Patent Application
20140248607
The ASCII file, entitled 59350SequenceListing.txt, created on May 5, 2014, comprising 409,600 bytes, submitted concurrently with the filing of this application is incorporated herein by reference.
The present invention, in some embodiments thereof, relates to Brucella phage nucleic acid sequences and uses thereof.
Brucella are Gram negative, small coccobacilli bacteria. They are animal pathogens causing abortions in the natural hosts, in the latest period of pregnancy. Genus Brucella includes 10 species divided to smooth and rough outer membrane LPS bearing organisms. Three smooth Brucella species, (B. melitensis, B. abortus and B. suis) associated with small ruminant, bovid and swine brucellosis, respectively, are zoonotic to humans. Less frequently, B. ceti and B. pinnipedialis associated with marine mammal brucellosis have been documented as causative agents of human brucellosis. In addition, B. canis that is associated with canine brucellosis is a rough organism that causes human infection. The disease in humans is presented as undulant fever also known as Malta fever, and it may sequel to a chronic disease or manifestation of meningitis, osteomyelitis, endocarditis and other complications. In rare occasions the disease may become fatal.
Brucella phages are bacterial viruses specific to Brucella species. A review of Brucella phages and their taxonomical relatedness was published in 1981. All contemporarily known Brucella phages were shown to comprise a similar icosahedral head and short tail morphology belonging to the family Podoviradae. The studied phages were shown to be closely related according to antigenic and physiological properties and resistance to chemical and physical agents. These findings have led the authors to include the summarized variants within a single species and propose phage Tb as type virus (Ackerman, H.-W., Simon, F., and Verger, J.-M. 1981. Intervirology 16: 1-7).
Brucella phages have linear double stranded DNA in size around 38 kilo base pairs. Restriction enzyme digestion analyses of phages Tb, Fi, Wb, Iz and R/C have shown similarity amongst the DNAs and little evidence has been found for lysogenic existence of the phages or presence of plasmid forms in the hosts.
Previous studies have established guanosine-cytosine content of 45.3-46.7% in phage Tb whereas a higher percentage of 48.9% was anticipated in other phages.
Use of phages as therapeutic agents of a pathogenic disease has been indicated by several researchers (Brüssow, H. 2005. Microbiol. 151: 2133-2140; Summers, W. C. 2001. Ann. Rev. Microbiol. 55: 437-451).
In addition, Brucella phages have been employed in Brucella typing and a phage susceptibility test has become instrumental in classification and establishing a taxonomical tree of genus Brucella. Specifically, it was suggested that division of genus Brucella into nomen-species is partly justified according to their species specific phage susceptibilities that also correlated well with host affiliation of the strains. Brucella phages have been divided into 7 groups according to their infectivity to Brucella spp. Phage Izatnagar (Iz1) represents group 6 that is infective to all smooth Brucella nomenspecies and partly to rough strains (Corbel and Tolari, 1988, Res Vet Sci; 44: 45-49).
According to an aspect of some embodiments of the present invention there is provided an isolated polynucleotide comprising a nucleic acid sequence of a Brucella phage, the nucleic acid sequence being specific to the Brucella phage and comprising a sequence selected from the group consisting of SEQ ID NOs: 396 and 387-393.
According to an aspect of some embodiments of the present invention there is provided an isolated polynucleotide being at least 15 nucleotides in length which hybridizes to the isolated polynucleotide of the present invention.
According to an aspect of some embodiments of the present invention there is provided a method of down-regulating expression of a gene of interest in a bacteria, the method comprising transforming bacteria with a nucleic acid construct which comprises a Brucella phage regulatory sequence, thereby down-regulating expression of the gene of interest.
According to an aspect of some embodiments of the present invention there is provided a nucleic acid construct comprising at least 100 nucleotides of a nucleic acid sequence as set forth in SEQ ID NO: 396.
According to an aspect of some embodiments of the present invention there is provided a nucleic acid construct comprising:
i. a polynucleotide encoding a gene of interest operationally fused to a Brucella promoter;
ii. a first Brucella phage sequence fused to a 5′ end of the promoter, the first sequence comprising at least 100 nucleotides of a nucleic acid sequence as set forth in SEQ ID NO: 394; and
iii. a second Brucella phage sequence fused to a 3′ end of the gene of interest, the second sequence comprising at least 100 nucleotides of a nucleic acid sequence as set forth in SEQ ID NO: 395.
According to an aspect of some embodiments of the present invention there is provided a recombinant Brucella phage which identifies Brucella bacteria by outputting a detectable signal.
According to an aspect of some embodiments of the present invention there is provided an isolated Brucella bacterial cell comprising the recombinant Brucella phage of the present invention.
According to an aspect of some embodiments of the present invention there is provided a method of diagnosing a Brucella infection in a subject, the method comprising contacting a sample of the subject with the recombinant Brucella phage of the present invention, thereby diagnosing the Brucella infection.
According to an aspect of some embodiments of the present invention there is provided a method of diagnosing a Brucella infection in a subject, the method comprising contacting a sample of the subject with the isolated Brucella bacterial cells of the present invention, thereby diagnosing the Brucella infection.
According to some embodiments of the invention, the isolated polynucleotide comprises at least 100 consecutive nucleotides of a nucleic acid sequence as set forth in SEQ ID NO: 396.
According to some embodiments of the invention, the isolated polynucleotide comprises the sequence as set forth in SEQ ID NO: 396.
According to some embodiments of the invention, the isolated polynucleotide comprises a nucleic acid sequence as set forth in SEQ ID NOs: 387-393.
According to some embodiments of the invention, the isolated polynucleotide has the nucleic acid sequence as set forth in SEQ ID NO: 1.
According to some embodiments of the invention, the isolated polynucleotide comprises at least one nucleic acid sequence being selected from the group consisting of SEQ ID NO: 394 and 395 in a forward or reverse orientation.
According to some embodiments of the invention, the isolated polynucleotide further comprises a heterologous nucleic acid sequence and a heterologous promoter sequence which directs expression of the heterologous nucleic acid sequence.
According to some embodiments of the invention, the nucleic acid sequence comprises a transcriptional regulatory region.
According to some embodiments of the invention, the transcriptional regulatory region comprises a brucella phage promoter.
According to some embodiments of the invention, the isolate polynucleotide comprises a sequence as set forth in SEQ ID NOs: 2-386.
According to some embodiments of the invention, the heterologous nucleic acid sequence encodes a detectable moiety.
According to some embodiments of the invention, the heterologous nucleic acid sequence encodes a polypeptide which is lethal to Brucella.
According to some embodiments of the invention, the bacteria comprises Brucella bacteria.
According to some embodiments of the invention, a strain of the Brucella bacteria comprises B. Suis or B. melitensis.
According to some embodiments of the invention, the gene is endogenous to the bacteria.
According to some embodiments of the invention, the gene is endogenous to a phage of the bacteria.
According to some embodiments of the invention, the regulatory sequence comprises at least 100 nucleotides of a nucleic acid sequence as set forth in SEQ ID NO: 396.
According to some embodiments of the invention, the regulatory sequence comprises the sequence as set forth in SEQ ID NO: 396.
According to some embodiments of the invention, the regulatory sequence further comprises the sequence as set forth in SEQ ID NO: 397.
