Patent Application
20100299781
The invention relates generally to modulating physiological responses to singlet oxygen in bacterial cells, algae or plants. Singlet oxygen (1O2) is produced by enzymes such as peroxidases and oxidases or as byproduct of various processes such as photosynthesis. Kochevar I, “Singlet oxygen signaling: from intimate to global,” STKE 204:pe7 (2004). In the photosynthetic process, input light energy converts water (H2O) and carbon dioxide (CO2) to oxygen (O2) and sugar. Cellular respiration subsequently converts some of the sugar into chemical energy in the form of ATP. The conversion is associated with chlorophyll, a green pigment common to all photosynthetic cells. Although O2 is a relatively non-reactive chemical, when exposed to high-energy or electron-transferring chemical reactions, it can be converted to highly reactive chemical forms collectively designated as “reactive oxygen species” (ROS). ROS are generally considered toxic to organisms because they oxidize carbohydrates, DNA, lipids and proteins, breaking down normal cellular, membrane and reproductive functions. Ultimately, at toxic ROS levels, a chain reaction of cellular oxidation can result in disease or lethality.
In phototrophs, including plants, light energy excites chlorophyll pigments in the light harvesting complexes to a triplet state. At some frequency, an energy transfer from the excited triple state chlorophyll pigments to ground-state O2 generates 1O2 which, as a strong oxidant, can destroy membrane integrity, abolish biomolecular function, and reduce photochemical activity by inactivating photosynthetic enzymes.
Because excited triplet-state chlorophyll pigments and ground-state oxygen are found in close proximity to one another, many phototrophs exhibit some natural defenses against 1O2. For example, carotenoids, fat-soluble, anti-oxidant pigments found within the photosynthetic apparatus, quench 1O2. Telfer A, “What is β-carotene doing in the photosystem II reaction centre,” Phil. Tans. R. Soc. Lond. 357:1431-1440 (2002). Carotenoids include, but are not limited to, β-carotene, zeaxanthin and tocopherols. If not completely quenched by carotenoids or other suitable compounds, 1O2 can specifically trigger upregulation of genes that encode proteins involved in the molecular defense against photo-oxidative stress. For example, a network of upregulated plant genes maintains a balance between ROS-scavenging proteins and ROS-producing proteins. Mittler R, “Reactive oxygen gene network of plants,” TRENDS in Plant Sci. 9:490-498 (2004). In bacteria, a set of sigma factors, interchangeable RNA polymerase subunits responsible for recognizing transcriptional promoters, maintain essential housekeeping functions and facilitate host response to specific environmental stresses, including ROS. A constitutively-expressed, principal sigma factor is responsible for transcribing essential housekeeping genes. Other sigma factors, transcriptionally- or post-translationally-activated in response to stresses, recognize promoters upstream of genes involved in the response to stresses. Sigma factors are themselves regulated by anti-sigma factors that bind to a specific sigma factor and inhibit that sigma factor's ability to recognize a promoter.
Activation of sigma factors has been studied, inter alia, in Rhodobacter sphaeroides, a member of the α-subdivision of Proteobacteria and a facultative phototroph. R. sphaeroides is among the most metabolically diverse organisms known, being capable of growth under a wide variety of growth conditions. In addition to being photosynthetic, R. sphaeroides possesses additional energy-acquiring mechanisms including lithotrophy, aerobic respiration and anaerobic respiration. SigmaE (σE), a 19.2 kDa alternative sigma factor encoded by rpoE and related to members of the extra-cytoplasmic function (ECF) subfamily of eubacterial RNA polymerase sigma factors, is increased following environmental stress in R. sphaeroides. σE directs transcription from rpoE P1, a promoter for the rpoEchrR operon, and from cycA P3, a promoter for cytochrome c2. Newman J, et al, “The Rhodobacter sphaeroides ECF sigma factor, σE, and the target promoters cycA P3 and rpoE P1,” J. Mol. Biol. 294:307-320 (1999), incorporated herein by reference as if set forth in its entirety. Basal σE activity, however, is quite low because it is complexed with a zinc-dependent anti-sigma factor, ChrR. ChrR loses its ability to inhibit σE if zinc is removed, or if a zinc-binding domain of the N-terminal domain is removed. Newman J, et al., “The importance of zinc-binding to the function of Rhodobacter sphaeroides ChrR as an anti-sigma factor,” J. Mol. Biol. 313:485-499 (2001), incorporated herein by reference as if set forth in its entirety.
GenBank Accession No. AAB17905 (SEQ ID NO:1), discloses the full-length R. sphaeroides ChrR sequence. ChrR with a C38R mutation prevented binding to σE. See Newman et al. (1999), supra. Likewise, ChrR with a C35S or a C38S mutation prevented binding to σE. See Newman et al. (2001), supra. Furthermore, a ChrR with a C187/189S mutation was shown to prevent binding to σE. Id. In addition, ChrR with a H6A mutation, a H31A mutation, a C35A mutation or a C38A mutation cannot bind zinc and ultimately cannot bind σE.
GenBank Accession No. AAB17906 (SEQ ID NO:2) discloses the full-length R. sphaeroides σE sequence. Mutations in region 2.1 (amino acids 22 to 46 of SEQ ID NO:2) of σE alter the interaction between ChrR and σE. Anthony J, et al., “Interactions between the Rhodobacter sphaeroides ECF sigma factor, σE, and its anti-sigma factor, ChrR,” J. Mol. Biol. 341:345-360 (2004), incorporated herein by reference as if set forth in its entirety. In particular, σE with a K38E mutation, a K38R mutation or a M42A mutation were less sensitive to ChrR both in vivo and in vitro.
Because 1O2 affects many organisms (including, but not limited to, bacteria, plants, animals and humans), the components of the biological response to 1O2 find application in medicine, agriculture, biotechnology and bioenergy production systems. Animals and plants use 1O2 to defend against microbial pathogens. Davies M, “Reactive species formed on proteins exposed to singlet oxygen,” Photochem. Photobiol. Sci. 3:17-25 (2004), incorporated herein by reference as if set forth in its entirety. For the foregoing reasons, there is a desire to manipulate physiological responses to 1O2 in animals, bacteria and plants. There are many advantages of studying responses to 1O2 in R. sphaeroides. First, one can control the formation of significant amounts of 1O2. Also, biochemical and genetic systems are available to study the response to 1O2 in vivo and in vitro, including an Affymetrix gene chip (Affymetrix; Santa Clara, Calif.), LC/MS proteomics and computation approaches.
The present invention relates to observations by the inventors relating to genes required for viability of R. sphaeroides in the presence of 1O2 which can be generated during photosynthesis. Specifically, changes in the interaction between alternative sigma factor σE and its anti-sigma factor ChrR affects expression of genes required for viability of R. sphaeroides in the presence of 1O2. Although homologs of σE and ChrR have been identified computationally in other bacteria, their involvement in a cellular response to 1O2 has not heretofore been noted.
