Extract from: http://www.bgsc.org/NewThisMonth.htm (Aug 2009)
A collaboration from groups at University of Maryland School of Medicine (J. Ravel and W. F. Fricke), Harvard Medical School (R. Kolter and A. Earl), and Harvard University (R. Losick) is currently aiming to sequence six environmental isolates belonging to the "Bacillus subtilis group" of related species:
We thank Ashlee Earl of Harvard Medical School for donating these strains to the BGSC collection, and we look forward to the availability of their genome sequences.
Earl, A. M., R. Losick, and R. Kolter. 2007. Bacillus subtilis Genome Diversity. J. Bacteriol. 189:1163-1170. (View paper)
Nakamura, L. K., M. S. Roberts, and F. M. Cohan. 1999. Relationship of Bacillus subtilis clades associated with strains 168 and W23: a proposal for Bacillus subtilis subsp. subtilis subsp. nov. andBacillus subtilis subsp. spizizenii subsp. nov. Int. J. Syst. Bacteriol. 49:1211-1215. (PubMed)
Targeted degradration of protein in B. subtilisFrom Kevin Griffith and Alan Grossmann at MIT come an exciting new collection of plasmids and strains that comprise a novel system for inducible protein degradation in Bacillus subtilis. With these tools, a user can rapidly deplete the concentration of a targeted protein and observe the phenotypic effects for the cell. For a more complete listing of the strains and plasmids in the collection and an introduction to their use, please see our product announcement. Our thanks to the Grossman lab for their generosity! Griffith, K. L. and A. D. Grossman. 2008. Inducible protein degradation in Bacillus subtilis using heterologous peptide tags and adaptor proteins to target substrates to the protease ClpXP. Mol. Microbiol. 70:1012-25. Integration & LacZ fusion vectorFrom 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. colihost 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 ofBacillus subtilis. J. Bacteriol. 185:973-982. (View the paper in PubMed.) Shuttle vector sequences
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. From Marie-Agnès 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) Protein expression and displayDr. 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, seeNguyen 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! E. coli - Bacillus shuttle vectors for protein expression
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 theBacillus genetics community.
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.
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.
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 also includes six control strains in which the cassettes, without inserts, have been integrated into the chromosomal loci.
Sixteen new Fluorescent Protein tagging vectors--The Bacillus Genetic Stock Center is pleased to offer 16 new vectors designed for constructing fluorescent protein fusions. Three of the vectors come from the laboratory of Wolfgang Schumann at the University of Bayreuth, Germany, while the remaining 13 come from Peter Lewis at the University of Newcastle, Australia.
Bacillus subtilis Integration Vectors with Inducible Expression of Cloned Inserts--From the laboratory of Wolfgang Schumann at the University of Bayreuth come two new expression vectors capable of integrating into the Bacillus subtilis chromosome at the lacA locus. Each allows for regulated expression of cloned inserts. |