1MgSO.sub.4.7H.sub.2 O 0.2 gl.sup.-1NaCl 40.0 gl.sup.-1Na.sub.2 CO.sub.3 10.0 gl.sup.-1Lactalbumin 10.0 gl.sup.-1Agar 20.0 gl.sup.-1MEDIUM DGlucose 10.0 gl.sup.-1Peptone (Difco) 5.0 gl.sup.-1Yeast Extract (Difco) 5.0 gl.sup.-1K.sub.2 HPO.sub.4 1.0 gl.sup.-1MgSO.sub.4.7H.sub.2 O 0.2 gl.sup.-1NaCl 40.0 gl.sup.-1Na.sub.2 CO.sub.3 10.0 gl.sup.-1Casein 20.0 gl.sup.-1Agar 20.0 gl.sup.-1MEDIUM ESoluble Starch 10.0 gl.sup.-1Peptone (Difco) 5.0 gl.sup.-1Yeast Extract (Difco) 5.0 gl.sup.-1K.sub.2 HPO.sub.4 1.0 gl.sup.-1MgSO.sub.4.7H.sub.2 O 0.2 gl.sup.-1NaCl 40.0 gl.sup.-1Na.sub.2 CO.sub.3 10.0 gl.sup.-1Lactalbumin 10.0 gl.sup.-1Agar 20.0 gl.sup.-1MEDIUM FSoluble Starch 10.0 gl.sup.-1Peptone (Difco) 5.0 gl.sup.-1Yeast Extract (Difco) 5.0 gl.sup.-1K.sub.2 HPO.sub.4 1.0 gl.sup.-1MgSO.sub.4.7H.sub.2 O 0.2 gl.sup.-1NaCl 40.0 gl.sup.-1Na.sub.2 CO.sub.3 10.0 gl.sup.-1Casein 20.0 gl.sup.-1Agar 20.0 gl.sup.-1MEDIUM GOxbile (Oxoid: Basingstoke, U.K.) 10.0 gl.sup.-1(NH.sub.4).sub.2 SO.sub.4 5.0 gl.sup.-1MgSO.sub.4.7H.sub.2 O 0.2 gl.sup.-1Yeast Extract (Difco) 0.5 gl.sup.-1Lactalbumin 10.0 gl.sup.-1Agar 20.0 gl.sup.-1Adjusted to pH 8.5 with 50% Na.sub.2 CO.sub.3 solutionMEDIUM HOxbile (Oxoid) 10.0 gl.sup.-1(NH.sub.4).sub.2 SO.sub.4 5.0 gl.sup.-1MgSO.sub.4.7H.sub.2 O 0.2 gl.sup.-1Yeast Extract (Difco) 0.5 gl.sup.-1Casein 20.0 gl.sup.-1Agar 20.0 gl.sup.-1Adjusted to pH 8.5 with 50% Na.sub.2 CO.sub.3 solutionMEDIUM ISabouroud Dextrose Agar (Oxoid) 65.0 gl.sup.-1NaCl 40.0 gl.sup.-1Na.sub.2 CO.sub.3 20.0 gl.sup.-1Cycloheximide 50.0 mgl.sup.-1Penicillin G 25000 IUl.sup.-1Streptomycin 25 mgl.sup.-1MEDIUM JBacto Potato Dextrose Agar (Difco) 39.0 gl.sup.-1NaCl 40.0 gl.sup.-1Na.sub.2 CO.sub.3 20.0 gl.sup.-1Novobiocin 50.0 mgl.sup.-1MEDIUM KBacto Potato Dextrose Agar (Difco) 39.0 gl.sup.-1NaCl 40.0 gl.sup.-1Na.sub.2 CO.sub.3 20.0 gl.sup.-1Cycloheximide 50.0 mgl.sup.-1Penicillin G 25000 IUl.sup.-1Streptomycin 25.0 mgl.sup.-1MEDIUM LGlucose 0.2 gl.sup.-1Peptone (Difco) 0.1 gl.sup.-1Yeast Extract (Difco) 0.1 gl.sup.-1K.sub.2 HPO.sub.4 1.0 gl.sup.-1MgSO.sub.4.7H.sub.2 O 0.2 gl.sup.-1NaCl 40.0 gl.sup.