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From Reindert Nijland of Newcastle University comes Bacillus licheniformis strain EI-34-6. This marine isolate forms a thick, red biofilm and produces bacitracin when grown at a medium-membrane interface in media containing glycerol and FeCl3. To learn more about this strain, you may read the paper describing its isolation. The strain is available from the BGSC as 5A37. We thank Dr. Nijland for this interesting new isolate! |
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From Thomas Wiegert at the Universität Bayreuth
comes a novel vector, pLacZ, designed to facilitate the construction of
lacZ transcriptional fusions and their subsequent integration into the
Bacillus subtilis amyE locus. Like other integration vectors,
pLacZ can replicate in E. coli but not in B. subtilis. It
contains the 5' and 3' ends from the amyE gene; sandwiched between
them is a kanamycin/neomycin resistance marker, used for selection, and the
complete lacZ coding sequence with convenient upstream sites for
inserting EcoRI and BamHI and compatible fragments. An E. coli host
containing pLacZ is available form the BGSC as strain ECE201; purified
plasmid DNA is available as ECE201P. A genetic and physical map of the
plasmid is available here. The sequence
of the plasmid is available here. The construction and use of pLacZ is
described in: Zellmeier, S., U. Zuber, W. Schumann, and T. Wiegert. 2003. The absence of FtsH metalloprotease activity causes overexpression of the σw-controlled pbpE gene, resulting in filamentous growth of Bacillus subtilis. J. Bacteriol. 185:973-982. (View the paper in PubMed.) Our thanks to Dr. Wiegert for his generous donation! |
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We are now pleased to offer Bacillus sp. NRRL B-14911, a marine bacterial strain isolated from ocean water at 10 m depth in the Gulf of Mexico. 16S rRNA sequence comparisons suggest that it is either a member of or closely related to Bacillus firmus (Siefert J. L., et al. 2000. Curr. Microbiol. 41:84-88; view paper in PubMed). A gapped genome sequence is available at AAOX01000000. NRRL B-14911 forms pink-pigmented colonies on LB, TBAB, or a variety of standard complete media. It can grow between 20°-40°C with optimal growth at 28°C. It is moderately halo-tolerant, capable of growth in in 0-5% (w/v) NaCl. This strain has been accessioned into the BGSC collection as 29A3. |
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From Dennis Claessen at the Jeff Errington lab at Newcastle University comes pLOSS*, a novel vector designed to screen for synthetic lethal or sick mutations in Bacillus subtilis and other gram-positive bacteria. Synthetic lethal mutations are those that individually are viable but in combination are lethal. These types of mutations can provide powerful insights into coordinated gene functions in cellular processes. For more information about pLOSS*, see our description here. Plasmid pLOSS* is available either as purified DNA (ECE200P) or in and E. coli host (ECE200). We thank Dr. Claessen for making this exciting new tool available through the BGSC! |
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The first generation general purpose shuttle vectors pMK3 and pMK4 are not new, but they are a tried and true tool for cloning in a wide variety of gram-positive bacteria, including species from the genera Bacillus, Listeria, and Staphylococcus. Recently, the BGSC determined the DNA sequence for these two plasmids. For details, see our description here. |
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From Marie-Agnčs Petit of the INRA in Jouy en Josas, France, comes a useful plasmid for tightening up regulation of Pspac promoter fusions in Bacillus subtilis and related organisms. Plasmid pMAP65 (Petit, M. A., et al. 1998. Mol. Microbiol. 29:261–273) is a LacI-overproduction plasmid based on the pUB110 replicon. Many of the most useful expression systems for gram-positive organisms are based on the Pspac system, composed of a hybrid SPO1/lac promoter and a constitutively expressed lacI repressor gene. This system, first developed by Yansura and Henner (1984. Proc. Natl. Acad. Sci. USA 81:439-443), allows for IPTG-inducible expression of gene fusions. It is still an expression system of choice in functional genomics projects. One limitation of Pspac, however, is that it is somewhat leaky; a significant basal level of expression still exists in the absence of IPTG, making the identification of essential genes, for example, somewhat problematic. Plasmid pMAP65 solves this problem by overexpressing the LacI repressor, virtually shutting down the expression of Pspac fusions in trans. Examples from the literature in which pMAP65 was used for this very purpose are listed below. We thank Dr. Petit for donating this useful tool. References citing the use of pMAP65: Amati, G., P. Bisicchia, and A. Galizzi. 2004. DegU-P Represses Expression of the Motility fla-che Operon in Bacillus subtilis. J. Bacteriol. 