According to some embodiments of the invention, the regulatory sequence is flanked by a transposon sequence.
According to some embodiments of the invention, the nucleic acid construct comprises a nucleic acid sequence as set forth in SEQ ID NO: 396.
According to some embodiments of the invention, the nucleic acid sequence is flanked by a transposon sequence.
According to some embodiments of the invention, the gene of interest encodes a therapeutic polypeptide.
According to some embodiments of the invention, the gene of interest encodes a detectable moiety.
According to some embodiments of the invention, the gene of interest is comprised in a Lux operon.
According to some embodiments of the invention, the detectable signal is a luminescent signal.
According to some embodiments of the invention, the recombinant Brucella phage comprise lytic activity.
According to some embodiments of the invention, a genome of the phage comprises a polynucleotide sequence which encodes the detectable signal.
Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.
In the drawings:
The present invention, in some embodiments thereof, relates to Brucella phage nucleic acid sequences and uses thereof.
Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.
The Brucella species are important zoonotic pathogens affecting a wide variety of mammals. In agriculturally important domestic animals, these bacteria cause abortion and infertility, and they are of serious economic concern worldwide. Brucella species that infect humans cause an undulating fever, which if untreated, can manifest as orchitis, osteoarthritis, spondylitis, endocarditis, and neurological disorders. In rare events it may be fatal.
The present inventors have sequenced the entire genome (38,254 base pairs) of the Brucella phage Iz1 as a genotype representative of all other Brucella phages. Two genomic Brucella phage Iz1 populations were identified, differing between C or A at nucleotide 5549. BLAST analysis of this sequence revealed 6 regions of homology with Ochrobactrum anthropi ATCC 49188 chromosome 1—see Table 2 in the Examples section herein below. Subtraction of these sequences from the sequence of the full length genome, leads the inventors to the discovery of novel polynucleotide sequences which are specific to Brucella phage (e.g. SEQ ID NOs: 387-393).
The information gleaned from the sequence has allowed the inventors to identify apparent sequence of minimal presence of ORFs, which can be used for inserting genes of interest (for example, those encoding detectable moieties) into the phage, without affecting its lytic activity. Thus, development of a recombinant Brucella phage has been sought based on recombinational replacement of this site in the phage genome with a detectable signal (e.g. Lux operon). Due to the outputting of this signal, such a phage could be used to identify Brucella bacteria. Phage carrier Brucella clones were generated in which phage Iz1 coresided in the cells in presence of plasmid pBBR1mcs4.1-II1053/luxCDABE/15B-18B (
In addition, the present inventors have identified regulatory regions in the phage which possess regulatory function and were shown to be capable of down-regulating light expression endowed by the plasmid indicating potential implementation of such a gene regulation mechanism within Brucella bacteria.
Thus, for example, the present inventors identified a fragment of phage DNA which can down-regulate a gene operatively linked thereto (SEQ ID NO: 396; 19630-18579) following transformation into Brucella bacteria. When this sequence was transformed into Brucella bacteria, together with an additional phage DNA fragment (SEQ ID NO: 397; 16509-15500), it conferred different lethal activities on Brucella species, being the most lethal to lethal to the B. abortus strain 544, less severely lethal to B. melitensis and non-lethal to the B. suis strain of Brucella. Correspondingly, the down-regulatory activity of this fragment was also shown to be species specific.
Knowledge of phage regulatory regions should add to computational identification of additional unrecognized regulatory sequences within the genome of Brucella. This approach has already been demonstrated in the legume endosymbiont Sinorhizobium meliloti (del Val C, et al., Mol Microbiol 2007; 66: 1080-1091), that is belonging to the alpha-proteobacteria class, as also Agrobactrum tumefaciens, the causative agent of crown-gall disease in plants and Brucella, indicating close relatedness and therefore possible shared functions between these organisms (Inon de Iannino N, et al., J Bacteriol 1998; 180: 4392-4400).
Thus, according to one aspect of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence of a Brucella phage, the nucleic acid sequence being specific to the Brucella phage and comprising a sequence selected from the group consisting of SEQ ID NOs: 387-393.
As used herein, the term “phage” (synonymous with the term “bacteriophage” refers to a virus that selectively infects prokaryotes—such as bacteria. Many bacteriophages are specific to a particular genus or species or strain of cell.
The phage is typically a lytic bacteriophage.
A lytic bacteriophage is one that follows the lytic pathway through completion of the lytic cycle, rather than entering the lysogenic pathway. A lytic bacteriophage undergoes viral replication leading to lysis of the cell membrane, destruction of the cell, and release of progeny bacteriophage particles capable of infecting other cells.
A lysogenic bacteriophage is one capable of entering the lysogenic pathway, in which the bacteriophage becomes a dormant, passive part of the cell's genome through prior to completion of its lytic cycle.
According to one embodiment, the phage is a Tb type phage, for example Phage Iz1.
A sequence specific to a Brucella phage is one which is present in the phage and not present in other organisms—i.e. unique to Brucella. Since the sequence of the Brucella phage genome is now known, Brucella phage specific sequences may be identified using BLAST or other similar programs.
According to one embodiment, the Brucella phage specific sequence does not comprise more than 70% identity with another nucleic acid sequence as verified using a sequence alignment software such as BLAST analysis.
According to one embodiment, the Brucella phage specific sequence does not comprise more than 60% identity with another nucleic acid sequence as verified using a sequence alignment software such as BLAST analysis.
As used herein the phrase “an isolated polynucleotide” refers to a single or double stranded nucleic acid sequences which is isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence and/or a composite polynucleotide sequences (e.g., a combination of the above).
As used herein the phrase “complementary polynucleotide sequence” refers to a sequence, which results from reverse transcription of messenger RNA using a reverse transcriptase or any other RNA dependent DNA polymerase. Such a sequence can be subsequently amplified in vivo or in vitro using a DNA dependent DNA polymerase.
As used herein the phrase “genomic polynucleotide sequence” refers to a sequence derived (isolated) from a chromosome and thus it represents a contiguous portion of a chromosome.
As used herein the phrase “composite polynucleotide sequence” refers to a sequence, which is at least partially complementary and at least partially genomic. A composite sequence can include some exonal sequences required to encode the polypeptide of the present invention, as well as some intronic sequences interposing therebetween. The intronic sequences can be of any source, including of other genes, and typically will include conserved splicing signal sequences. Such intronic sequences may further include cis acting expression regulatory elements.
According to one embodiment, the isolated polynucleotide comprises at least 15, at least 20, at least 40, at least 50, at least 100, at least 200, at least 500 or at least 1000 consecutive nucleotides of the sequences as set forth in SEQ ID NOs: 387-393.
Thus, the polynucleotides of the present invention may be from 15-38,254 nucleotides long.
It will be appreciated that homologues of the sequences described hereinabove are also envisaged by the present invention. Accordingly, the polynucleotides of this aspect of the present invention may have a nucleic acid sequence at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 89%, at least 90% at least 91%, at least 93%, at least 95% or more say 100% identical to the sequences derived from SEQ ID NOs: 387-393, as determined using BlastN software of the National Center of Biotechnology Information (NCBI) using default parameters.