As the inventors detail below, 1O2 typically has detrimental effects upon cells, but cells can avoid or overcome the effects by increasing σE, which is ordinarily complexed with ChrR. In the presence of 1O2, ChrR and σE dissociate and synthesis of σE increases, allowing free σE to bind to a core RNA polymerase, facilitating transcription of a regulon involved in attenuating physiological effects of 1O2. This observation suggests that σE or ChrR can be manipulated to exploit the response of cells and organisms to 1O2. Even though some of the observations were made in R. sphaeroides, the invention is not intended to be limited to this single prokaryote, as responses to 1O2 are present in many other species, including both photosynthetic and non-photosynthetic prokaryotes and eukaryotes.
The observation can be exploited to inhibit or prevent microbial survival, by preventing dissociation between ChrR and σE or by reducing the extent of dissociation in the microbes. In the presence of 1O2, the microbes would succumb to damage caused by increased oxidative stress.
The observation can alternatively be exploited to increase efficiency of microbially-catalyzed commercial processes for generating commodity chemicals such as, but not limited to, acetic acid and other organic acids, acetone, acrylamide, butanol, ethanol, glycerol, isoprenoids, quinines, and pigments as well as strategic chemicals with potential use as lubricants or biofuels since some of the gene products involved in this conserved response modify hydrocarbons within the cell membrane. Nagasawa T & Yamada H, “Microbial production of commodity chemicals,” 67 Pure & Appl. Chem. 1241-1256 (1995), incorporated herein by reference as if set forth in its entirety. In particular, photosynthetic organisms for use in such processes can be engineered to inhibit or eliminate binding between ChrR and σE, such that when the microbe finds itself in the presence of 1O2, it readily overcomes any toxic effects by mobilizing its increased available supply of σE to initiate transcription of the protective regulon, ensuring robust production from the process, notwithstanding the presence of 1O2. Increasing production of these or other commodity chemicals involves inhibiting the interaction between ChrR and σE in the presence of 1O2 so that the microbe continues to produce a desired commodity chemical notwithstanding oxidative stress. In one approach, the microbe can be engineered either to contain a mutated ChrR that cannot bind σE, or to lack ChrR entirely. A similar effect can be obtained by engineering a microbe for use in the process where the microbe contains a mutated σE relative to wild-type σE such that the mutated sigma factor cannot bind ChrR, or binds ChrR only weakly. In some embodiments, the photosynthetic organism is a bacterium, an alga or a plant. In some embodiments, the photosynthetic organism is R. sphaeroides. In some embodiments, σE is modified relative to wild type by engineering a K38E mutation, a K38R mutation or a M42A mutation in σE. In some embodiments, ChrR is modified relative to wild type by engineering a C35S mutation, a C38S mutation, a C38R mutation or a C187/189S in ChrR.
In another aspect, the observation can be exploited by protecting the phototroph from toxic effects of 1O2 by looking beyond the direct interaction of σE and ChrR, to the genes transcribed directly by σE or genes whose expression is increased by a σE-dependent transcription factor. For example, where the available supply of σE is not, or cannot, be increased as noted above, the phototroph, especially a plant, can be engineered to increase expression of genes that encode protective proteins. For example, CfaS, shown herein to be upregulated by σE, encodes cyclopropane-fatty-acyl-phospholipid synthase which catalyzes the generation of cyclopropane fatty acids by adding a methylene bridge across a double bond of a fatty acid. In lipid bilayers, 1O2 can hydroxylate unsaturated fatty acids and membrane-destabilizing lipid peroxides can form. On the other hand, 1O2 cannot hydroxylate cyclopropane fatty acids in the bilayers, so lipid peroxides cannot form and the phototrophs are protected from oxidative stress. Conversely, unwanted or invasive plant species can be made more susceptible to oxidative stress by engineering the phototroph to downregulate genes that encode protective proteins, such as CfaS, which is known to modify the fatty acid composition of the membrane and result in accumulation of hydrocarbons of strategic value as biofuels, lubricants, etc.
In another aspect, the present invention is summarized in that a consensus promoter responsive to σE is disclosed as SEQ ID NO:3. In some embodiments, the nucleic acid residues at positions 2, 6, 12, 16, 17 and 20 are G; at position 11 is A; and at positions 19, 22 and 26 are C. The isolated nucleic acid sequence of SEQ ID NO:3 can be operably linked to a heterologous reporter gene or gene of interest to produce a genetic construct suitable for transfer into cells of a phototroph. Expression of the operably linked gene can thereby be placed under the control of σE. In so doing, not only can protective proteins be produced in the presence of 1O2, but any other protein, polypeptide, peptide or oligonucleotide of interest can be induced under such conditions. Similarly, a reporter gene can be provided so that the presence of 1O2 can be detected, observed and monitored.
It is an advantage of the present invention that it provides the skilled person with the tools for efficiently producing products of this response in phototrophic or non-phototrophic organisms while avoiding longstanding issues arising from the presence of 1O2 or allowing skilled persons to express gene products in microbes to produce such commodity or strategic chemicals either in the presence or absence of this ROS.
A second advantage is that the methods and compositions are non-toxic to the environment.
These and other features, aspects and advantages of the present invention will become better understood from the description that follows as well as from Dufour et al., J. Mol. Biol. 383:713-730 (2008), incorporated herein by reference as if set forth in its entirety. In the description, reference is made to the accompanying drawings, which form a part hereof and in which there is shown by way of illustration, not limitation, embodiments of the invention. The description of preferred embodiments is not intended to limit the invention to cover all modifications, equivalents and alternatives. Reference should therefore be made to the claims recited herein for interpreting the scope of the invention.
Abbreviations used in the drawings: PS—photosynthetically grown cells, Aero—cells grown by aerobic respiration, WT—wild type, ΔChrR—cells lacking the anti-sigma factor ChrR, ΔσE—cells lacking both σE and ChrR.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.
As used herein, a “phototroph” or “photosynthetic organism” refers to any organism that is capable of photosynthesis.
Methods
Bacterial strains and plasmids: R. sphaeroides 2.4.1 (wild-type, WT), R. sphaeroides with a mutant ChrR (ΔChrR) (chrR-1::drf, see Newman et al., supra.) or R. sphaeroides with both a mutant σE (ΔσE) and ΔChrR (TF18; rpoEchrR-1::drf, see Schilke B & Donohue T, “ChrR positively regulates transcription of the Rhodobacter sphaeroides cytochrome c2 gene,” J. Bacteriol. 177:1929-1937 (1995), incorporated herein by reference as if set forth in its entirety) were grown at 30° C. in Sistrom's succinate-based minimal medium A. Media used for growth of strains containing low-copy lacZ reporter plasmids was supplemented with 25 μg/ml kanamycin.
Growth conditions: For aerobic respiratory growth, 500 ml of media was bubbled with a mixture of 69% N2, 30% O2 and 1% CO2 in the dark. Conversely, for photosynthetic growth, 500 ml of media was bubbled with a mixture of 95% N2 and 5% CO2 in front of an incandescent light source (10 W/m2 as measured with a Yellow-Springs-Kettering model 6.5 A radiometer through a Corning 7-69, 620 to 110 nm filter).