-1Na.sub.2 CO.sub.3 10.0 gl.sup.-1Casein 20.0 gl.sup.-1Agar 20.0 gl.sup.-1MEDIUM M (pH 9.6)Oxbile (Oxoid) 2.0 gl.sup.-1(NH.sub.4).sub.2 SO.sub.4 1.0 gl.sup.-1MgSO.sub.4.7H.sub.2 O 0.04 gl.sup.-1Yeast Extract (Difco) 0.1 gl.sup.-1Olive Oil 10.0 ml l.sup.-1Na.sub.2 CO.sub.3 6.1 gl.sup.-1Agar 20.0 gl.sup.-1MEDIUM N (pH 9.6)Oxbile (Oxoid) 2.0 gl.sup.-1(NH.sub.4).sub.2 SO.sub.4 1.0 gl.sup.-1MgSO.sub.4.7H.sub.2 O 0.04 gl.sup.-1Yeast Extract (Difco) 0.1 gl.sup.-1Olive Oil 10.0 ml l.sup.-1Na.sub.2 CO.sub.3 6.1 gl.sup.-1Tergitol 7 (Fluka: Buchs, CH) 500 ppmAgar 20.0 gl.sup.-1MEDIUM O (pH 9.6)Oxbile (Oxoid) 2.0 gl.sup.-1(NH.sub.4).sub.2 SO.sub.4 1.0 gl.sup.-1MgSO.sub.4.7H.sub.2 O 0.04 gl.sup.-1Yeast Extract (Difco) 0.1 gl.sup.-1Olive Oil 10.0 ml l.sup.-1Na.sub.2 CO.sub.3 6.1 gl.sup.-1NaCl 40.0 gl.sup.-1Agar 20.0 gl.sup.-1MEDIUM P (pH 9.6)Oxbile (Oxoid) 10.0 gl.sup.-1(NH.sub.4).sub.2 SO.sub.4 0.2 gl.sup.-1MgSO.sub.4.7H.sub.2 O 0.2 gl.sup.-1Yeast Extract (Difco) 0.5 gl.sup.-1Olive Oil 10.0 ml l.sup.-1NaCl 40.0 gl.sup.-1Agar 20.0 gl.sup.-1Adjusted to pH 9.6 with 50% Na.sub.2 CO.sub.3 solutionMEDIUM O (pH 9.6)Oxbile (Oxoid) 10.0 gl.sup.-1(NH.sub.4).sub.2 SO.sub.4 5.0 gl.sup.-1MgSO.sub.4.7H.sub.2 O 0.2 gl.sup.-1Yeast Extract (Difco) 0.5 gl.sup.-1Olive Oil 10.0 ml l.sup.-1Agar 20.0 gl.sup.-1Adjusted to pH 9.6 with 50% Na.sub.2 CO.sub.3 soutionMEDIUM R (pH 9.5)Fresh Yeast 82.5 gl.sup.-1Glucose 3.3 gl.sup.-1K.sub.2 HPO.sub.4 1.6 gl.sup.-1K.sub.2 CO.sub.3 0.6 gl.sup.-1KHCO.sub.3 1.76 gl.sup.-1CaCl.sub.2 0.05 gl.sup.-1MgSO.sub.4.7H.sub.2 O 0.05 gl.sup.-1FeSO.sub.4 0.005 gl.sup.-1MnSO.sub.4 0.0066 gl.sup.-1MEDIUM SFresh Yeast 8.25 gl.sup.-1Glucose 1.32 gl.sup.-1K.sub.2 HPO.sub.4 1.6 gl.sup.-1CaCl.sub.2 0.05 gl.sup.-1MgSO.sub.4.7H.sub.2 O 0.05 gl.sup.-1FeSO.sub.4 0.005 gl.sup.-1MnSO.sub.4 0.0066 gl.sup.-1NaCl 40.0 gl.sup.-1Adjusted to pH 10.5 with 40% Na.sub.2 CO.sub.3 solutionMEDIUM T (pH 10.1)Glucose 10.0 gl.sup.-1Peptone (Difco) 5.0 gl.sup.-1Yeast Extract (Difco) 5.0 gl.sup.-1K.sub.2 HPO.sub.4 1.0 gl.sup.-1MgSO.sub.4.7H.sub.2 O 0.2 gl.sup.-1NaCl 40.0 gl.sup.