Thus, the present invention encompasses nucleic acid sequences described hereinabove; fragments thereof, sequences hybridizable therewith, sequences in the opposite orientation thereto, sequences homologous thereto, sequences encoding similar polypeptides with different codon usage, altered sequences characterized by mutations, such as deletion, insertion or substitution of one or more nucleotides, either naturally occurring or man induced, either randomly or in a targeted fashion.
Exemplary polynucleotides contemplated by the present invention are those that comprise the nucleic acid sequence as set forth in SEQ ID NOs: 1 and 387-393.
Other exemplary polynucleotides contemplated by the present invention are those that comprise regulatory regions within the sequences as set forth in SEQ ID NOs: 387-393, such as at least 100 consecutive nucleotides of the regulatory region as set forth in SEQ ID NO: 395 shown by the present inventors to possess down-regulating activity of a gene operationally linked thereto, when inserted in the opposite orientation (i.e. SEQ ID NO: 396). Another regulatory sequence includes SEQ ID NO: 394, both in the forward or reverse orientation. This is also present in the complete phage genome possibly affecting regulatory functions of phage Iz1 genes.
Using bioinformatic tools, the present inventors identified additional regulatory regions within the full length sequence of the phage genome which may serve as promoter sequences (i.e. transcriptional regulatory regions) in the phage (SEQ ID NOs: 2-386). Such promoter sequences may be placed upstream of a heterologous nucleic acid sequence so as to promote transcription thereof. Moreover, the downregulatory activity might be used to identify important chemicals that change the activity of the transcriptional regulatory regions, thereby facilitating development of novel drugs.
Other sequences which encode putative polypeptides are also provided and also considered to be in the realm of the present invention. Such sequences are provided in Table 1 herein below. Polynucleotide sequences encoding such polypeptides may be used for various purposes. Thus, for instance a polynucleotide sequence encoding a putative lysin or holin may be used to selectively kill Brucella cells. The advantages of lysin-based therapy are numerous: they can be prepared with high purity and possess high specific activity; they exhibit rapid lethal action; they are nontoxic; and apparently, antibodies that form against these proteins do not neutralize their lytic activity. Lastly, no bacterial resistance develops to these proteins, probably because they possess multiple domains for cell wall binding and hydrolysis.
Examples of additional important polypeptides are:
1. Secretory proteins including flagellar proteins (secretion Type III, This apparatus is related to the injectisome used by many gram-negative pathogens and symbionts to transfer effector proteins into host cells) and VirB (Type IV, The translocation of DNA across biological membranes is an essential process for many living organisms. In bacteria, type IV secretion systems (T4SS) are used to deliver DNA as well as protein substrates from donor to target cells. The T4SS are structurally complex machines assembled from a dozen or more membrane proteins in response to environmental signals The translocation of DNA across biological membranes is an essential process for many living organisms. In bacteria, type IV secretion systems (T4SS) are used to deliver DNA as well as protein substrates from donor to target cells. The T4SS are structurally complex machines assembled from a dozen or more membrane proteins in response to environmental signals)
2. Phage integrase—catalyses the site specific integration and excision of the bacteriophage in the lysogenic cycle.
3. Toxins, phospholipase—degrade phospholipids, protease—degrade proteins.
palustris DX-1]
leguminosarum bv.
trifolii
leguminosarum bv.
viciae 3841]
palustris DX-1]
palustris DX-1]
salmonicida
salmonicida
Typhi
Typhi
damselae subsp.
damselae subsp.
Javiana
Javiana
Virchow
Virchow
Typhi
Typhi
Agona
Agona
Kentucky
Kentucky
odorifera DSM 4582]
odorifera DSM 4582]
turicensis z3032]
turicensis z3032]
furnissii CIP 102972]
furnissii CIP 102972]
Hadar
Hadar
profundum SS9]
profundum SS9]
pseudotuberculosis IP
pseudotuberculosis IP
pestis KIM 10]
pestis KIM 10]
profundum 3TCK]
profundum 3TCK]
pseudotuberculosis
pseudotuberculosis
cholerae CT 5369-93]
cholerae CT 5369-93]
carotovorum
carotovorum
cancerogenus ATCC
cancerogenus ATCC
parahaemolyticus
parahaemolyticus
mimicus VM603]
mimicus VM603]
proteamaculans 568]
proteamaculans 568]
mimicus MB-451]
mimicus MB-451]
cholerae
cholerae
enterocolitica
enterocolitica
cholerae 12129(1)]
cholerae 12129(1)]
cholerae TM 11079-
cholerae TM 11079-
cholerae 1587]
cholerae 1587]
cholerae TMA 21]
cholerae TMA 21]
coli 83972]
coli 83972]
cholerae 623-39]
cholerae 623-39]
mimicus VM223]
mimicus VM223]
coli CFT073]
coli CFT073]
coli B354]
coli B354]
coli B185]
coli B185]
coli IAI39]
coli IAI39]
orientalis CIP 102891]
orientalis CIP 102891]
coli ED1a]
coli ED1a]
alginolyticus 40B]
alginolyticus 40B]
fergusonii
fergusonii
cholerae bv. albensis
cholerae bv. albensis
wasabiae
wasabiae
aquaeolei VT8]
aquaeolei VT8]
cholerae V52]
cholerae V52]
cholerae O1 biovar El
cholerae O1 biovar El
cholerae BX 330286]
cholerae BX 330286]
mirabilis ATCC
mirabilis ATCC
mirabilis HI4320]
mirabilis HI4320]
amylovora CFBP1430]
amylovora CFBP1430]
atrosepticum
atrosepticum
coralliilyticus ATCC
coralliilyticus ATCC
parahaemolyticus
parahaemolyticus
ananatis LMG 20103]
ananatis LMG 20103]
nasoniae]
nasoniae]
pneumoniae subsp.
pneumoniae subsp.
pneumoniae 342]
pneumoniae 342]
alcalifaciens DSM
alcalifaciens DSM
carotovorum subsp.
brasiliensis
carotovorum subsp.
brasiliensis
asymbiotica]
asymbiotica]
splendidus 12B01]
splendidus 12B01]
luminescens subsp.
laumondii
luminescens subsp.
laumondii
frigidimarina
frigidimarina
salexigens
salexigens
oneidensis MR-1]
oneidensis MR-1]
benthica KT99]
benthica KT99]
violacea DSS12]
violacea DSS12]
cholerae V51]
cholerae V51]
halifaxensis
halifaxensis
ingrahamii 37]
ingrahamii 37]
hydrophila subsp.
hydrophila subsp.
putrefaciens 200]
putrefaciens 200]
putrefaciens
putrefaciens
denitrificans
denitrificans
salmonicida subsp.
salmonicida A449]
salmonicida subsp.
salmonicida A449]
haloplanktis TAC125]
haloplanktis TAC125]
atlantica
atlantica
akajimensis
akajimensis
amazonensis SB2B]
amazonensis SB2B]
bacteriovorus HD100]
bacteriovorus HD100]
carotovorum subsp.
carotovorum WPP14]
carotovorum subsp.
carotovorum WPP14]
lithotrophicus ES-1]
lithotrophicus ES-1]
rustigianii DSM 4541]
rustigianii DSM 4541]
metschnikovii CIP
metschnikovii CIP
mimicus VM573]
mimicus VM573]
palustris DX-1]
leguminosarum bv.
viciae 3841]
leguminosarum bv.
trifolii
medicae WSM419]
medicae WSM419]
leguminosarum bv.
viciae 3841]
opportunistum
leguminosarum bv.
trifolii
Typhi
denitrificans PD1222]
pseudomallei NCTC
pseudomallei 668]
autotrophicum
macleodii ‘Deep
macleodii ‘Deep
nodulans ORS 2060]
nodulans ORS 2060]
nodulans ORS 2060]
Marseille]
nodulans ORS 2060]
palustris DX-1]
nodulans ORS 2060]
nodulans ORS 2060]
nodulans ORS 2060]
abortus bv. 1 str. 9-
nodulans ORS 2060]
Typhi
melitensis 16M]
nodulans ORS 2060]
botulinum E1 str.