To test the effects of 1O2, photosynthetic cultures were exposed to aerobic growth conditions (69% N2, 30% O2 and 1% CO2) in the presence or in the absence of light (10 W/m2). Where indicated, light was passed through a 1283 filter (Kopp Glass; Pittsburgh, Pa.) that impedes >99% of light at wavelengths <770 nm, but transmits >45% of light at 830 nm and >80% of light at 900 nm. When using methylene blue (Sigma-Aldrich; St. Louis, Mo.) to generate 1O2, a final concentration of 1 μM was added to aerobic cultures in the presence or in the absence of incandescent light (10 W/m2). To test the effects of other ROS, 0.5 mM H2O2, 1 mM diamide or 1 mM paraquat (Sigma-Aldrich) was added to aerobic cultures.
All experiments were initiated when cultures reached ˜2×108 cfu/ml to minimize light or O2 limitation to photosynthetic and aerobic cells, respectively. To measure cell viability, samples were removed, diluted and plated in media supplemented with 25 μg/ml kanamycin to select for the rpoE P1::lacZ reporter plasmid. The whole cell abundance of carotenoids was measured as described in Cohen-Bazire G, et al., “Kinetic studies of pigment synthesis by non-sulfur purple bacteria,” J. Cell Physiol. 49:25-68 (1957).
Determining promoter activity: In vivo promoter activity was determined by measuring β-galactosidase activity from a low copy rpoE P1::lacZ reporter plasmid or a trxA::lacZ reporter plasmid. The promoter for the thioredoxin gene (trxA, −214 to +27 relative to the known transcription initiation site) was fused to lacZ and mobilized into R. sphaeroides.
β-galactosidase activity (units/ml of culture) was calculated as follows: (A420×1000)/(Cell volume in assay (ml)×Time of assay (min)). Culture density was typically monitored by measuring A600 in a BioSpec 1601 spectrophotometer (Schimatzu; Columbia, Md.). The density of cultures treated with methylene blue was monitored at 500 nm because methylene blue absorbs light between 609-668 nm. The differential rate of β-galactosidase synthesis was determined by calculating the slope from plots of enzyme activity (units/ml of culture) against optical density. All experiments were repeated a minimum of three times with differential rates of β-galactosidase synthesis typically deviating less than two-fold between experiments.
In vitro promoter activity was determined by using plasmid templates in which a test promoter was cloned into a plasmid (pRKK96) containing a known transcriptional terminator. Standard assay conditions mixed recombinant His6-σE (50 nM) with R. sphaeroides core RNA polymerase (50 nM) for 30 minutes at 30° C. in transcription buffer (40 mM Tris-HCl pH 7.9, 200 mM KCl, 10 mM Mg acetate, 1 mM DTT, and 62.5 mg/ml acetylated bovine serum albumin). Next, 20 nM of plasmid DNA was added and incubated for 30 minutes before transcription was initiated by adding nucleotide triphosphates. Newman et al., supra. Samples were incubated for 20 minutes before RNA products were analyzed by 6% (wt/vol) denaturing polyacrylamide gel electrophoresis, and products were visualized on a phosphorimager (Molecular Dynamics, Sunnyvale, Calif.
Identification of σE target genes: Triplicate cultures of WT and ΔChrR were grown aerobically to ˜2−3×108 CFU/ml. RNA was isolated and cDNA was synthesized, labeled and hybridized to R. sphaeroides GeneChip Custom Express microarrays (Affymetrix). After data extraction using Affymetrix MAS 5.0 software, data sets were imported into GeneSpring software (Silicon Genetics; Redwood City, Calif.) for normalization and analysis (Gene Expression Omnibus (GEO) accession number GSE2219).
Candidate σE promoters (extending ˜200 bp upstream of the predicted start of translation, Table 1) were amplified from 20 ng of WT chromosomal DNA in EasyStart PCR tubes (Molecular BioProducts; San Diego, Calif.) with 2.5 units Pfu Turbo (Stratagene; La Jolla, Calif.). PCR products were cloned into pRKK96 for in vitro assays or into a lacZ reporter plasmid (pRKK200) for determining activity in vivo.
1ΔChrR: R. sphaeroides WT with trimethoprim cartridge inserted into ChrR.
2Increase in RNA abundance from comparing transcript levels in WT and ΔChrR cells. Data has been deposited at GEO under accession number GSE2219.
3Function known or predicted by genome annotation.
Results
Conditions that generate 1O2 within the photosynthetic apparatus increase R. sphaeroides activity: Mutations that inactivate an early enzyme in carotenoid biosynthesis, CrtB, cause a small increase in σE activity (data not shown). Since carotenoids play a protective role against 1O2, it was determined whether 1O2 directly affected σE activity.
To determine if R. sphaeroides σE activity responds to 1O2, we examined the differential rate of β-galactosidase synthesis from a σE-dependent rpoE P1::lacZ reporter fusion after anaerobic, photosynthetic cells were exposed to O2 in the presence of light. After exposure to O2, the cells maintain approximately the same doubling rate, since O2 is used as a respiratory electron acceptor. However, after exposure to O2, the differential rate of β-galactosidase synthesis from the σE-dependent promoter increased ˜10-fold (from 6 to 65) when compared to a control culture grown under either a steady state photosynthetic condition (light in the absence of O2) or a respiring condition (30% O2) (
1O2
1Aero = cells grown by aerobic respiration (30% O2),
2PS = cells grown photosynthetically.
This transcriptional response was maintained throughout the experiment, suggesting that σE activity was sustained. There was less than a two-fold increase in the differential rate of β-galactosidase synthesis from the rpoE P1::lacZ reporter fusion when photosynthetic cells were shifted to aerobic conditions in the dark (Table 2). This was expected since little 1O2 is made under this condition due to lack of light needed to produce triplet state chlorophyll molecules. From these results, one can conclude that the combination of light and O2, conditions known to generate 1O2 within the photosynthetic apparatus, are required for this transcriptional response.
Control experiments indicated that this response was dependent on σE since the differential rate of a β-galactosidase synthesis from the rpoE P1::lacZ reporter fusion in ΔσE cells (<1 unit) did not increase upon exposure to 1O2. ΔσE cells grow under these conditions, presumably because the carotenoids within the photosynthetic apparatus quench 1O2. In addition, it appears that 1O2 does not fully induce σE activity since the differential rate of β-galactosidase synthesis from the rpoE P1::lacZ reporter fusion in WT cells exposed to 1O2 was 10-fold less than that seen in ΔChrR cells (65 versus 650).
Wavelengths of light that excite chlorophyll pigments are sufficient to increase σE activity: If production of 1O2 by the photosynthetic apparatus was responsible for this transcriptional response, then wavelengths of light known to generate triplet state chlorophyll molecules within the light harvesting complexes should increase σE activity. R. sphaeroides contains two light harvesting complexes, B800-850 and B875, named for their absorption maxima in the near infrared. To determine if light absorbed by the light harvesting complexes could cause this response, we looked at the action spectrum of this transcriptional response. Under photosynthetic conditions with light that was filtered to remove wavelengths <830 nm, the differential rate of β-galactosidase synthesis from the σE-dependent promoter was an ˜4-fold lower than that observed with cells grown in white light (Table 2), presumably because the cells grow slower when light <830 nm is removed. However, there was an ˜17-fold increase in the differential rate of β-galactosidase synthesis when cultures illuminated with >830 nm light were exposed to O2 (Table 2). The magnitude of this response was similar to that observed when photosynthetic cells were exposed to O2 and white light (˜17-fold versus ˜10-fold, Table 2). Thus, wavelengths of light that excite the light harvesting complexes are sufficient to increase σE activity.