-1Na.sub.2 CO.sub.3 10.0 gl.sup.-1MEDIUM UOxbile 10.0 gl.sup.-1(NH.sub.4).sub.2 SO.sub.4 5.0 gl.sup.-1MgSO.sub.4.7H.sub.2 O 0.2 gl.sup.-1Yeast extract (Difco) 0.5 gl.sup.-1Casein 10.0 gl.sup.-1Adjusted to pH 9.8 with 40% Na.sub.2 CO.sub.3 solutionMEDIUM VTryptone Soya Broth (Oxoid) 30.0 gl.sup.-1Adjusted to pH 9.9 with 40% Na.sub.2 CO.sub.3 solutionMEDIUM W (pH 10.1)Soluble starch 10.0 gl.sup.-1Peptone (Difco) 5.0 gl.sup.-1Yeast extract (Difco) 5.0 gl.sup.-1KH.sub.2 PO.sub.4 1.0 gl.sup.-1MgSO.sub.4.7H.sub.2 O 0.2 gl.sup.-1NaCl 40.0 gl.sup.-1Na.sub.2 CO.sub.3 10.0 gl.sup.-1MEDIUM XSkim milk (Difco) 100.0 gl.sup.-1Adjusted to pH 10.8 with 25% Na.sub.2 CO.sub.3 solutionMEDIUM YYeast Extract (Difco) 1.0 gKNO.sub.3 10.0 gKH.sub.2 PO.sub.4 1.0 gMgSO.sub.4.7H.sub.2 O 0.2 gNa.sub.2 CO.sub.3 10.0 gNaCl 40.0 gSoluble starch (Merck) 20.0 gDemineralised water 1 literMEDIUM ZBrain Heart Infusion (Difco) 20.0 gNa.sub.2 EDTA (Komplexion III, Siegfried AG, 1.0 gSwitzerland)FeSO.sub.4.7H.sub.2 O 0.006 gMnSO.sub.4.H.sub.2 O 0.003 gCaCl.sub.2.2H.sub.2 O 1.0 gMgSO.sub.4.7H.sub.2 O 0.25 gTween 80 5.0 gSoya oil 5.0 gDistilled water to 1 liter, pH of medium adjustedto pH 9.6 with 25% Na.sub.2 CO.sub.3 solution.MEDIUM AAYeast Extract (Difco) 20.0 gKH.sub.2 PO.sub.4 5.0 gFeSO.sub.4.7H.sub.2 O 0.006 gMnSO.sub.4.H.sub.2 O 0.003 gCaCl.sub.2.2H.sub.2 O 1.0 gMgSO.sub.4.7H.sub.2 O 0.25 gTween 80 5.0 gSoya oil 5.0 gDistilled water to 1 liter, pH of medium adjustedto pH 9.6 with 25% Na.sub.2 CO.sub.3 solution.MEDIUM BBBrain Heart Infusion (Difco) 20.0 gFeSO.sub.4.7H.sub.2 O 0.006 gMnSO.sub.4.H.sub.2 O 0.003 gCaCl.sub.2.2H.sub.2 O 1.0 gMgSO.sub.4.7H.sub.2 O 0.25 gTween 80 5.0 gSoya oil 5.0 gDistilled water to 1 liter, pH of medium adjustedto pH 9.6 with 25% Na.sub.2 CO.sub.3 solution.______________________________________ *(when required for a solid medium)
APPENDIX B
Methods for Unit Tests
1. Character numbers 1 to 5
Colony color, form, elevation, margin, size
A suspension of bacteria was spread over an alkaline nutrient agar (Medium A) and cultivated at 37.degree. C. Colonies were examined after 48 hours.