Typhi
nodulans ORS 2060]
cholerae RC385]
cholerae RC385]
intermedium LMG
ovis ATCC 25840]
melitensis 16M]
Marseille]
Marseille]
Typhi
magneticum AMB-1]
magneticum AMB-1]
autotrophicus Py2]
abortus bv. 9 str. C68]
anthropi ATCC 49188]
thermodenitrificans
thermodenitrificans
parahaemolyticus
cryptum JF-5]
cryptum JF-5]
opportunistum
Typhi
Typhi
vadensis ATCC BAA-
vadensis ATCC BAA-
bethesdensis
bethesdensis
cereus Rock4-2]
cereus Rock4-2]
lacuscaerulensis ITI-
intestinalis M50/1]
extorquens PA1]
extorquens PA1]
palustris DX-1]
denitrificans ATCC
thuringiensis serovar
berliner ATCC
thuringiensis serovar
berliner ATCC
inulinivorans DSM
termitidis ATCC
populi BJ001]
populi BJ001]
Typhi
Typhi
thuringiensis serovar
israelensis
thuringiensis serovar
israelensis
thuringiensis serovar
israelensis
thuringiensis serovar
israelensis
nodosus VCS1703A]
acetiphilus DSM
acetiphilus DSM
chloromethanicum
chloromethanicum
anthropi ATCC 49188]
prausnitzii L2-6]
denitrificans ATCC
cereus AH676]
cereus AH676]
saphenum ATCC
aromaticivorans DSM
denitrificans PD1222]
vulgatus PC510]
vulgatus PC510]
influenzae R3021]
influenzae R3021]
monocytogenes EGD-
nasoniae]
nasoniae]
pseudomallei NCTC
pasteurianus IFO
pasteurianus IFO
pseudomallei 668]
influenzae 3655]
influenzae 3655]
hygroscopicus ATCC
hygroscopicus ATCC
rhamnosus
methylpentosum DSM
botulinum B1 str.
innocua Clip11262]
innocua Clip11262]
merdae ATCC 43184]
merdae ATCC 43184]
coprophilus DSM
botulinum A3 str.
gonorrhoeae F62]
minutum
distasonis ATCC
distasonis ATCC
faecalis Fly1]
faecalis Fly1]
Accumulibacter
cellulosilytica DSM
cellulosilytica DSM
saccharolyticum-like
uniformis]
acidovorans SPH-1]
vannielii ATCC
asymbiotica]
prausnitzii L2-6]
Liberibacter asiaticus
parahaemolyticus
parahaemolyticus
medicae WSM419]
medicae WSM419]
radiotolerans JCM
radiotolerans JCM
pseudomallei NCTC
magneticum AMB-1]
alginolyticus 12G01]
alginolyticus 12G01]
pseudomallei 668]
leguminosarum bv.
viciae 3841]
meningitidis alpha153]
baumannii AB0057]
baumannii AB0057]
meningitidis MC58]
acidovorans SPH-1]
testosteroni CNB-2]
baumannii ATCC
baumannii ATCC
Typhi
Typhi
baumannii ACICU]
baumannii ACICU]
meningitidis FAM18]
pamelaeae 7-10-1-bT]
meningitidis serogroup
gryphiswaldense
aeruginosa LESB58]
petrii DSM 12804]
petrii DSM 12804]
radioresistens SK82]
denitrificans PD1222]
concisus 13826]
concisus 13826]
meningitidis 8013]
aeruginosa LESB58]
antarcticus 307]
haemolytica PHL213]
palustris TIE-1]
Marseille]
leguminosarum bv.
trifolii
palustris DX-1]
crispatus MV-3A-US]
radioresistens SK82]
radioresistens SK82]
succiniciproducens
faecalis E1Sol]
faecalis E1Sol]
influenzae HK1212]
influenzae HK1212]
silvestris BL2]
carboxidovorans
pneumoniae subsp.
bronchiseptica RB50]
succinogenes 130Z]
influenzae 6P18H1]
bacterium KLH11]
influenzae HK1212]
influenzae HK1212]
albicans WO-1]
albicans WO-1]
timonensis CRIS 5C-
timonensis CRIS 5C-
hamburgensis X14]
pertussis Tohama I]
influenzae HK1212]
influenzae HK1212]
albicans WO-1]
albicans WO-1]
influenzae HK1212]
influenzae HK1212]
influenzae HK1212]
influenzae HK1212]
influenzae HK1212]
denitrificans ATCC
crispatus JV-V01]
haemolytica PHL213]
pseudomallei
pseudomallei
avium 104]
avium 104]
agalactiae H36B]
agalactiae H36B]
crispatus MV-3A-US]
graminis C4D1M]
enterocolitica subsp.
dubliniensis CD36]
dubliniensis CD36]
marinum DSM 15272]
phytofirmans PsJN]
xenovorans LB400]
parascrofulaceum
parascrofulaceum
influenzae PittAA]
influenzae HK1212]
diazotrophicus PAl 5]
diazotrophicus PAl 5]
phymatum STM815]
crispatus MV-1A-US]
influenzae 3655]
formatexigens DSM
formatexigens DSM
marinum M]
marinum M]
extructa W1219]
extructa W1219]
bacterium
bacterium
campestris pv.
vesicatoria
fuscans subsp.
aurantifolii
axonopodis pv. citri
bacterium D7]
bacterium D7]
Typhi
glumae BGR1]
intermedium LMG
Liberibacter asiaticus
botulinum
erythraeum IMS101]
influenzae HK1212]
influenzae HK1212]
haemolytica
haemolytica
avium 197N]
avium 197N]
nodulans ORS 2060]
nodulans ORS 2060]
vulnificus YJ016]
guilliermondii ATCC
guilliermondii ATCC
cenocepacia J2315]
elongisporus NRRL
multivorans CGD1]
parvula ATCC 17745]
parvula ATCC 17745]
vannielii ATCC
influenzae HK1212]
influenzae HK1212]
nodulans ORS 2060]
shibae DFL 12]
guilliermondii ATCC
guilliermondii ATCC
peptidovorans DSM
testosteroni KF-1]
testosteroni KF-1]
vietnamiensis G4]
cholerae B33]
faecium E980]
diazotrophicus PAl 5]
hansenii ATCC 23769]
extorquens AM1]
degradans 2-40]
flavefaciens
flavefaciens
Typhi
nodulans ORS 2060]
cholerae 1587]
mitsuokai DSM
mitsuokai DSM
vietnamiensis
vietnamiensis
cholerae CT 5369-93]
intermedium LMG
cholerae 12129(1)]
cholerae NCTC 8457]
winogradskyi Nb-255]
anthropi
pyogenes MGAS5005]
meliloti 1021]
denitrificans PD1222]
multivorans CGD1]
cholerae MZO-3]
As mentioned herein above, the present invention also contemplates isolated polynucleotides which hybridize to the isolated polynucleotides described herein above. Such polynucleotides may be used to monitor Brucella phage gene expression, eventually allowing detection of Brucella strains (i.e. diagnosing) in a bacterial contaminated environment.