Continued exposure to conditions that generate 1O2 in the photosynthetic apparatus are needed to sustain this response: The half-life of 1O2 in cells is less than 100 ns and was used to further test if σE activity was responding to 1O2. For example, if increased σE activity required 1O2, then placing photosynthetic cells that had previously been exposed to O2 in the dark should terminate this transcriptional response. When the cells were shifted to aerobic conditions in the presence of light, we observed an expected increase in the differential rate of β-galactosidase synthesis from the σE-dependent promoter (˜10-fold,
1O2
R. sphaeroides σ
E activity is increased by formation of O2 in the absence of the photosynthetic apparatus: If 1O2 was responsible for the observed σE transcriptional response, then other conditions that generate this ROS should also increase σE activity. To test this hypothesis, one can generate 1O2 by illumination of methylene blue in the presence of O2 to produce a similar response. When aerobically grown WT cells were exposed to 1 μM methylene blue in the presence of light and O2, cell growth continued and the differential rate of β-galactosidase synthesis from the rpoE P1::lacZ reporter fusion increased ˜20-fold compared to aerobic cells grown in the absence of methylene blue (Table 4). Control experiments indicated there was less than a two-fold increase in the rate of β-galactosidase synthesis when aerobic cultures were exposed to methylene blue in the dark (Table 4). The lack of a comparable increase in σE activity in aerobic cells exposed to methylene blue in the dark is expected since both light and O2 are required for this compound to generate 1O2.
1O2
1Differential rates of β-galactosidase synthesis from the σE-dependent rpoE::lacZ fusion when WT cells are grown aerobically under conditions that either do or do not generate 1O2.
For these experiments, cells were grown in the presence of 30% O2, a condition where pigment-protein complexes of the photosynthetic apparatus are not detectable. Therefore, the transcriptional response to 1O2 can occur in cells that either contain or lack the photosynthetic apparatus.
Other ROS do not produce a similar increase in σE activity: The damaging effects of 1O2 on many biomolecules could stimulate the formation of other ROS. To test if other ROS could produce an increase in σE activity, the differential rate of β-galactosidase synthesis from a rpoE P1::lacZ reporter fusion was monitored in aerobic cells treated with concentrations of hydrogen peroxide (H2O2), paraquat (to stimulate superoxide (O2−) formation) or diamide (to alter the oxidation-reduction state of the cytoplasmic thiol pool), previously shown to generate an oxidative stress response in R. sphaeroides. Li K, et al., “Expression of the trxA gene for thioredoxin 1 in Rhodobacter sphaeroides during oxidative stress,” Arch. Microbiol. 180:484-489 (2003). For these experiments, the differential rate of β-galactosidase synthesis was monitored from a control trxA::lacZ reporter fusion, since the trx promoter has previously been shown to respond to oxidative stress in R. sphaeroides.
The addition of paraquat or H2O2 to aerobic cells produced increases in the differential rate of β-galactosidase synthesis from the trxA::lacZ reporter gene that are consistent with changes in abundance of trxA transcripts produced by these compounds in previous studies (Table 5). However, the differential rate of β-galactosidase synthesis from the σE-dependent reporter fusion either decreased (paraquat) or increased no more than 1.2-fold (H2O2) when compared to untreated cells (Table 5). Any observed increase in σE activity in the presence of these ROS was below the 10-fold increase in σE activity seen when cells are exposed to 1O2.
1Differential rates of β-galactosidase synthesis from the indicated promoters when WT cells are grown aerobically under conditions that either do or do not generate indicated ROS.
σE activity in the presence of diamide was not monitored because previous work has shown that σE activity does not increase upon exposure to this compound. Based on these results, the transcriptional response observed when 1O2 is generated does not occur in the presence of other ROS.
When carotenoids are low, cells require σE to mount response to O2: While cells ΔσE cells are unable to mount this transcriptional response to 1O2 (
Exponential growth of aerobically grown WT cells continued after exposure to 1O2 (
Additional members of the σE regulon: To identify genes that are part of this transcriptional response to 1O2, we compared RNA levels in aerobically grown (30% O2) WT cells and in a ΔChrR mutant. Because ChrR inhibits σE activity, one looks for RNA that is more abundant in the ΔChrR mutant. As expected, global gene expression analysis showed an increase (˜12-fold) in rpoE-specific RNA from ΔChrR cells.
RNA from ˜180 genes (˜60 operons) was >3-fold more abundant in cells that contained increased σE activity (Table 1). In contrast, the ˜35-fold increase in cycA P3 activity that occurs in ΔChrR cells in vivo causes only an ˜1.6-fold increase in total cycA-specific RNA (Table 1). The smaller increase in cycA-specific RNA levels reflects the fact that cycA contains additional strong promoters that are recognized by other sigma factors. This suggests that a global gene expression microarray approach might miss other σE-dependent genes that also contain multiple promoters.
To test if any of these candidate operons contained a σE-dependent promoter, we tested DNA upstream of the first gene in each of twenty-eight potential operons for transcription by EσE. (Table 6) These operons were chosen either because of their increased levels of expression in cells with elevated σE activity or because of a potential role of their gene products in the photosynthetic apparatus (a source of 1O2). It was observed that rpoHII, which encodes one of two R. sphaeroides heat shock sigma factors (Rsp0601), is transcribed by σE. Production of the rpoHII transcript is inhibited by addition of ChrR, as is the case with other σE-dependent promoters like rpoE P1 and cycA P3 (
1Function known or predicted by genome annotation. Genes were chosen based on increased RNA abundance in cells that have elevated σE activity or for their known role in photosynthetic growth.
2Increase in RNA abundance from comparing transcript levels in WT and ΔChR cells. Data has been deposited at GEO under accession number GSE2219. NC- no change.
3Coordinates are numbered relative to the start site of translation.
4Based on the ability to detect a σE-dependent transcript in vitro (see FIG. 4A).
Each gene is predicted to be part of a polycistronic operon that encodes uncharacterized proteins. The level of transcripts produced from the rpoHII, Rsp1087 and Rsp2143 promoters are comparable to that of rpoE P1 (within 1.1-fold), suggesting that these 4 promoters are of similar strength. In contrast, the abundance of the σE-dependent transcript produced by Rsp1409 in vitro is comparable to the σE-dependent promoter, cycA P3, which has ˜80-fold less activity than rpoE P1.