2. Character number 6 and 7
Cell morphology, Gram's strain reaction
Bacteria cells grown in alkaline nutrient broth (Medium A, without agar) for 24 hours were spun down in a centrifuge and resuspended in a small amount of alkaline nutrient broth and allowed to air-dry on a microscope slide. Or, bacteria were cultivated for 24-48 hours on an alkaline nutrient agar (Medium A) so as to form colonies. Colonies of bacteria were suspended in physiological saline and a few drops allowed to air-dry on a microscope slide. The Gram's staining test was performed using the Dussault modification (Journal of Bacteriology, 70, 484-485, 1955) with safranin as counterstain.
3. Character number 8
Oxidase reaction
Filter paper moistened with a 1% aqueous solution of N,N,N',N'-tetramethyl-p-phenylenediamine or oxidase identification discs (bioMerieux: Charbonieres-les-Bains, France) were smeared with a young bacterial culture from alkaline nutrient agar. A purple color within 1 minute was recorded as a positive reaction. E. coli, used as a control, did not give a positive reaction within one minute.
4. Character number 9
Skim milk test
A minimal medium composed (g/l distilled water) of yeast extract, 1.0; KNO.sub.3, 10.0; K.sub.2 HPO.sub.4, 1.0;MgSO.sub.4.7H.sub.2 O, 0.2; NaCl, 40.0; Na.sub.2 CO.sub.3, 10.0; agar, 20.0 was supplemented with 5.0 g/l skim milk powder, sterilised by autoclaving and poured into Petri dishes. Bacteria were inoculated and incubated at 37.degree. C. Areas of clearing around bacterial colonies in an otherwise opaque agar were recorded as a positive reaction. Non-alkaliphilic reference strains were tested in an identical fashion using media of the same composition but without Na.sub.2 CO.sub.3 so as to give a pH of 6.8-7.0.
4. Character number 10
Gelatin hydrolysis
Charcoal-gelatin discs (bioMerieux) or "chargeis" (Oxoid) were incubated at 37.degree. C. in an alkaline nutrient broth (Medium A) together with bacteria. A black sediment indicated a positive reaction.
6. Character number 11
NaCl tolerance
Two methods were applied.
(a) Bacterial strains were cultivated at 37.degree. C. on an alkaline nutrient agar (Medium A) containing 0%, 4%, 8%, 12% or 15% (w/v) NaCl. The agar plates were examined for bacterial growth after 48 hours.
(b) Bacterial strains were cultivated at 37.degree. C. in an alkaline nutrient broth (Medium A) containing 0%, 4%, 8%, 12%, 15% or 25% NaCl. Bacterial growth was monitored by optical density measurements using a Klett meter (green filter) at 0, 12, 24, 48, 72 and 144 hours.
7. Character number 12
Minimum pH for growth
Nutrient agar, pH 6.8-7.0 (Medium A without sodium carbonate) was poured into square Petri dishes. A strip of solidified agar was removed from one end and replaced with molten 4% (w/v) agar containing 3.6% (w/v) Na.sub.2 CO.sub.3 and 0.8% (w/v) NaOH. A pH gradient from pH 10.5 to pH 7 across the plate was allowed to develop overnight. Bacteria were inoculated by streaking along the pH gradient and cultivated at 37.degree. C. for 48 hours. The pH at the point where bacterial growth ceased was measured with a flat head electrode and with "Alkalite" pH strips (Merck: Darmstadt, W. Germany).
8. Character numbers 13-21
Carbohydrate utilisation
A minimal medium composed (g/l distilled water) of yeast extract, 1.0; KNO.sub.3, 10.0; K.sub.2 HPO.sub.4, 1.0; MgSO.sub.4,7H.sub.2 O, 0.2; NaCl, 40.0; NarCO.sub.3, 10.0; agar, 20.0 was supplemented with 2.0 g/l of the carbohydrate under test and poured into square Petri dishes. Bacteria were inoculated, using a 25 point multi-point inoculator, from 1.0 ml of a bacterial suspension cultivated for 48 hours in an alkaline nutrient broth (Medium A). The agar-plates were incubated at 37.degree. C. for 48 hours. The results were recording by comparing bacterial growth on minimal nutrient medium containing a carbohydrate supplement with growth on a minimal medium without the carbohydrate under test. Non-alkaliphilic reference strains were tested in an identical fashion using media of the same composition but without NA.sub.2 CO.sub.3 so as to give a pH of 6.8-7.0.