Such polynucleotides typically comprises a region of complementary nucleotide sequence that hybridizes under experimental conditions to at least about 8, 10, 13, 15, 18, 20, 22, 25, 30, 40, 50, 55, 60, 65, 70, 80, 90, 100, 120 (or any other number in-between) or more consecutive nucleotides to the sequence of the Brucella phage.
The polynucleotide (or plurality thereof) may be fixed to a solid support (e.g. in an array) and may be used to monitor phage expression in a Brucella sample.
Alternatively, the polynucleotide may serve as a primer in a primer pair and may be used in an amplification reaction (e.g. PCR) to identify Brucella phage.
The conditions are selected such that hybridization of the polynucleotide to the Brucella phage sequence is favored and hybridization to other non Brucella phage nucleic acid sequences is minimized.
By way of example, hybridization of short nucleic acids (below 200 bp in length, e.g. 13-50 bp in length) can be effected by the following hybridization protocols depending on the desired stringency; (i) hybridization solution of 6×SSC and 1% SDS or 3 M TMACl, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5% SDS, 100 μg/ml denatured salmon sperm DNA and 0.1% nonfat dried milk, hybridization temperature of 1-1.5° C. below the Tm, final wash solution of 3 M TMACl, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5% SDS at 1-1.5° C. below the Tm (stringent hybridization conditions) (ii) hybridization solution of 6×SSC and 0.1% SDS or 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5% SDS, 100 μg/ml denatured salmon sperm DNA and 0.1% nonfat dried milk, hybridization temperature of 2-2.5° C. below the Tm, final wash solution of 3 M TMACl, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5% SDS at 1-1.5° C. below the Tm, final wash solution of 6×SSC, and final wash at 22° C. (stringent to moderate hybridization conditions); and (iii) hybridization solution of 6×SSC and 1% SDS or 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5% SDS, 100 μg/ml denatured salmon sperm DNA and 0.1% nonfat dried milk, hybridization temperature at 2.5-3° C. below the Tm and final wash solution of 6×SSC at 22° C. (moderate hybridization solution).
The polynucleotides may further be labeled with detectable moieties. Methods for labeling nucleic acid molecules are well-known in the art. For a review of labeling protocols, label detection techniques, and recent developments in the field, see, for example, L. J. Kricka, Ann. Clin. Biochem. 2002, 39: 114-129; R. P. van Gijlswijk et al., Expert Rev. Mol. Diagn. 2001, 1: 81-91; and S. Joos et al., J. Biotechnol. 1994, 35: 135-153.
As mentioned, the present inventors have identified a region within the Brucella phage genome which serves as a regulatory sequence in Brucella and other bacteria—see Table 3 of the Examples section herein below.
Thus, according to another aspect of the present invention there is provided a method of down-regulating expression of a gene of interest in bacteria, the method comprising transforming bacteria with a nucleic acid construct which comprises a Brucella phage regulatory sequence, thereby down-regulating expression of the gene of interest.
The phrase “Brucella bacteria” as used herein, refers to all strains of Brucella including, but not limited to B. abortus strain 544, B. Suis strain 1330 and B. melitensis strain 16M. According to a particular embodiment, the downregulating is effected in the B. Suis strain or the B. melitensis of Brucella.
Examples of bacterial constructs include the pET series of E. coli expression vectors [Studier et al. (1990) Methods in Enzymol. 185:60-89). An example of a bacterial construct which allows expression in Brucella bacteria is the plasmid pBBR1mcs-4 (Kovach et al., 1995, Gene 1995; 166: 175-176), the contents of which are incorporated herein by reference and the pNSGroE plasmid (Seleem et al., BioTechniques 37:740-744 (November 2004), the contents of which are incorporated by reference herein.
It will be appreciated that the method of this aspect of the present invention may be used to down-regulate expression of a gene which is endogenous to the bacteria or endogenous to a phage which is comprised in the bacteria.
The gene of interest is preferably downregulated by at least 10%. According to one embodiment, the gene of interest is downregulated by about 50%. According to another embodiment, the gene of interest is downregulated by about 90%.
Examples of genes of interest include genes that encode polypeptides important for survival of the bacteria. By down-regulating such genes, the method may be used to kill the brucella bacteria, thereby treating a brucella infection.
The present invention contemplates insertion of transposon sequences on either side of the regulatory region such that it can be randomly inserted via a transposition event into the bacterial genome or site specific designed mutation.
As used herein, the term “transposition event” refers to the movement of a transposon from a donor site to a target site.
As used herein, the term “transposon” refers to a genetic element, including but not limited to segments of DNA or RNA that can move from one chromosomal site to another.
An exemplary transposon sequence is provided in SEQ ID NO: 398 (ME1 transposon sequence) and SEQ ID NO: 399 (ME2 transposon sequence). For directed down-regulation of a particular gene, bacterial sequences may be added on either side of the regulatory region, to facilitate a recombination event.
According to one embodiment, the regulatory region comprises from 100 to all the nucleotides of the nucleic acid sequence as set forth in SEQ ID NO: 396 (19630-18579).
Optionally, the nucleic acid construct comprises additional regulatory regions such as the one set forth in SEQ ID NO: 397 (16509-15500).
According to a particular embodiment, the nucleic acid construct further comprises a heterologous nucleic acid sequence and upstream thereto, a promoter sequence which directs expression of the heterologous nucleic acid sequence. The promoter sequence is selected such that it allows transcription of the heterologous nucleic acid sequence in the bacteria. Thus an exemplary promoter that may be used in Brucella is one set forth in SEQ ID NO: 400. Another promoter that may be used to express a heterologous nucleic acid sequence in Brucella include the groE promoter [Saleem et al., BioTechniques 37:740-744 (November 2004)]. Additional prokayotic promoters are also contemplated by the present inventors which are known in the art.
The regulatory region (for example SEQ ID NO: 396) is typically placed immediately downstream to the heterologous sequence in order to down-regulate expression thereof.
An exemplary construct contemplated by the present invention that may be used to show that SEQ ID NO: 396 comprises regulatory activity may comprise as follows:
i. a polynucleotide encoding a gene of interest (e.g. detectable moiety) operationally fused to a Brucella promoter; and
ii. a Brucella phage sequence fused to a 3′ end of the gene of interest, the regulatory sequence comprising from 100 nucleotides to all the nucleotides of the nucleic acid sequence as set forth in SEQ ID NO: 396.
Optionally, the construct may also comprise:
iii. a Brucella phage sequence fused to a 5′ end of the promoter, the sequence comprising from 100 nucleotides to all the nucleotides of the nucleic acid sequence as set forth in SEQ ID NO: 397.