186:6003–6014. (PubMed) Pellegrini, O., J. Nezzar, A. Marchfelder, H. Putzer, and C. Condon. 2003. Endonucleolytic processing of CCA-less tRNA precursors by RNase Z in Bacillus subtilis. EMBO J. 22:4534–4543. (PubMed) Petit, M. A. and S. D. Ehrlich. 2000. The NAD-dependent ligase encoded by yerG is an essential gene of Bacillus subtilis. Nucleic Acids Res. 28:4642–4648. (PubMed) Petit, M. A. and S. D. Ehrlich. 2002. Essential bacterial helicases that counteract the toxicity of recombination proteins. EMBO J. 21:3137–3147. (PubMed) Saxild, H. H., K. Brunstedt, K. I. Nielsen, H. Jarmer, and P. Nygaard. 2001. Definition of the Bacillus subtilis PurR Operator Using Genetic and Bioinformatic Tools and Expansion of the PurR Regulon with glyA, guaC, pbuG, xpt-pbuX, yqhZ-folD, and pbuO. J. Bacteriol. 183:6175–6183. (PubMed) Uicker, W. C., L. Schaefer, M. Koenigsknecht, and R. A. Britton. 2007. The Essential GTPase YqeH Is Required for Proper Ribosome Assembly in Bacillus subtilis. J. Bacteriol. 189:2926–2929. (PubMed) Wegscheid, B., C. Condon, and R. K. Hartmann. 2006. Type A and B RNase P RNAs are interchangeable in vivo despite substantial biophysical differences. EMBO Rep. 7:411–417. (PubMed) Yao, S., J. B. Blaustein, and D. H. Bechhofer. 2007. Processing of Bacillus subtilis small cytoplasmic RNA: evidence for an additional endonuclease cleavage site. Nucleic Acids Res. 35:4464–4473. (PubMed) |
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Also from Dr. Petit come three novel gene knockouts in a Bacillus subtilis 168 trpC2 background, all described in Noirot-Gros, M.-F., et al. 2002. Mol. Genet. Genom. 267:391-400. We once again extend our thanks for these strains.
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Dr. George Ordal of the University of Illinois at Urbana-Champaign has donated three additional chemotaxis mutants, with knockouts in cheC, chedD, or both. The mutants are described in Rosario MML, et al. (1995) Biochemistry 34:3823 and Kirby JR, et al. (1997) Mol Microbiol 24:869. We thank Dr. Ordal for these strains.
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Dr. Michiko Nakano of the OGI School of Science and Engineering at Oregon Health and Science University has kindly deposited two additional knockout mutants, constructed in a JH642 background. The knockouts affect yjbI, which encodes a truncated hemoglobin, and ypmQ, which functions in delivering copper to cytochrome oxidase. We thank Dr. Nakano for these interesting new mutants.
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Dr. Wolfgang Schumann has donated two plasmids, pNDH09 and pNDH10, and a Bacillus subtilis host, NDH03, designed for the inducible expression of foreign proteins and their subsequent attachment to the host cell surface. Plasmid pNDH10 carries a xylose-inducible cassette and a sortase-mediated cell anchoring motif. B. subtilis NDH03 expresses sortase A, making it a suitable host for plasmids based on pNDH10. The sortase gene can also be integrated into the chromosome of other B. subtilis strains to create hosts by means of the integration vector pNDH09. For more details, see Nguyen HD, Schumann W (2006) J Biotechnol 122:473 and our product announcement for pNDH10. BGSC strains 1A857, ECE196, and ECE197 are B. subtilis NDH03, E. coli DH5α(pNDH09), and DH5α(pNDH10), respectively. We thank Dr. Schumann for this useful set of gene expression tools! |
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Dr. Patricia S. Vary of Northern Illinois has donated 81 auxotrophic, antibiotic-resistant, and temperature-sensitive germination mutants of Bacillus megaterium QM B1551 from the James C. Vary collection. Strain QM B1551 (available from the BGSC as 7A16) has been carefully studied as a bacterial genetic system by several labs during the past three decades. The large dimensions of the B. megaterium cell have made it an attractive organism for studies in development and subcellular localization of expressed proteins. QM B1551 is now the subject of a whole-genome sequencing project that is nearing completion (www.bios.niu.edu/b_megaterium/). The availability of genome sequence data and a collection of genetically well-characterized legacy strains should make B. megaterium an exciting topic for future research. |
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From Brunella Perito at the Universitŕ degli Studi de Firenze, Italy, comes a recent set of strains constructed to analyze the process of calcium carbonate precipitation in Bacillus subtilis. These strains are described in Barabesi, C., A. Galizzi, G. Mastromei, M. Rossi, E. Tamburini, and B. Perito. 2007. Bacillus subtilis Gene Cluster Involved in Calcium Carbonate Biomineralization. J. Bacteriol. 189:228-235. They represent knockout insertions in five genes within the lcfA operon. Four of the five knockouts are deficient in precipitation on B4 medium (0.4% yeast extract, 0.5% dextrose, 0.25% calcium acetate, 1.5% agar). We thank Dr. Perito for donating these strains to the BGSC!