The same putative rpoHII and Rsp1087 promoter regions were fused to lacZ to test for σE-dependent activity in vivo. Expression was not detectable from these reporter fusions in WT R. sphaeroides cells, but it was comparable to that of rpoE P1 in ΔChrR cells (
Generation of ChrR mutants to irreversibly bind to σE. The N-terminal anti-sigma domain of ChrR (ChrR-ASD) appears important in binding between ChrR and σE (data not shown). The skilled artisan is familiar with methods for delivering genetically engineered antimicrobial agents to microbes by phage therapy. Westwater C, et al., “Use of genetically engineered phage to deliver antimicrobial agents to bacteria: an alternative therapy for treatment of bacterial infections,” Antimicrob. Agents Chemother. 47:1301-1307 (2003), incorporated herein as if set forth in its entirety. Phage delivery systems are advantageous because they allow for targeting specific bacterial cells at a high frequency. Accordingly, a phage DNA is modified to contain a coding sequence that codes for at least amino acids 1-85 from GenBank Accession No. AAB17905 (SEQ ID NO:1), which discloses the R. sphaeroides full-length ChrR sequence. The N-terminal portion of ChrR encoded by this construct is sufficient to irreversibly bind zinc and σE. However, cells containing this or similar N-terminal ChrR variants are not able to mount a response to 1O2, resulting in a condition where cells have increased sensitivity to this reactive oxygen species.
Bacterial cells are grown under standard culture conditions. Once an adequate concentration of bacterial cells is present, they are infected with a phage modified to express at least amino acids 1-85 of SEQ ID NO:1. Following exposure to the phage, oxidative stress ensues, but the cells do not express genes regulated by σE. Consequently, the concentration of bacterial cells decreases.
Alternatively, bacterial cells are infected with a phage modified to express at least amino acids 1-85 of SEQ ID NO:1. They are then grown under standard culture conditions; however, the concentration of bacterial cells does not increase upon oxidative stress because the cells do not express genes regulated by σE.
Other methods for reducing availability of σE can include using RNAi directed against σE, mutating the promoter that directs transcription of σE (see Newman et al. (1999), supra), and engineering the cells to put σE under control of a regulatable promoter or repressor.
Bacterial cells with a modified ChrR that cannot bind σE are grown under standard culture conditions. However, growth and, consequently, production of a commodity chemical are increased because the cells are protected against the deleterious effects of 1O2.
Likewise, bacterial cells with a modified σE that cannot be bound by ChrR are grown under standard culture conditions. However, growth and consequently production of a commodity chemical are increased because the cells are protected against the deleterious effects of 1O2.
Methods of manipulating plant genes are known to the skilled artisan. For example, Constabel C, et al., “Transgenic potato plants overexpressing the pathogenesis-related STH-2 gene show unaltered susceptibility to Phytophthora infestans and potato virus X,” Plant Mol. Biol. 22:775-782 (1993), incorporated herein by reference as if set forth in its entirety. Accordingly, a plant is modified such that the plant exhibits a high level of cyclopropane-fatty-acyl-phospholipid synthase (CfaS) relative to an unmodified plant. The plant is grown under standard conditions; however, growth is increased because the plant is protected against the deleterious effects of 1O2.
Methods of inserting a gene of interest into a plasmid are known to the skilled artisan. Schilke & Donohue, supra. A gene encoding a product of interest is inserted into a plasmid under regulation of a σE-dependent promoter selected from the consensus sequence (SEQ ID NO:3) in
Using a combination of clustering analysis, promoter motif predictions, and functional assays, the inventors revealed that σE directly transcribed 9 operons with a total of 15 genes and that the promoter motif of these operons has a high information content (as defined in paragraph [00099]). The R. sphaeroides rpoEchrR operon, which encodes σE and ChrR, contains a σE-dependent promoter. Expression from this promoter increases in cells with a mutated ChrR (ΔChrR) due to the high σE activity in those cells. To identify potential members of the σE-ChrR regulon, the inventors clustered transcript levels from 67 R. sphaeroides global gene expression datasets collected from the Gene Expression Omnibus database, available at the NCBI GEO website, as of May 2005. The datasets contain RNA abundance measurements from 67 GeneChip Custom Express microarrays (Affymetrix, Santa Clara, Calif.) obtained from wild-type or mutant strains grown in a succinate-based minimal medium under various conditions (GSM1670, GSM1671, GSM1672, GSM1673, GSM2416, GSM2417, GSM2418, GSM2419, GSM2420, GSM2421, GSM2422, GSM2423, GSM2424, GSM2425, GSM2426, GSM2427, GSM2429, GSM2430, GSM2450, GSM25295, GSM25296, GSM25297, GSM25298, GSM25299, GSM25300, GSM25301, GSM25302, GSM25303, GSM26242, GSM26243, GSM26244, GSM26245, GSM26260, GSM26262, GSM26263, GSM26265, GSM26266, GSM27348, GSM27349, GSM27350, GSM27351, GSM27352, GSM27353, GSM3030, GSM3031, GSM3032, GSM3258, GSM3260, GSM3262, GSM3272, GSM3273, GSM3274, GSM38774, GSM38775, GSM38776, GSM38777, GSM38778, GSM38779, GSM38780, GSM38781, GSM38782, GSM38783, GSM38810, GSM40560, GSM8107, GSM8108, and GSM8109). RNA levels in all datasets were normalized using the Robust Multichip Average method to log2 scale. Irizarry et al., Biostatistics 4:249-264 (2003). To limit spurious correlation in the clustering steps, the inventors selected only those RNA species that were twofold or more abundant (FDR=0.1) in ΔchrR cells compared to wild type cells as determined by EBarrays software (Kendziorski et al., Stat Med. 22:3899-3914 (2003)). Approximately 100 loci were selected based on these criteria. Hierarchical clusters were constructed with the R statistical software environment, available from the Department of Statistics and Mathematics of the WU Wien, using (1-Pearson's correlation coefficient)/2 as the distance between expression patterns and the “complete” method for cluster linkage. The Pearson's correlation coefficient indicates the degree of association between two variables. A positive correlation value indicates positive association and a negative correlation value indicates negative or inverse association.
The Pearson's correlation coefficient between the abundance of rpoE transcripts and that of chrR transcripts was high (0.95) because these two genes are cotranscribed. In addition, the correlation coefficient between transcript levels for rpoE and another known σE target gene (rpoHII) was 0.88, suggesting that clustering genes with expression patterns that correlated with that of rpoE could predict other potential members of the σE-ChrR regulon. RNA abundance levels from the resulting 110 genes were clustered in a hierarchical tree based on the Pearson's correlation coefficient of their respective transcript patterns. In this tree, a cluster of transcript patterns containing rpoE and some other known σE target genes was identified, indicating that this method successfully predicts other members of the σE-ChrR regulon. Likewise, genes that are likely to belong to the same transcription unit, based on computational predictions of R. sphaeroides operons, were present in the rpoE-containing transcript cluster, providing further confidence in the predictive nature of the output. This rpoE-containing cluster contains one gene that had not previously been identified as a potential σE target (RSP1852), suggesting the existence of additional yet-to-be-identified members of this regulon.