It will be appreciated that when the heterologous nucleic acid sequence encodes a detectable moiety, it may be used to determine a strain of Brucella. The present inventors have shown that a plasmid construct comprising SEQ ID NO:396 placed immediately downstream of a detectable moiety can downregulate its expression in a strain specific manner. Thus, expression of the detectable moiety was almost completely down-regulated in B. suis and only partially down-regulated in B. melitensis. Such a construct can also be used to determine which bacteria are sensitive to the brucella phage regulatory region and engineer these bacteria by gene down-regulation. In addition, the construct may be used as a tool to decipher novel factors that modify promoter activity by analysis of the detectable signal.
The detectable moiety is typically comprised in a reporter polypeptide which emits a detectable signal. It may be a fluorescent signal (e.g. green fluorescent protein (GFP) red fluorescent protein (RFP) or yellow fluorescent protein (YFP)); a luminescent signal (e.g. luciferase—LUX) or a color signal (e.g. β-glucuronidase (GUS) and β.-galactosidase). In addition, transcribed RNAs of the polypeptides can be used as reporter products of the system.
According to a specific embodiment of this aspect of the present invention, the heterologous nucleic acid sequence encodes a LUX operon. Such an operon is encoded by the sequence as set forth in SEQ ID NO: 401. Further information regarding LUX operons may be found in Winson M K, Swift S, Hill P J, Sims C M, Griesmayr G, Bycroft B W, Williams P, Stewart GSAB. 1998, Engineering the luxCDABE genes from Photorhabdus luminescens to provide a bioluminescent reporter for constitutive and promoter probe plasmids and mini-Tn5 constructs. FEMS Microbiol Letteres 163: 193-202; Craney A Hohenauer T, Xu Y, Navani N K, Li Y, Nodwell J. 2007. A synthetic luxCDABE gene cluster optimized for expression in high-GC bacteria. Nuc Acid Res 35: No. 6 e46, both of which are incorporated herein by reference.
The present inventors identified sequences in the Brucella phage genome which were devoid of open reading frames and generated constructs which facilitated insertion of genes of interest (for example, those encoding detectable moieties) into the Brucella phage at those positions, so as not to affect the vital life cycle of the phage.
Thus, according to yet another aspect of the present invention there is provided a nucleic acid construct comprising:
i. a polynucleotide encoding a gene of interest operationally fused to a Brucella promoter;
ii. a first Brucella phage sequence fused to a 5′ end of the promoter, the first sequence comprising from 100 nucleotides to all the nucleotides of the nucleic acid sequence as set forth in SEQ ID NO: 394; and
iii. a second Brucella phage sequence fused to a 3′ end of the gene of interest, the second sequence comprising from 100 nucleotides to all the nucleotides of the nucleic acid sequence as set forth in SEQ ID NO: 395.
Since the flanking sequences around the gene of interest (i.e. SEQ ID NO: 394 and SEQ ID NO: 395) are Brucella phage sequences, such a construct may be used to insert the gene of interest by recombination into the Brucella phage genome.
If a phage is required which may be used to identify Brucella bacteria (and diagnose an infection), the gene of interest may encode a detectable moiety. Detectable moieties are further described herein above.
If a phage is required which may be used to kill Brucella bacteria, the gene of interest may encode a polypeptide which is lethal to Brucella bacteria. Such polypeptides may include anti-bacterial toxins (bacteriocins) and the like. In addition, non-translated sequences may be used to down-regulate important bacterial functions and factors that affect these sequences could be exploited to control bacterial functions.
Examples 3 and 4 of the Example section herein below describe a method of generating Brucella bacteria which carry the phage as co-residence of recombinant strains. Such carrier Brucella clones provide a means of unlimited chances to achieve direct recombinantional events between harbored foreign DNA and Brucella phage.
It will be appreciated that the phage which identifies Brucella bacteria by outputting a detectable signal (or carrier Brucella clones comprising same) may be used to diagnose a Brucella infection in a subject.
According to this aspect of the present invention, the method of diagnosing comprises contacting a sample of the subject with the recombinant Brucella phage described herein above. Infection of the Brucella bacteria with the recombinant Brucella phage would result in an increase in expression of the detectable moiety, thereby providing a signal that the infection is due to Brucella bacteria. The subject is typically a mammalian subject, e.g. sheep, cows, goats and humans.
Typically, the sample which is analyzed is a cellular sample derived from blood, urine, faeces, uterine, fetus membranes and placental membranes and fluids, mammary glands, lymph nodes, granuloma, sperm, testes, brain, cardio and renal organs, Cerebrospinal fluid (CSF), milk, dairy products, of the subject. Environmental samples (soil, aerosols, water) are also contemplated.
The terms “comprises”, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to”.
The term “consisting of” means “including and limited to”.
The term “consisting essentially of” means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.
As used herein the term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.
Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.
Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.
Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, “Molecular Cloning: A laboratory Manual” Sambrook et al., (1989); “Current Protocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide to Molecular Cloning”, John Wiley & Sons, New York (1988); Watson et al., “Recombinant DNA”, Scientific American Books, New York; Birren et al. (eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis, J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique” by Freshney, Wiley-Liss, N.Y. (1994), Third Edition; “Current Protocols in Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J., eds. (1985); “Transcription and Translation” Hames, B. D., and Higgins S. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide to Molecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317, Academic Press; “PCR Protocols: A Guide To Methods And Applications”, Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategies for Protein Purification and Characterization—A Laboratory Course Manual” CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.
Purification of Phage Iz1 and Extracting Genome DNA:
Phage Iz1 was consecutively propagated on Brucella abortus reference strain 544 by inoculating drops of a phage Iz1 suspension at routine test dilution (RTD) concentration on tryptic soy agar plates on which a 0.1 ml aliquot of a B. abortus strain 544 suspension was spread. After overnight growth at 37° C. in 5% CO2 atmosphere plaques were collected into tryptic soy broth and enumerated for further preparations of RTD suspensions used in phage typing of Brucella isolates.
The same procedure was applied in order to purify phage Iz1 particles for DNA extractions. However, phage suspension was prepared in TMGS buffer and filtered twice, first through 0.45 μm and subsequently through 0.2 μm filters in order to achieve a non-Brucella contaminated phage suspension.
2 ml of phage suspensions were centrifuged per tube using Sorvall RC M100 ultracentrifugation (Du-Pont). Ultracentrifugation was carried out at 10° C., for 4 hours at 60,000 rpm and DNA was extracted from phage pellets using QIAamp DNA Mini Kit, according to manufacturer's instructions (Qiagen GmbH, Hilden, Germany).
Cloning Phage Iz1 HindIII DNA Fragments—Construction of Phage Iz1 HindIII Fragment Clones in E. coli Plasmid pBluescript:
Phage Iz1 DNA was digested by HindIII, according to manufacturer's instructions (Fermentas Inc., Maryland, USA). The HindIII digestion profile concurred with previously published data (Rigby et al., Can J Vet Res. 1989; 53: 319-325). The DNA fragments were purified from agarose gel using Wizard® SV Gel system (Promega). Plasmid pBlueScript was digested by HindIII and purified from agarose gel using Wizard® SV Gel system (Promega). The purified pBlueScript plasmid and Phage Iz1 DNA fragments were mixed, ligated and transformed to E. coli JM109 (Promega). Plasmid DNAs were extracted using HiYield Plasmid Mini Kit (RBC Bioscience, Taipei County, Taiwan).