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Dr. Brian Federici at the University of California, Riverside, has donated a recombinant strain that produces the mosquitocidal Bacillus sphaericus binary toxin in the B. thuringiensis subsp. israelensis plasmid-cured host 4Q7. With transcription of the toxin genes driven by the cyt1A promoters and protected by the STAB-SD sequence, the toxin proteins themselves accumulate as large crystals in sporulating cells. We have deposited this strain, 4Q7(pPHSP-1), under the accession 4Q11 in our collection. We thank Dr. Federici for making the strain available to us! |
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The laboratory of Alessandro Galizzi at the University of Pavia have donated a set of strains (listed below) in which the DegU-mediated repression of motility in Bacillus subtilis has been relieved by mutations in the regulatory region of the fla-che motility operon.
Also from the Galizzi laboratory come two other mutants affected in motility. Strain 1A842 (originally PB5250) has a knockout in the flagellin hag locus. Strain 1A850 (originally PB5249) has a hypermotility phenotype due to a mutation in the ifm locus. Both mutants are described in Senesi, S., et al. 2004. J. Bacteriol. 186:1158-1164. |
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Allesandra Albertini, also from the Pavia Bacillus subtilis group, has kindly donated a knockout mutant in the mutS2 paralog yshD. Strain 1A845 (originally PB5266) is described in Rossolillo, P. and A. M. Albertini. 2001. Mol. Gen. Genet. 264:809-818. |
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From Dr. Arthur I. Aronson comes a Bacillus thuringiensis subsp. kurstaki plasmid cured mutant that produces only Cry1Ab crystals. The mutant was isolated during the classic plasmid-curing studies that demonstrated the plasmid location of cry genes in this organism (Gonzalez, J. M., H. T. Dulmage, and B. C. Carlton. 1981. Correlation between specific plasmids and delta-endotoxin production in Bacillus thuringiensis. Plasmid 5:351-365). Strain HD1-1-9 is missing only its 165-kb megaplasmid, but as a result has retained only cry1Ab from its complement of crystal toxin genes. As demonstrated in the Aronson lab, Cry1Ab production in this strain is temperature sensitive, requiring temperatures below 30°C (Minnich, S. A. and A. I. Aronson. 1984. regulation of protoxin synthesis in Bacillus thuringiensis. J. Bacteriol 158:447-454). We thank Dr. Aronson for providing this mutant. |
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Dr. Rainer Borriss of Humboldt University in Berlin has deposited in the BGSC collection three additional mutants of Bacillus amyloliquefaciens FZB42. The wild type isolate stimulates plant growth and suppresses pathogens in the rhizosphere. The mutants show a reduction in plant growth promotion activity due to reduced production of the hormone indole-3-acetic acid, a biochemical process that requires tryptophan as a substrate. Here are the three new strains, with their BGSC accession numbers:
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New Gram-Positive-E. coli expression vectors featuring high structural stability--Expression of foreign proteins in Bacillus subtilis and other gram-positives has been a technically challenging problem, due in part to the inherent instability of the rolling-circle replicating plasmids on which most shuttle vectors are based. From the Wolfgang Schumann lab come three new expression vectors, pHCMC02 (weakly constitutive), pHCMC04 (xylose inducible), and pHCMC05 (IPTG inducible). We thank Dr. Schumann for donating this set of vectors and anticipate that they will prove very useful to the Bacillus genetics community. | |||||||||||||||
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Dr. Rainer Borriss of Humboldt University in Berlin has deposited in the BGSC collection four strains of Bacillus amyloliquefaciens. Strain FZB42 is a rhizosphere colonizing strain that stimulates plant growth and suppresses plant pathogenic organisms (Idriss EE, et al. (2002) Microbiology 148:2097). Like many B. subtilis isolates, it displays natural competence for transformation during stationary phase. A survey of the genome revealed sx large gene clusters encoding nonribosomal peptide synthetases (NRPS) and polyketide synthases (PKS). The Borriss lab constructed knockout mutants in two of these clusters, fen and bmy, encoding fengycin and bacillomycin D. Single mutants retained most of their ability to kill a fungal plant pathogen, but double mutants were severely impaired in this activity. Here are the strains, with their BGSC accession numbers:
