News

From Brunella Perito at the Università 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!

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.

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.

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!

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 1981). 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 (Minich 1984). We thank Dr. Aronson for providing this mutant.

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:

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.

New Gram-Positive-E. coli expression vectors featuring high structural stability

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:

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.

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

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.

Integration vectors allow improved expression of Cyan and Yellow Fluorescent Proteins in Bacillus

From two sources--the ARS collection at the US Department of Agriculture and the T. Leighton laboratory at Children’s Hospital Oakland Research Institute--come a collection of 14 strains from underrepresented species in our collection. Included are nine type strains. We appreciate the kindness of Alex Rooney at the ARS and Katie Wheeler at CHORI in helping us acquire these strains:

1. 80A1T Aneurinibacillus aneurinilyticus NRRL NRS-1589T
2. 81A1T Aneurinibacillus migulanus NRRL NRS-1137T
3. 11A2T Bacillus atrophaeus NRRL NRS-213T
4. 6A17 Bacillus cereus ATCC 13472
5. 6A18 Bacillus cereus ATCC 15816
6. 61A1T Bacillus coagulans ATCC 7050T
7. 7A36T Bacillus megaterium ATCC 14581T
8. 6A20 Bacillus mycoides ATCC 11986
9. 6A19 Bacillus mycoides ATCC 31101
10. 2A9T Bacillus subtilis subsp. spizizenii NRRL B-23049T
11. 2A10 Bacillus subtilis subsp. spizizenii NRRL B-14472
12. 34A1T Paenibacillus thiaminolyticus NRRL B-4156T
13. 41A1T Brevibacillus borstelensis NRRL NRS-818T
14. 42A1T Brevibacilus centrosporus NRRL NRS-664

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.

New ectopic integration vectors for Bacillus subtilis

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 integration vector for high level, constitutive expression of cloned inserts

Dr. Len Peruski from the Indiana University School of Medicine in Gary, Indiana, has donated several strains to fill holes in our collection, including four strains of Bacillus mycoides (BGSC Codes 6A11-6A14), two strains of Bacillus firmus (BGSC 29A1 and 29A2), and one strain each of Bacillus lentus (60A1), Brevibacillus brevis (26A6), and Bacillus circulans (16A4). For more information about any of these strains, enter the BGSC code or species name on our improved search page! Our thanks to Dr. Peruski for his generosity.

Anne K. Dunn, a student in the Jo Handlesman lab at the University of Wisconsin, has constructed pAD123 (see map and sequence), a new shuttle vector optimized for fluorescence-assisted cell sorting. This vector can be a powerful tool for isolating sets of promoters that all respond to certain environmental or physiological stimuli. These GFP fusions can also serve to localize proteins within cells. Request strain ECE165 for pAD123 or strain ECE166 for pAD43-25 (see map and sequence), a derivative carrying a constitutive B. cereus promoter.

Amanda J. Ozin, currently at the Max Planck Institute for Infection Biology in Berlin, has donated a safA (formerly yrbA) knockout mutant in B. subtilis. The gene product, SpoVID-associated factor (SafA), is required during the early stages of spore coat assembly. Mutants produce abnormal spores lacking several coat proteins. To obtain this mutant, request our strain 1S117.

Peter Zuber of the OGI School of Science & Engineering has donated a surfactin-producing strain of Bacillus subtilis, ATCC 21332, our strain 3A22. Surfactin is a cyclic lipopeptide with a fascinating array of properties. At micromolar concentrations, it lowers the surface tension of water from 72 mN m-1 to 27 mN m-1, suggesting many possible \"environmentally friendly\" applications in industry. Anti-clotting, antibacterial, antitumoral, and hypocholesterolemic properties have all been described as well. For an interesting minireview, read Peypoux, F., J. M. Bonmatin and J. Wallach. 1999. Recent trends in the biochemistry of surfactin. Appl. Microbiol. Biotechnol. 51:553-563. The Zuber lab has extensively published research elucidating the molecular genetics and biochemistry of surfactin synthesis and its relation to developmental processes in B. subtilis.