DNA-DNA Hybridizations:
Hybridization was executed using DIG nonradioactive nucleic acid labeling and detection system, according to manufacturer's instructions (Roche Diagnostics GmbH, Mannheim, Germany).
Construction of Plasmid pBBR1mcs-43-II1053LuxCDABE:
Plasmid pBBR1mcs-41-II1053LuxCDABE was constructed by inserting the Photorhabdus luminescens luxCDABE operon (Winson et al., 1998, FEMS Microbiol Letters 1998; 163: 193-202, incorporated herein by reference) into plasmid pBBR1mcs-4 (Kovach et al., 1995, Gene 1995; 166: 175-176).
Construction of Plasmids pBBR1mcs-4.1-II1053LuxCDABE (15B-18B and 15A-18A, Respectively)
Specific Phage Iz1 sequences were used as scaffold of the plasmid constructs. Primers were designed to include both Tn5 mosaic ends, 1 and 2, respectively. The KpnI::PstI sequence was added 5′ and PvuII::ME-1::KpnI sequence added to the 3′ end of fragment 15500 to 16509 of Phage Iz1 amplicon. The SacI::ME-2::PvuII sequence was added 5′ and SalI::SacI added 3′ to 18579 to 19630 fragment of Phage Iz1 amplicon (PvuII, KpnI, SacI, SalI are nucleotide sequences of the restriction endonuclease restriction sites of these enzymes; The position of the primers used to generate these constructs is illustrated in
Phage Iz1 naked DNA was used as the substrate in separate PCR DNA amplification reactions to obtain the desired amplicons. The first fragment [SacI::ME-2::PvuII-Iz(18579-19630)-SalI::SacI] was digested with SacI then ligated into a SacI linerized plasmid pBBR1mcs-4.1-II1053LuxCDABE to generate an intermediate plasmid pBBR1mcs-4.1-II1053LuxCDABE::SacI::ME-2::PvuII-Iz(18579-19630)-SalI::SacI, in orientations A and B (
Establishing Phage Iz1 Pre-Infected-Electro-Competent Brucella suis Strain 1330 Cells:
Brucella suis strain 1330 was grown at 37° C. in 5% CO2 atmosphere for 2 days on Trypticase soy agar supplemented with Serum-dextrose (Alton G G, Jones L M, Angus R D, Verger J M. Techniques for the brucellosis laboratory. Institute National de la Recherche Agronomique, Paris. 1988). Cells were collected by a plating loop and transferred to 6 ml Tryptic soy broth (TSB) establishing cell suspension at a concentration of around 107-108 cells/ml. Then, 1 ml of the cell suspension was inoculated into 22 ml of TSB in a 250 ml Erlenmeyer vessel and laid down to chill in the refrigerator for 2 hours. In total, 4 such Brucella suis strain 1330 cell suspensions were prepared. Incubation was stopped by taking each vessel out from the incubator to ambient temperature and 1 ml phage Iz1 in TSB at ×104 concentration of the routine test dilution (RTD, Alton et al., 1988) were added to the Brucella cell suspension. Taken that Brucella complete a single cycle of cells replication by 4 hours Brucella phage infection was allowed to a minimal period of 2 hours by incubating the cell suspension at 37° C. in 5% CO2 atmosphere and chilling the cell suspension immediately after by incubation in ice. Then, the 4 cell suspensions were centrifuged for 13 minutes at 6500 rpm, in a fixed angle rotor at 4° C., each in a separate tube. The supernatants were spilled and every two cell pellets were pooled together and resuspended in 12 ml of 10% glycerol solution in double distilled water pre-cooled to 4° C. Washing in the cold (including pre-cooled pipettes and micro-tips) was repeated 4 times, each carried out by resuspending the pellet and repeated centrifugation for 13 minutes at 8000 rpm. The two cell pellets were resuspended and pooled together in 3 ml 10% glycerol and spun down at 8000 rpm for 10 minutes, at 4° C. The final cell pellet was then resuspended in 0.5 ml of pre-cooled 10% glycerol solution and further divided to aliquots of 50 μl each in pre-cooled eppendorf tubes that were immediately cooled to freezing using liquid nitrogen and then stored at −80° C. until use.
Results
The complete genome sequence of phage Iz1 has been deciphered using 454 Life Sciences™ Roche GS-FLX sequencing platform (DYN Labs, LTD, Israel). The largest contig that was identified includes 38,254 bp (SEQ ID NO: 1 and SEQ ID NO: 2). Within this contig, the present data identified two Brucella phage Iz1 genome populations differing by an SNP or a heterozygote nucleotide (nucleotide 5546 was recorded as N but in fact it was conclusively identified as C, and the polymorphism was distributed equally in 8 contigs between C or A at nucleotide 5549, respectively (
To further corroborate the sequence of the phage, 8 HindIII DNA digest segments (1.1, 2.1; 3.1; 4.1, 5.1; 5.2; 5.3; 7.3) from phage Iz1 were sub-cloned into plasmid pBS and sequenced corroborating the established sequences of identical overlapping fragments in phage Iz1 genome. In concordance with these results, whole genomic naked DNA of phage Iz1 hybridized with each of these clones. Two additional clones, e.g., 5.4 and 71—3 and 71—5_I, included partial sequencing (
The sequence of the phage was analyzed using BLAST ((Basic Local Alignment Search Tool) software.
The results are displayed in Table 2, herein below.
Ochrobactrum
anthropi ATCC
Ochrobactrum
anthropi ATCC
Ochrobactrum
anthropi ATCC
Ochrobactrum
anthropi ATCC
Ochrobactrum
anthropi ATCC
Ochrobactrum
anthropi ATCC
Results from the analysis of CpG islands show that although the observed/expected ratio>0.60, the actual percent C+percent G>50.00, indicating that the phage comprises sequences other than Brucella.
An inverted repeat was found at positions 5088-5179 and 5405-5310 suggesting a putative site of the origin of replication.
Using an internet based promoter finding tool (worldwidewebdotfruitflydotorg/seq_tools/promoter) the present inventors identified 183 potential promoters on the forward strand and 201 potential promoters on the reverse strand. The sequences of these promoters are set forth in SEQ ID NOs: 3-185 for the forward strand and 186-386 for the reverse strand.
Plasmid constructs that include selected sequences from Phage Iz1 genome were designed and transformed to E. coli JM109 including those indicated in
Further, light activity was completely silenced in B. suis strain 1330 whereas it was partially expressed in B. melitensis strain 16M (Results could not be shown with B. abortus as the plasmid was lethal to this strain, as explained above). When arguing for silencing activities by Phage Iz1 sequences this could be demonstrated by adding external n-decanal to Brucella suspensions. The pentacistronic Lux operon consists of a luxAB component that encodes for a bacterial luciferase that oxidizes FMNH2 and a long-chain aliphatic aldehyde (n-decanal substrate) in the presence of molecular oxygen to yield a 490-nm optical signature. The aldehyde is subsequently regenerated by a multi-enzyme reductase complex encoded by the luxC, luxD, and luxE genes. Accordingly, the Lux operon is encoding two separate functions, expression of luciferase by genes A and B and the substrate, by genes, C, D and E, respectively. External N-decanal could be used as a substitute for the native substrate. Because the addition of external n-decanal to the cell suspension fully restored light in both B. suis strain 1330 pBBRImcs4.1 II1053LuxCDABE/15A-18A and B. melitensis strain 16M pBBRImcs4.1 II1053LuxCDABE/15A-18A, this indicates that luciferase was present in the reaction mixture at the time, inferring it was fully expressed under the promoter that resides upstream to Phage Iz1 15A and LuxC and Lux D, sequences. It is most likely therefore that gene LuxE, downstream of LuxA and LuxB was under unique regulation from Phage Iz1 18A sequence under the 3′ orientation. This is further supported by the fact that this regulation was exerted at different intensities between B. suis strain 1330 (null Lux activity) and B. melitensis strain 16M (partial Lux activity). As phage Iz1 fully lyses B. abortus and B. suis strains but has only partial lysis on B. melitensis strains, our data corroborate the historical Brucella species phage typing method and support our invention that the 18A Phage Iz1 sequence regulates Brucella gene expression.