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From the lab of Diego de Mendoza at the IDCM in Rosario, Argentina come two knockout mutants in Bacillus subtilis. The first, MAΔK (our accession 1A834) has its cysK gene disrupted by a kanamycin cassette. It grows at a reduced rate on sulfate and requires the addition of yeast extract and casamino acids on cysteine as a sulfur source. The second, LC5 (our 1A835) has a similar cassette disrupting its des locus. This mutant is unable to perform the Δ5 desaturation on its fatty acids in response to cold shock. We thank Drs. Cecilia Mansilla, Larisa Cybulski, and Diego de Mendoza for these mutant strains. |
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Dr. Jeff Errington has graciously deposited a set of 14 new mutants generated in his lab as part of the Bacillus subtilis genome consortium. Each mutant has a pMUTIN plasmid insertion into the target gene, placing expression of that gene under the control of the IPTG-inducible Spac promoter. With this strategy, essential genes can be distinguished from non-essential by the requirement of IPTG for growth and viability. For a description of this phase of the genome project, see Kobayashi K, et al. (2003) PNAS 100:4678. We thank Dr. Errington for his generosity and invite other members of the genome consortium to similarly deposit new mutants in the BGSC collection.
*Mutant requires IPTG for growth on LB |
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Integration vectors allow improved expression of Cyan and Yellow Fluorescent Proteins in Bacillus--Jan-Willem Veening of the University of Groningen has kindly donated a set of integration vectors that greatly facilitate the construction of fusions to either the Cyan or Yellow Fluorescent Proteins in Bacillus subtilis. The original CFP and YFP proteins were engineered for expression in eukaryotic organisms, not gram-positives. These improved variants contain several additional codons at the 5' end, allowing for much higher levels of expression in B. subtilis and potentially a host of other gram-positive bacteria. The large multiple cloning site should make construction of fusions a simple matter. We thank Dr. Veening and colleagues for their generosity. Look for a paper describing the plasmids to appear in an upcoming issue of Applied and Environmental Microbiology. |
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New integration vector for high level, constitutive expression of cloned inserts--Dr. Brian Jester of Trinity College, Dublin, Ireland has kindly donated a novel vector, pBCJ164.3, to our collection. The plasmid contains the 5' and 3' ends of the Bacillus subtilis rpsD gene, together with its promoter and transcription terminator. An NdeI site within this cassette allows for inserted fragments to be placed under the control of the strong rpsD promoter. Like other integration vectors, pBCJ164.3 replicates in E. coli but not in B. subtilis. When a recombinant plasmid is isolated from E. coli and transformed into a recombination-proficient B. subtilis host with selection for chloramphenicol resistance, a non-mutagenic Campbell-type insertion even should take place within the host chromosomal rpsD locus. |
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New ectopic integration vectors for Bacillus subtilis--Rebecca Middleton of the University of California, Berkeley, has generously donated to the BGSC a set of novel integration vectors. The vectors integrate into the Bacillus subtilis chromosome “ectopically,” that is, at a locus targeted by homologous sequences within the vector itself, rather than by sequences within a cloned insert. Each vector contains an integration cassette consisting of the 5’ and 3’ ends of a non-essential chromosomal gene, interrupted by a selectable antibiotic resistance marker and a multiple cloning site. When the vectors are introduced into a host strain by transformation with selection for antibiotic resistance, a double-crossover event replaces the chromosomal locus with the plasmid-borne cassette, including any fragments that have been inserted into the cloning sites. The six plasmids within the collection allow the user to target any of three loci—gltA, pyrD, or sacA—with selection for either kanamycin or chloramphenicol resistance. The collection als |