From Fred Cohan at Wesleyan University in Middletown, Connecticut come three strains belonging to Bacillus mojavensis, a species closely related to B. subtilis. Our strains 28A1, 28A2, and 28A3 were originally described as RO-H-1, RS-A-2, and RO-C-2, respectively, in Roberts, M. S., L. K. Nakamura, and F. M. Cohan. 1994. Bacillus mohavensis sp. nov., Distinguishable from Bacillus subtilis by Sexual Isolation, Divergence in DNA Sequence, and Differences in Fatty Acid Composition. Int. J. Syst. Bacteriol. 44:256-264. The Cohan lab has used these and other relatives of B. subtilis to investigate the relationship between DNA sequence divergence and sexual isolation in bacteria.

Also from the Schumann laboratory come three vectors designed to tag a gene of interest with either the FLAG, cMyc, or HA epitopes, greatly simplifying the detection and purification of proteins in gram-positive organisms.

Three new Epitope-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.

Sixteen new Fluorescent Protein tagging vectors

Jan van der Ploeg at the University of Zurich has kindly donated four Bacillus subtilis mutants with knockouts in one or more sulfur utilization genes: ssuA, ssuC, ssuD, cysI, or cysJ.

New Bacillus subtilis sulfur source utilization gene knockout mutants

From F. Denizot at the INRS in Marseille, France, comes pDG148-Stu a shuttle vector, capable of replicating in E. coli from the pBR322 origin and in Bacillus from the pUB110 origin. It allows inducible expression of foreign inserts cloned into its unique StuI site. Oriented, ligation-independent cloning of PCR fragments is possible using the proper primers and a prepared template. Erratum - The original Adobe Acrobat file describing pDG148-Stu was in error. Instead of treating StuI-linearized vector with T4 polymerase in the presence of dATP, one must use dTTP. The file above has been corrected.

Gram-Positive - E. coli Shuttle Vector Featuring Ligation-Independent Cloning and Inducible Expression

New from the Patrick Piggot lab at Temple University School of Medicine: a Bacillus subtilis integration vector that inserts into the dif site at about 166° on the chromosome. Integration takes place via the host system for resolving chromosome dimers and does not require the action of the standard recombination pathways. Even recA mutants can be transformed with this vector!

RecA-independent Integration Vector for Bacillus subtilis

A high titer PBS1 transducing lysate is now available, a gift from Brooke Murphy of the Tina Henkin lab here at the Ohio State University. The BGSC accession number for PBS1 is 1P1. Please note that this phage is heat labile and requires a motile strain of Bacillus subtilis as a host.

Vasant K. Chary from the Patrick Piggot lab at Temple University School of Medicine has kindly donated a pair of novel antibiotic-cassette switching vectors, pVK71 and pVK73, for use in Bacillus subtilis and other Gram-positive organisms.

Antibiotic Switching Vectors

The Bacillus Genetic Stock Center is pleased to offer an isogenic set of Bacillus subtilis mutants deficient in the major autolysins. Philippe Margot, from the Dimitri Karamata group at the Institut de Génétique et de Biologie Microbiennes in Lausanne, Switzerland, kindly donated the collection.

Bacillus subtilis autolysin-deficient mutants

From the laboratory of Samuel Singer, who retired in 1997 from Western Illinois University, comes a collection of environmental bacterial isolates demonstrating activity against a variety of invertebrate species.

Novel Strains Showing Insecticidal, Nematicidal, and Molluscicidal Activity

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.

Bacillus subtilis Integration Vectors with Inducible Expression of Cloned Inserts

pMUTIN4 should allow the researcher to produce knockout or conditional expression mutations in any unknown coding sequence.

A Vector Useful for Gram-Positive Genomics

From the laboratory of Chet Price at UC Davis comes an important set of mutants altered with the regulation of late stationary phase events. PrpC is a member of the PPM family of serine/threonine protein phosphatases; it removes phosphates from PrkC, a serine/threonine kinase. The pair is believed to regulate the activity of Elongation factor G (EF-G) during stationary phase. In prpC knockouts, such as PB702 (=BGSC 1A961), stationary phase cultures grow to a much greater density in rich, non-sporulation media. In contrast, stationary phase cultures of prkC knockouts, such as PB705 (=BGSC 1A962), grow to a significantly lower density. In double mutants, such as PB722 (=BGSC 1A964), prkC is epistatic to prpC (1). We thank Tatiana Gaidenko and Chet Price for donating this set of strains to the BGSC.

Neil Welker has donated plasmid pNW33N, a fifth generation vector that stably replicates in Bacillus subtilis, Geobacillus stearothermophilus and Escherichia coli

Cloning Vector for Thermophilic Bacillus Strains