Table 3, herein below provides additional plasmids comprising phage Iz1 sequences that are capable of down-regulating genes placed immediately downstream thereto in both brucella and other bacteria.
E. coli
B. suis
B. melitensis
E. coli
E. coli
B. suis
E. coli
E. coli
E. coli
E. coli
B. suis
B. suis
B. melitensis
B. melitensis
B. abortus
pII1053 is strongly expressed in a constitutive manner in the three Brucella species, B. suis, B. melitensis and B. abortus. This promoter is expressed less intensively in E. coli.
The construct 18A downregulates Lux expression in both E. coli and Brucella, most likely by silencing LuxE.
In this example, the goal was to develop a method that will extend existence of phage Iz1 infection of Brucella to an un-limited period of time in order to enable phage genome engineering at that time by recombinant DNA technology.
Naked phage DNA exists within bacterial cytosol immediately after phage infection following intrusion of the bacterial envelop by the phage DNA. Phage replication further ensues due to controlling bacterial gene expression and gearing the bacterial DNA replication machinery to a phage system. Prevention of DNA packaging into intact phage particles will therefore allow gene engineering of the phage genome by electroporation of the bacterial host during this period with recombinant DNA constructs that facilitate gene transposition or gene recombination. Accordingly, the present inventors hypothesized that phage infection could be arrested by chilling the Brucella host cells immediately after infection, then washing the Brucella cells several times using water-glycerol and freezing the cells at −80° C. until needed for electroporation.
Results
One eppendorf tube that contained phage arrested infection as described above, was taken out from the −80° C. freezer, and the cells were thawed on ice, and diluted to 1 ml by adding 0.95 ml of SOC-B solution (Lai F, Microb Pathog 1990; 9:363-368). Then, 1:10 dilutions of the cell suspension were prepared in cold physiological saline solution, up to 10−7. Drops of 10 μl were then inoculated on B. abortus strain 544 that was spread (0.1 ml of a TSB heavy cell suspension) on a TSA plate by a bacterial Drigalski spreader.
For comparison, an aliquot of the SOC-B cell suspension was passed through 0.45 μm syringe filter in order to ascertain that free Iz1 phage particles did not exist in the cell suspension. The filtrate was similarly diluted 1:10 in physiological saline solution and 10 μl drops from each dilution were inoculated on B. abortus strain 544 plate (see above).
The two plates were incubated over night at 37° C. in 5% CO2 atmosphere. The next day, plaques were sought in each dilution of whole cell suspension and cell-filtrate. The last dilution of the cell filtrate that yielded phage plaques was 10−2 in which only two plaques were identified. In agreement with the dilution around 200 plaques were identified at 10−1 dilution, indicating a minimal presence of free phage particles in the whole cell suspension prior to filtration. In contrast, the last dilution that yielded phage plaques by the whole cell suspension was 10−6, at which a single plaque was identified. In agreement with the dilutions, 10 plaques were identified at dilution 10−5 and concentrated plaques were found at 10−4 and below, indicating existence of close to a 4 logarithmic higher magnitude of phage particles in the cell suspension compared to the cell filtrate. All together, these data support the working hypothesis that despite interfering with phage replication by cooling off the cell suspension, cold washings and freezing at −80° C., the phage infection was fully restored when the cells were thawed and re-cultured. Similar results were achieved when phage infection was stopped after 1 hour and 15 minutes.
The present example describes how a pre-phage infection state could be used to develop a recombinant phage Iz1 clone that induces Lux activity in Brucella species. Such a recombinant clone could be used as a highly sensitive reporter to indicate presence of living Brucella cells in a suspected sample by light measurements in a host, its tissues (such as aborted placenta and fetus membranes and fluids) or milk samples.
Method
The method involves two steps (See
A short denaturation and annealing process between equal amounts of phage Iz1 and plasmid pBBR1mcs4.1-II1053LuxCDABE/15B-18B DNAs was applied, the latter shares homologous sequences with the phage genome (
DNA denaturation and annealing was carried out in total volume of 25 μl reaction mixture using Biometra, T-Gradient, thermocycler, Germany. The thermocycling reactions were as follows:
6 cycles of: 95° C.-1.15′, 55.5° C.-2.00′, 72° C.-2.00′
Final annealing at 55.5° C.-7.00′
Electrocompetent pre-phage Iz1 infected B. suis cells were used. 40 μl of electrocompetent cells were electroporated by adding 2 μl of the final thermocycling reaction mixture using bacterial mode of MicroPulser™, BIO-RAD, Hercules, Calif., USA (2.49 Kv, 4.9 ms). Immediately after electroporation, cells were suspended in 1 ml SOC-B (see above) and incubated with shaking at 37° C. for 1 hour and 30 min. A 1:10 dilution of the SOC-B electroporated cell suspension was prepared and 10 μl aliquots from both undiluted and 1:10 diluted SOC-B suspensions were inoculated on TSA plates that included 50 μg/ml ampicillin as the selecting antibiotic.
As a control, 10 μl drops of the electroporated cells in SOC-B and 1:10 dilution were inoculated on B. abortus strain 544 cells that were pre-spread on TSA agar plate in order to demonstrate phage Iz1 infection of the electroporated B. suis strain 1330 cells.
Results
Non-electroporated cell suspension successfully grew on plain TSA plates but did not grow on TSA plates that included 50 μg/ml ampicillin. After electroporation, about 90 colonies grew on selective agar from the non-diluted SOC-B cell suspension and about 9 colonies grew from the 1:10 cell dilution. Four colonies from each dilution were selected for further analysis, each transferred on a TSA plate that included 50 μg/ml ampicillin. Table 4 herein below summarizes the luminescence and phage activity of electroporated clones.
These results indicate the following: 1. Plasmid pBBR1mcs4.1-II1053LuxCDABE was successfully transformed into these clones. 2. Smooth clones (2, 3, and 4) did not secret phage activity and rough clones were phage carriers (
Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.
All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.
This application is a division of U.S. patent application Ser. No. 13/500,360 filed on Apr. 5, 2012, which is a National Phase of PCT Patent Application No. PCT/IL2010/000812 having International filing date of Oct. 7, 2010, which claims the benefit of priority of U.S. Provisional Patent Application No. 61/272,574 filed on Oct. 7, 2009. The contents of the above applications are all incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 61272574 | Oct 2009 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13500360 | Apr 2012 | US |
| Child | 14274724 | US |