9. Character numbers 22-53
Growth on carbon substrates
Use was made of the commercially available test strip ATB 32 GN (API-bioMerieux: La Balme les Grottes, France). The strips were used according to the manufacturer's instructions but with an addition of 1.0 ml of a solution containing 4% NaCl and 1% Na.sub.2 CO.sub.3 to the vials of basal medium provided. The strips were incubated at 37.degree. C. for 48 hours. Non-alkaliphilic reference strains were incubated in the standard basal medium.
10. Character numbers 54-72
Enzymatic activities
Use was made of the commercially available test strip APIZYM (API-bioMerieux) which was used according to the manufacturer's instructions, except that the alkaliphilic bacterial cells were suspended in alkaline nutrient broth (Medium A). The strips were incubated at 37.degree. C. for 4 hours.
11. Character numbers 73-82
Amino acids as carbon and nitrogen source
The same technique was employed as for tests 14-21 except that KNO.sub.3 was omitted from the minimal nutrient medium.
12. Character numbers 83-104
Antibiotic sensitivity
A light suspension of bacteria in alkaline nutrient broth was spread on the surface of alkaline nutrient agar (Medium A) and allowed to dry. Commercially available antibiotic susceptibility test discs (Oxoid or Mast Laboratories: Merseyside, U.K.) were applied to the agar surface. The bacteria were cultivated at 37.degree. C. for 48 hours. Clear zones around the antibiotic disks indicated sensitivity.
______________________________________Appendix CUnit Tests for Analysis by Numerical TaxonomyCHARACTER TEST COMPUTERNUMBER DESCRIPTION CODE______________________________________1 Colony color white = 1 cream = 2 beige = 3 yellow = 4 orange = 5 pink = 6 brown = 7 red = 82 Colony form circular = 1 irregular = 2 punctiform = 3 filamentous = 43 Colony elevation convex = 1 raised = 2 umbonate = 3 flat = 44 Colony margin entire = 1 undulate = 2 lobate = 3 fimbriate = 45 Colony size diameter in millimeters6 Cell morphology rod = 1 coccus = 27 Gram's stain negative = 1 positive = 28 Oxidase test negative 1 positive = 29 Skim milk test negative = 1 positive = 210 Gelatin hydrolysis negative = 1 positive = 211 NaCl tolerance growth at 0%-4% = 1 growth at 0%-8% = 2 growth at 0%-12% = 3 growth at 0%-15% = 4 growth only at 0% = 5 growth only at 4%-15% = 612 Minimum pH for pH 7.5 = 7.5 growth on nutrient agar pH 8.0 = 8.0 pH 8.5 = 8.5 pH 9.0 = 9.0 pH 9.5 = 9.5 pH 10.0 = 10.0 pH 10.5 = 10.513-21 Carbohydrate utilisation13 Formate14 Fumarate15 Succinate16 Galactose enhanced growth = 217 Pyruvate equal growth = 118 Fructose growth inhibited = 019 Lactose20 Xylose21 Starch22-53 Growth on carbon substrates22 Rhamnose23 N-acetylglucosamine24 D-ribose25 Inositol26 D-saccharose27 Maltose28 Itaconate29 