The inventors hypothesized that operons directly transcribed by σE should contain a conserved promoter motif. To test this hypothesis, DNA sequences upstream of the potentially clustered operons were queried for a common sequence element. The BioProspector algorithm was used to search for a conserved bipartite motif within 300 by upstream of the predicted translation starting site in sets of candidate promoter sequences. The parameters were set to search for two blocks of 6 by and for a gap between 13 by and 16 bp. Sequence logos were generated using WebLogo, available at the Berkeley University, California website. Sequence logos are generated by aligning sequences and graphically representing the respective frequency of a nucleotide at a given position. Crooks et al., Genome Research, 14:1188-1190 (2004); Schneider and Stephens, Nucleic Acids Res. 18:6097-6100 (1990), each of which is incorporated herein by reference as if set forth in its entirety. The taller a nucleotide symbol, the greater its frequency at a particular position. Also, the taller the overall stack of nucleotide symbols, the greater the conservation at a particular position of the aligned sequences.
A conserved motif was found upstream of all the potential operons contained within the rpoE-containing cluster (
To search for additional candidate members of the σE-ChrR regulon that were not detected by analyzing the global gene expression patterns, a library of putative R. sphaeroides promoters was queried for sequences related to the conserved sequence motif. The conserved sequence motif was converted into a position-specific weighted matrix (PSWM), which represents motifs in biological sequences, that was used to score the promoter library based on the content information of the best match in each sequence. To relax the stringency of the query, spacer lengths of 13 by or 14 by between the −35 region and the −10 region were allowed without penalty. Six of the best fifteen matches (Table 7) were known σE target operons and one was a candidate σE promoter upstream of a gene within the rpoE-containing cluster (RSP1852). None of the promoter regions for any of the other 110 differentially expressed genes analyzed above contained a match to the motif with a score ≧75% of the maximum. Thus, it appears that the other differentially expressed operons are not likely to contain σE promoters.
RSP1087-
20.14
1.00
TGATCCG
ccttgggcgacag
TCCGTAT
0.91
1091
RSP0601
rpoH11
19.50
0.97
TGATCCG
gacatgtgttttt
TCCGTAG
0.88
RSP2143-
phrBcfaS
18.95
0.91
TGATCCG
ggaagcgggcccg
CGCGTAA
0.85
2144
RSP1092-
rpoEchrR
18.32
0.91
TGATCCA
gactggcccggcc
GCCGTAA
1
1093
RSP1409
18.14
0.90
TCATCCG
ccggagccgccttc
TGCGTAG
0.84
RSP0296
cycA
16.83
0.84
TGATCCG
gaacgcgcggccc
GCAGTAG
To determine if the candidate sequences (Table 7) contain functional σE promoters, the regions spanning from −200 by to +80 by relative to the predicted transcription initiation site were analyzed for function in vitro and in vivo, as described in Example 1. The inventors determined the relative strength of these candidate promoters by comparing their activity to known GE target genes and analyzed the ability of ChrR to inhibit the candidate promoter's function.
In vitro transcription using R. sphaeroides core RNA polymerase reconstituted with recombinant σE produced a product of the predicted size from the candidate RSP1852 promoter at a level similar to that obtained from the strong σE-dependent promoter (RSP1092) upstream of rpoE. Addition of the anti-σ factor ChrR prevented the accumulation of the σE transcript from the known σE SP1092 promoter and the candidate RSP1852 promoter. In contrast, the levels of transcript obtained from the candidate RSP3336 and RSP6222 promoters were lower, and detection of these products required a σE concentration that was 5- to 10-fold (250-500 nM recombinant protein) in excess over R. sphaeroides core RNA polymerase (50 nM). Similar increases in the concentration of σE do not generate a higher amount of product from strong promoters such as RSP 1092 in multiple-round transcription assays under these conditions. Thus, DNA binding by EσE holoenzyme may be a limiting kinetic step for transcription at the RSP3336 and RSP6222 promoters. The inventors could not assess whether ChrR inhibits transcription from the RSP3336 and RSP6222 candidate promoters because the anti-σ factor could not be obtained at concentrations sufficiently high to inhibit all the GE in the assay. All other candidate promoters shown in Table 7 failed to produce detectable in vitro transcripts, regardless of the levels of σE added to core RNA polymerase (data not shown).
Specific bases at these positions of the −10 and −35 elements are associated with σE-dependent transcription. The tested promoters active with σE in vitro contain a GTA motif in their −10 region, whereas those that were inactive in this assay have one or more substitutions to any other nucleotide in this motif. The RSP1207 promoter, also not transcribed by σE in vitro, contains a GTA motif in its putative −10 region but contains a substitution in place of the first T of the −35 region that is conserved in all active promoters.
To test for promoter activity in vivo, these and other candidate promoter sequences were fused to a promoterless β-galactosidase gene on a low-copy plasmid. The activity of these reporter genes was tested in wild-type R. sphaeroides cells (low σE activity), ΔChrR cells (high σE activity), and cells lacking the rpoEchrR operon (no σE activity). Consistent with the results of in vitro transcription assays, the inventors observed high reporter gene activity in ΔChrR cells from known σE target genes (RSP601, RSP1091, RSP1092, RSP1409, and RSP2143) and RSP1852, while both RSP6222 and RSP3336 showed lower activity (
To identify possible additional members of the σE-ChrR regulon, the inventors assessed genome-wide interactions of GE or the β′ subunit of RNA polymerase with DNA using chromatin immunoprecipitation and microarray (ChIP-chip) analysis. For immunoprecipitation, specific polyclonal antibodies were used to enrich DNA bound by σE or the (3′ subunit in ΔChrR cells (high σE activity). ChIP-chip hybridization was performed essentially as described in Lee et al., Nat. Protoc. 1:729-748 (2006), incorporated herein by reference as if set forth in its entirety. R. sphaeroides ΔchrR cells (increased σE activity) were grown by bubbling 500 ml of cell culture with 1% CO2, 30% O2, and 69% N2. At midexponential phase (˜2×108 colony-forming units/ml), formaldehyde and sodium phosphate were added to a final concentration of 1% and 10 mM, respectively. This mixture was incubated at 30° C. for 4 minutes before glycine was added to 100 mM, and the solution was placed on ice for 30 minutes with gentle agitation to quench excess formaldehyde. Cells were centrifuged at 3000 g, washed twice with chilled phosphate-buffered saline, centrifuged, and flash-frozen at −80° C. About 2×1010 cells were suspended in 0.5 ml of 100 mM Tris (pH 8.0), 300 mM NaCl, 2% Triton X-100, and 1 mM phenylmethylsulfonyl fluoride, and sonicated eight times for 20 seconds with a Branson Sonifier (Branson Ultrasonics Corp., Danbury, Conn.) set to level 6 and 50% output using a 3-mm microtip. A mixture of micrococcal nuclease (50 U) and RNase A (0.5 μg) in 200 μM CaCl2, 1.2 mM KCl, 6 mM sucrose, and 10 μM DTT was added. The mixture was incubated for 1 hour at 4° C.; and then nuclease activity was inhibited by adding 10 mM ethylenediaminetetraacetic acid (EDTA). Cell debris was removed by centrifuging for 10 minutes at 12,000 g, and an aliquot was removed to analyze DNA fragmentation by agarose gel eletrophoresis (desired size of ˜200-1000 by with enrichment for ˜500-bp molecules). The supernatant was incubated with gentle mixing with 20 μl of Staphylococcus aureus protein A Sepharose beads (Sigma-Aldrich, St. Louis, Mo.) for 3 hours at 4° C. as pretreatment to remove potential nonspecific binding to the beads. After the beads had been removed by centrifugation (5 minutes at 3000 g), one-tenth of the sample was removed and used as non-antibody-treated control. Two microliters of the anti-R. sphaeroides σE rabbit polyclonal antibody serum was added, and the mixture was incubated overnight at 4° C. with gentle mixing before being incubated with protein A Sepharose beads (30 μl) for 2 hours at 4° C. The beads were recovered by centrifugation and then washed once at 4° C. with 250 mM LiCl, 100 mM Tris (pH 8.0), and 2% Triton X-100, followed by two washes in 600 mM NaCl, 100 mM Tris (pH 8.0), and 2% Triton X-100; two washes in 300 mM NaCl, 100 mM Tris (pH 8.0), and 2% Triton X-100; and two washes in TE buffer (10 mM Tris pH 8.0 and 1 mM EDTA). Protein-DNA complexes were eluted from the beads by incubation at 65° C. for 30 minutes in 50 mM Tris (pH 8.0), 10 nM EDTA, and 1% sodium dodecyl sulfate. The beads were removed by centrifugation, and protein-DNA cross-linking was reversed by incubating the samples for 12 hours at 65° C.