Suberate30 Malonate31 Acetate32 DL-lactate positive = 233 L-alanine negative = 134 Mannitol35 D-glucose36 Salicin37 D-melibiose38 L-fucose39 D-sorbitol40 L-arabinose41 Propionate42 Caprate43 Valerate44 Citrate45 Histidine46 5-ketogluconate47 Glycogen48 3-hydroxybenzoate49 L-serine50 2-ketogluconate51 3-hydroxybutyrate52 4-hydroxybenzoate53 L-proline54-72 Enzymatic activities54 Alkaline phosphatase55 Esterase (C4)56 Esterase lipase (C8)57 Lipase (C14)58 Leucine arylamidase59 Valine arylamidase60 Cystine arylamidase positive = 261 Trypsin negative = 162 Chymotrypsin63 Acid phosphatase64 Naphthol-AS-BI-phosphohydrolase65 .alpha.-galactosidase66 .beta.-galactosidase67 .beta.-glucuronidase68 .alpha.-glucosidase69 .beta.-glucosidase70 N-acetyl-.beta.-glucosaminidase71 .alpha.-mannosidase72 .alpha.-fucosidase73-82 Aminoacids as carbon and nitrogen source73 Serine74 Proline75 Asparagine76 Arginine enhanced growth = 277 Alanine equal growth = 178 Lysine no growth = 079 Methionine80 Phenylalanine81 Glycine82 Valine 83-104 Antibiotic sensitivity83 Gentamycin 10 .mu.g84 Nitrofurantoin 50 .mu.g85 Ampicillin 25 .mu.g86 Nalidixic Acid 30 .mu.g87 Sulphamethoxazole 50 .mu.g88 Trimethoprim 2.5 .mu.g89 Penicillin G 1 IU antibiotic sensitive90 Chloramphenicol 25 .mu.g inhibition of growth = 291 Erythromycin 5 .mu.g92 Fusidic Acid 10 .mu.g antibiotic insensitive,93 Methicillin 10 .mu.g no growth inhibition = 194 Novobiocin 5 .mu.g95 Streptomycin 10 .mu.g96 Tetracycline 25 .mu.g97 Sulphafurazole 100 .mu.g98 Oleandomycin 5 .mu.g99 Polymixin 300 IU100 Rifampicin 2 .mu.g101 Neomycin 30 .mu.g102 Vancomycin 30 .mu.g103 Kanamycin 30 .mu.g104 Bacitracin 10 IU______________________________________
______________________________________Appendix DScreening for Proteolytic, Amylolytic and Lipolytic ActivitySTRAIN LACTALBUMIN CASEIN BLOOD GELATIN______________________________________Proteolytic ActivityCluster 11E.1.sup.CT + + + +2E.1 - - - -wB2 - - - +wB5 - - - -wBs4 - + + +10B.1 + + + +20N.1 + + - +27M.1 - + - -wNk2 - + - +Cluster 239E.3 n.t. n.t. n.t. +41E.3 n.t. n.t. n.t. +45E.3.sup.CT n.t. n.t. n.t. +47E.3 n.t. n.t. n.t. +51N.3 n.t. n.t. n.t. +52N.3 n.t. n.t. n.t. +42E.3 n.t. n.t. n.t. +50N.3 n.t. n.t. n.t. +Cluster 36B.1 + + - +7B.1 + + - -8B.1 - + - +38E.2 n.t. n.t. n.t. -56E.4 + n.t. n.t. -25B.1 n.t. + n.t. +26N.1 - + - +11C.1 + + - +wB.1 - - - +12C.1 - + - -28N.1.sup.CT - - - -61N.4 + n.t. n.t. -36E.2 n.t. n.t. n.t. -40E.3 n.t. n.t. n.t. +65B.4 + n.t. n.t. -94LM.4 n.t. n.t. n.t. +19N.1 - + - +24B.1 + + - +21M.1 + + + +29C.1 - - - -35E.2 n.t. n.t. n.t. -37E.2 n.t. n.t. n.t. -48E.3 n.t. n.t. n.t. +78LN.4 n.t. + n.t. +73aC.4 n.t. + n.t. +75C.4 n.t. + n.t. +73bC.4 n.t. + n.t. +74C.4 n.t + n.t. -77LN.4 n.t. + n.t. -wN1 - - - -49N.3 n.t. n.t. n.t. +44E.3 n.t. n.t. n.t. +58E.4 n.t. n.t. n.t.57E.4 + n.t. n.t. +Cluster 4wE5 - - - +wB4.sup.CT - - - -wNk1 - - - +wEl1 - - - -wEl2 - - - +Cluster 59B.1 - + - +16N.1 + n.t. n.t. +17N.1.sup.CT + + - +22M.1 + + - +Cluster 618N.1 + + - +59E.4 + n.t. n.t. +64B.4.sup.CT + n.t. n.t. +63N.4 + n.t. n.t. +53E.4 + n.t. n.t. +Non-Clustering StrainswN.2 - - - +4E.1 - - - -5E.1 - - - +92LM.4 n.t. n.t. n.t. +wBn5 - - - +______________________________________ n.t. = not tested
LIPASE ESTERASE LIPASE STARCH ACTIVITY LIPASE ACTIV-STRAIN HYDROLYSIS ON OLIVE OIL ACTIVITY ITY______________________________________Amylolytic and Lipolytic Activity*Cluster 11E.1.sup.CT + - + +2E.1 + - + -wB2 - - + -wB5 - - + -wBs4 - - + -10B.1 + - + -20N.1 - - + -27M.1 - - + -wNk2 + - + -Cluster 239E.3 + + + -41E.3 + + + -45E.3.sup.CT + + + +47E.3 + + - -51E.3 + + + +52E.3 + + + +42E.3 + + + +50N.3 + + + +Cluster 36B.1 - - + -7B.1 + - + +8B.1 + + + +38E.2 - - + -56E.4 - + + +25B.1 - - + +26N.1 - - + -11C.1 - - + -wB1 - - + -12C.1 + - - -28N.1.sup.CT + - + -61N.4 + - - -36E.2 - - - -40E.3 + + + +65B.4 + - + -94LM.4 + - + -19N.1 + - + +24B.1 - - + -21M.1 + - - -29C.1 + - + +35E.2 - - + -37E.2 - - + -48E.3 - + + -78LN.4 - - + -73aC.4 - - + -75C.4 + - - -73bC.4 + - + -74C.4 + - + -77LN.4 - - + +wN1 + - + -49N.3 + + + -44E.3 + + + +58E.4 + + + -57E.4 + - + -Cluster 4wE5 + - - -wB4.sup.CT - - + -wNk1 + - + -wEl1 - - + -wEl2 - - + -Cluster 59B.1 + - + -16N.1 + - + -17N.1.sup.CT + - + -22M.1 + - + -Cluster 618N.1 + - + -59E.4 + - + -64B.4.sup.CT + - + -63N.4 + - - -53E.4 + - + -Non-Clustering StrainswN2 + - + +4E.1 - - + -5E.1 + - + -92LM.4 + + + -wBn5 - - + -______________________________________ *Starch Hydrolysis determined according to Character 21 (Appendix B) Lipase Activity (Olive Oil) determined on media M P (Appendix A) Esterase Lipase Activity determined according to Character 56 (Appendix B Lipase Activity determined according to Character 57 (Appendix B)
______________________________________Appendix EPercentage Positive States for Characters in Clusters CLUSTERCHARACTER 1 2 3 4 5 6______________________________________�6! Cell morphology 9 0 12 0 0 0�7! Gram's stain 0 0 0 0 0 0�8! Oxidase test 55 100 35 80 0 20�9! Skim milk test 36 11 6 20 0 20�10! Gelatin 67 100 56 60 100 100�13! Formate 0 0 15 40 0 0�14! Fumarate 45 78 74 80 75 60�15! Succinate 45 89 85 40 67 50�16! Galactose 45 11 12 40 25 0�17! Pyruvate 64 89 88 40 75 0�18! Fructose 45 11 68 60 50 100�19! Lactose 9 0 0 0 0 0�20! Xylose 18 11 15 20 0 0�21! Starch 73 100 91 20 100 100�22! Rhamnose 9 0 15 20 0 0�23! N-acetylglucosamine 9 0 26 20 0 100�24! D-ribose 27 0 50 0 0 20�25! Inositol 9 0 9 0 50 0�26! D-saccharose 27 0 74 20 25 100�27! Maltose 9 11 74 40 50 100�28! Itaconate 0 11 6 0 0 0�29! Suberate 9 67 53 40 0 0�30! Malonate 0 11 