DNA was purified using the QIAquick PCR Purification Kit (QIAGEN, Inc., Valencia, Calif.) and amplified via ligation-mediated PCR. DNA fragments were treated with T4 DNA polymerase, purified by phenol extraction and ethanol precipitation (adding 20 μg of glycogen), and ligated with T4 DNA ligase to 4 μM annealed oligonucleotide linkers (oJW102: GCGGTGACCCGGGAGATCTGAATTC; oJW103: GAATTCAGATC) at 16° C. overnight. DNA was ethanol-precipitated before amplification (22 cycles; annealing temperature of 60° C.) with Taq DNA polymerase (New England Biolabs) and 1 μM oJW102 linker (50-μl reaction). The amplification reaction was repeated using 15 μl of the first amplification reaction product as template. After the products had been purified using a QIAquick Kit (QIAGEN), triplicates of control and sample DNA (concentrated to ˜250 ng/μl) were pooled to obtain ˜4-μg quantities and hybridized to a custom-made NimbleGen microarray (NimbleGen Systems, Madison, Wis.). For each antibody, triplicate microarrays were analyzed.
The custom-made NimbleGen microarray used for ChIP-chip analysis was designed to tile the R. sphaeroides 2.4.1 genome (two chromosomes and five plasmids) with overlapping isothermal probes that ranged from 35 to 65 bases, with an average spacing of 12 bp. The probes on the array alternate between coding and noncoding DNA sequences.
For analysis of each ChIP-chip microarray, quantile normalization was used to obtain the same empirical distribution across the Cy3 and Cy5 channels and across arrays to correct for dye intensity bias and to minimize microarray-to-microarray absolute intensity variations. Bolstad et al., Bioinformatics 19:185-193 (2003). The log2 of the ratio of experimental signals (Cy3) to control signals (Cy5) was calculated. The data from the biological replicates were averaged for visualizations in SignalMap 1.9 software (NimbleGen Systems). Regions of the genome enriched for occupancy by σE were identified using TAMALPAIS, available from the UC Davis Genome Center, at p≦0.01 for a threshold set at the 98th percentile of the log2 ratio for each chip. Only enriched regions that achieved the specified statistical significance in all three replicates were considered. The peaks were ranked by order of intensities calculated from the average of the 10 highest consecutive probe signals for each peak.
Regions containing strong σE-dependent promoters (RSP2143-2144, RSP0601, RSP1091-1087, RSP1092-1093, RSP1852, and RSP1409) were significantly enriched by immunoprecipitation of (Table 8). In addition, each of these operons showed enrichment for the β′ subunit of RNA polymerase (
Most of the σE occupancy sites detected by this assay that were not also detected by the clustering or motif-scanning analysis, were within protein coding sequences or large intergenic regions (Table 8). Further, there is no evidence that these other σE occupancy sites are functional σE-dependent promoters in vivo because most of the conserved motifs were oriented in the opposite direction of the nearby open reading, RNA polymerase occupancy could not be found beyond the putative σE binding sites (with the exception of RSP1612 (
The inventors identified a set of genes that potentially constitute the core of σE-dependent biological response to 1O2 and that is potentially conserved across distantly related bacterial species. To determine if the response to singlet oxygen is regulated in other bacteria in a manner similar to the R. sphaeroides σE-ChrR regulon system, the inventors tested for the presence of this response across the bacterial kingdom. The inventors analyzed the genomes of 84 bacteria that contain a gene encoding a group IV alternative a factor adjacent to one coding for a ChrR homolog. The analysis was limited to 73 bacteria with almost complete genome sequences to probe for both σE-ChrR as well as members of the σE-ChrR regulon. All genomic DNA sequences were obtained from the Integrated Microbial Genomes system on Jan. 10, 2007, available at the Department of Energy Joint Genome Institute website. Groups of orthologous genes were determined using the OrthoMCL software set to the default parameters. The protein sequences of orthologous gene products were aligned with CLUSTAL W, a multiple sequence alignment computer program, using the default parameters. Phylogenetic trees were constructed from the protein sequence alignments with the MrBayes 3.1.2 software over 4×106 generations (first 25% as burn-in) using the General Time-Reversible model including an estimated proportion of invariable sites and a γ-shaped distribution of rate variation across sites. Ronquist, F. & Huelsenbeck, J. P. 2003 Bioinformatics 19, 1572-1574.
Chromosome 1
744077
745110
35.09
19.02
RSP2143, upstream
Chromosome 1
2342719
2344030
34.25
14.78
RSP0601, upstream
Chromosome 1
2848771
2853927
30.86
13.37
RSP1091-1092, upstream
Chromosome 1
445724
446747
26.36
10.93
RSP1852, upstream
Chromosome 1
3186065
3186861
18.73
1.05
RSP1409, upstream
A test promoter library for each bacterium was constructed by extracting 300 by upstream of each protein coding sequence. Each test promoter sequence was scanned for its best match to the defined PSWM according to its information content (Iseq):
where i is the position in the matrix, b is one of the four bases, fb,i is the observed frequency of each base at that position, pb is the background frequency of each base in the target genome (pA+pT=0.31 and pG+pC=0.69 for R. sphaeroides). Higher information content is designated by values closer to 1, lower information content is designated by lower values. The skilled person will appreciate that this is an empirical determination made on a case-by-case basis, such that no bright line distinctions between the two are appropriate, although the skilled artisan can evaluate the values as needed. The motif was defined by two matrices (6 by for the −35 region and 4 by for the −10 region). Alternative spacer lengths of 15-17 by were considered without penalties. Promoter sequences containing a match of at least 0.75 times the maximal information content that could be obtained from the PSWM were considered positive and assigned a value of 1.0. Genes directly downstream and transcribed in the same direction of genes with a positive promoter were assigned a score of 0.8. The next gene downstream was assigned a score of 0.6 and so on, until either the end of a potential operon was reached (five genes) or another gene transcribed in the opposite was found. The score for the each group of orthologous gene products was calculated by summing individual gene scores.
Bacteria having σE-ChrR homologs were distributed almost evenly between α-proteobacteria and γ-proteobacteria, with only one encoded by a β-proteobacterium (Acidovorax anenae subsp. citrulli) or a 8-proteobacterium (Myxococcus xanthus).
To test for lateral transfer of the σE-ChrR structural genes, the inventors constructed a species tree based on the amino acid sequence of RuvB, RpoD, and GyrB. These proteins were selected to construct the species tree because they are encoded by single copy genes that are only rarely subject to lateral gene transfer in the selected bacteria. The phylogenetic trees constructed with each protein supported the notion that no lateral gene transfer occurred. The inventors concatenated the RuvB, RpoD, and GyrB protein sequences to build a species phylogenetic tree. Individual phylogenetic trees constructed for σE and ChrR also suggested their coinheritance, so their amino acid sequences were concatenated to build a σE-ChrR phylogenetic tree.
As evident from
To characterize the putative σE-ChrR regulon across these bacteria, the inventors analyzed the 73 bacterial genomes for possible orthologs of genes within the R. sphaeroides σE-ChrR regulon and genes predicted to contain σE promoters. Some of these 73 genomes were not completely assembled, so not all potential σE target genes might be captured. A two-step process was used to predict members of the σE regulon in these bacteria based on the R. sphaeroides σE promoter motif and the known target genes.
The first step was to identify candidate promoters in these 73 genomes. This prediction was based on amino acid conservation across the 73 R. sphaeroides σE homologs. The inventors predicted the residues of the 73 R. sphaeroides σE homologs that are involved in the −35 promoter element recognition based on the known Escherichia coli σE residues that contact the −35 promoter element. Because these residues were conserved among the 73 R. sphaeroides σE homologs, the inventors hypothesized that the sequence of the −35 promoter element recognized by these proteins is conserved. While information about interactions between domains 2.3-2.4 and the −10 promoter element was not available for group IV σ factors, alignments of the 73 homologs revealed a high degree of amino acid sequence conservation in this region (
To identify σE-ChrR regulon orthologs, the inventors performed a de novo search for the R. sphaeroides σE promoter motif upstream of rpoEchrR in other bacteria. This query identified a sequence upstream of a putative σE structural gene in 57 of these 73 microbes that is almost identical with that of the R. sphaeroides σE motif (
To determine if groups of orthologous genes, constructed using OrthoMCL, were consistently associated with a predicted σE promoter, orthologs identified downstream of a candidate σE promoter were scored to reflect their potential membership in the σE-ChrR regulon. To account for the existence of σE-dependent operons, genes likely to be cotranscribed with one containing a match to the σE promoter motif were considered positive. After each protein coding sequence had been scored, ortholog group-specific values were calculated and ranked by summing individual values within each group.
The dataset of ranked orthologous proteins was divided into proteins found in α-proteobacteria or γ-proteobacteria (data derived from one δ-proteobacterium and one β-proteobacterium were removed, since they did not exhibit any informative pattern). The σE (protein ID 21307) and ChrR (protein ID 20362) homologs ranked highest in both α-proteobacteria and γ-proteobacteria as expected, since their expression should be positively autoregulated (see above). The remaining highly ranked groups from both α-proteobacteria and γ-proteobacteria contain orthologs of known members of the R. sphaeroides σE-ChrR regulon (RSP1087; protein ID no. 20348; RSP1088, protein ID no. 21508; RSP1090, protein ID no. 20013; RSP1091, protein ID no. 21520; RSP2143, protein ID no. 21876; RSP2144, protein ID no. 254; Table 9 (manuscript 3)). The presence of a σE promoter motif upstream of these genes supports their assignment to a core set of genes for this regulon and suggests that some part of the biological response controlled by σE and ChrR is conserved in α-proteobacteria and γ-proteobacteria. Except for RSP2143 and RSP2144 (DNA photolyase and cyclopropane fatty acid synthase, respectively), the biological functions of most of these conserved proteins are unknown.
Some ortholog groups appear to be specific to the α-proteobacteria or the γ-proteobacteria. For example, RSP0601 (rpoHII, protein ID no. 20947), which encodes a second member of the σ32 family, is part of the R. sphaeroides σE-ChrR regulon. It is therefore not surprising to find rpoHII orthologs that appear to contain a σE promoter in α-proteobacteria such as Roseovarius sp. 217, Hyphomonas neptunium ATCC 15444, Oceanicaulis alexandrii HTCC2633, Jannaschia sp. CCS1, Loktanella vestfoldensis SKA53, Maricaulis maris MCS10, and Silicibacter pomeroyi DSS3. However, other α-proteobacteria contain an rpoHII gene that lacks elements related to the conserved σE promoter motif even though this sequence is present upstream of other predicted core members of this regulon. In addition, the predicted σE-ChrR regulons in these a-proteobacteria do not appear to be extended to compensate for the loss of any RpoHII target gene, raising questions about the existence of a second tier of 1O2-responsive genes in these species and the exact role of RpoHII in bacteria containing this gene product.
In the γ-proteobacteria that contain rpoEchrR operons, no rpoHII orthologs were found. On the other hand, the motif search revealed that a putative polyketide cyclase specific for the γ-proteobacteria often possesses a σE binding motif in its promoter region. In addition, this gene is generally found in the genomic neighborhood of orthologs of the R. sphaeroides σE-regulated genes in these γ-proteobacteria.
Several species of α-proteobacteria and γ-proteobacteria do not have the conserved σE binding motif associated with any of the predicted conserved regulon members. To determine if the σE promoter motif evolved into a different sequence, the inventors queried the promoter regions of possible regulon members in each species individually for a conserved sequence that related to a σ factor binding site. No conserved motif with high information content could be found. It is possible that σE is not functional or that both the σE binding motif and the regulon composition have diverged in these bacteria. However, no evidence for such divergence was evident in sequence alignments of their σE homologs in regions 2.3-2.4 and 4.2.
The invention has been described in connection with what are presently considered to be the most practical and preferred embodiments. However, the present invention has been presented by way of illustration and is not intended to be limited to the disclosed embodiments. Accordingly, those skilled in the art will realize that the invention is intended to encompass all modifications and alternative arrangements within the spirit and scope of the invention as set forth in the appended claims.
This application is a continuation-in-part of U.S. patent application Ser. No. 11/410,431, filed Apr. 25, 2006, which claimed the benefit of U.S. Provisional Patent Application Ser. No. 60/674,470, filed Apr. 25, 2005. Each application is incorporated herein by reference as if set forth in its entirety.
This invention was made with United States government support awarded by the following agencies: National Institute of General Medical Science, GM37509 & GM075273; and Department of Energy, DE-FG02-05ER15653 & ER63232-1018220-0007203. The United States has certain rights in this invention.
| Number | Date | Country | |
|---|---|---|---|
| 60674470 | Apr 2005 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 11410431 | Apr 2006 | US |
| Child | 12410066 | US |