65 0 25 40�31! Acetate 18 100 100 80 25 100�32! DL-lactate 27 56 100 100 0 40�33! L-alanine 18 89 82 100 0 100�34! Mannitol 0 0 41 20 0 80�35! D-glucose 9 11 71 60 0 60�36! Salicin 0 0 3 20 0 60�37! D-melibiose 0 0 3 0 0 0�38! L-fucose 0 0 0 20 0 20�39! D-sorbitol 18 0 41 0 0 40�40! L-arabinose 9 0 3 40 0 0�41! Propionate 9 100 94 80 0 80�42! Caprate 0 78 32 40 0 80�43! Valerate 9 100 97 80 0 40�44! Citrate 9 56 94 20 50 100�45! Histidine 0 0 71 0 0 80�46! 5-ketogluconate 0 0 21 0 0 0�47! Glycogen 9 22 26 20 25 100�48! 3-hydroxybenzoate 9 0 38 0 0 0�49! L-serine 0 0 68 60 0 0�50! 2-ketogluconate 0 0 56 80 25 20�51! 3-hydroxybutyrate 18 33 94 100 0 100�52! 4-hydroxybenzoate 0 0 71 80 0 0�53! L-proline 45 100 100 80 25 80�54! Alkaline phosphatase 100 44 94 40 100 60�55! Esterase (C4) 100 100 100 100 100 100�56! Esterase lipase (C8) 100 89 85 100 100 100�57! Lipase (C14) 9 67 26 0 0 0�58! Leucine arylamidase 91 67 94 60 50 0�59! Valine arylamidae 91 33 65 100 25 0�60! Cystine arylamidase 73 0 15 0 0 0�61! Trypsin 64 11 9 60 0 0�62! Chymotrypsin 73 0 3 20 75 0�63! Acid phosphatase 91 11 94 100 100 60�64! Naphthol 91 22 3 20 100 40 phosphohydrolase�65! .alpha.-galactosidase 0 11 3 20 100 0�66! .beta.-galactosidase 9 0 0 20 100 0�67! .beta.-glucuronidase 9 0 3 20 25 0�68! .alpha.-glucosidase 27 0 79 60 100 40�69! .beta.-glucosidase 9 0 9 80 75 0�70! N-acetyl-.beta.- 27 0 0 0 0 0 glucosaminidase�71! .alpha.-mannosidase 0 0 0 0 0 0�72! .alpha.-fucosidase 9 0 0 20 0 0�73! Serine 22 13 29 60 100 50�74! Proline 56 63 65 80 100 100�75! Asparagine 67 38 61 60 75 100�76! Arginine 56 25 53 80 50 50�77! Alanine 67 100 62 40 100 50�78! Lysine 44 75 71 80 75 100�79! Methionine 60 50 53 nc 50 50�80! Phenylalanine 89 100 76 100 100 100�81! Glycine 44 13 29 80 50 50�82! Valine 44 50 41 0 50 25�83! Gentamycin 36 67 3 20 0 0�84! Nitrofurantoin 18 33 21 0 0 0�85! Ampicillin 45 67 56 0 100 80�86! Nalidixic Acid 36 78 76 0 0 20�87! Sulphamethoxazole 18 33 38 0 0 0�88! Trimethoprim 45 22 35 0 75 60�89! Penicillin G 27 11 29 0 75 100�90! Chloramphenicol 100 100 100 100 100 100�91! Erythromycin 91 100 100 100 100 100�92! Fusidic Acid 18 0 3 20 100 100�93! Methicillin 45 11 50 0 100 100�94! Novobiocin 18 0 3 0 25 0�95! Streptomycin 100 78 97 100 75 100�96! Tetracycline 45 22 3 20 100 100�97! Sulphafurazole 40 13 20 nc 0 0�98! Oleandomycin 100 38 94 100 100 100�99! Polymixin 89 63 78 100 0 0�100! Rifampicin 100 38 78 100 100 100�101! Neomycin 0 13 0 0 0 0�102! Vancomycin 22 13 0 20 100 75�103! Kanamycin 11 13 0 0 0 0�104! Bacitracin 33 13 0 20 100 100______________________________________

	Number	Date
Parent	903786	Jun 1992
Parent	719307	Jun 1991
Parent	562863	Aug 1990

Gram-negative alkaliphilic microorganisms

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