News

Phil Brumm has deposited Paenibacillus lautus strain Y4.12MC10, a novel environmental isolate with a recently completed genome sequence (GenBank CP001793). We have assigned the BGSC accession number 36A2 to this strain. Although P. lautus 36A2 was isolated from a geothermal pool in Yellowstone National Park, Montana, it is a mesophile, with a growth optimum of 37°C. The 7.12 Mb genome lacks nitrogen fixation, antibiotic production and social interaction genes reported for other Paenibacillus isolates, but does show a high degree of similarity to an organism detected in the Human Microbiome Project. Analysis of predicted carbohydrate active enzymes suggests that the isolate is unable to degrade cellulose or hemicellulose, but is instead rich in enymes that attack dietary fiber that is resistant to most ruminant bacteria. These observations raise the possibility that P. lautus 36A2 could be adapted to life in the digestive tract, perhaps of bison or other wildlife observed near the pool.

We thank Dr. Brumm for kindly donating this interesting isolate to us!

One of the more complex issues facing the growing bacterium is how to replicate and partition a millimeter-length chromosome inside a micrometer-length cell. Endospore-forming bacteria, like Bacillus subtilis, must be able to partition chromosomes correctly with respect to a symmetrical septum during vegetative growth or an asymmetric one during sporulation. There has been much progress in elucidating the details of these processes in bacterial model systems (see Toro and Shapiro for a recent review), due in no small part to the clever use of fluorescent proteins for labeling the origin of replication, oriC, so that it can be visualized in living cells.

Heath Murray, from the Centre for Bacterial Cell Biology at Newcastle University, has donated just such a tool for visualizing B. subtilis oriC. Strain HM1049 contains the gene for a xylose-inducible TetR repressor protein that is fused to the mCherry fluorescent label. It also contains an array of ~167 tetO operator sites for the repressor, located near the replication origin between spo0J and yyaC. When xylose is added to a culture of HM1049, then, the origin region is strongly labeled with a red fluorescent protein, allowing it to be visualized using epifluorescence microscopy techniques. Strain HM1049 is similar to strain HM355 (see Murray and Errington), except that it contains the wild type allele of soj.

We thank Dr. Murray and Dr. Jeff Errington for donating this useful tool to the BGSC.

BGSC Code: 1A1087
Original Code: Bacillus subtilis HM1049
Description: trpC2 spo0J::(tetO~167 neo) amyE::(Pxyl-tetR-mCherry spc) Km Sp
Maintenance: Grow in the presence of spectinomcyin (50 µg/ml) and kanamycin (2 µg/ml) to maintain stability of the tetR fusion and tetO array.

The Thorsten Mascher lab (Ludwig-Maximilians-University Munich) has constructed a novel protein expression system for Bacillus subtilis that features both tight repression and high levels of induction. This system is based on the liaI promoter, which is controlled in B. subtilis by the antibiotic-inducible LiaRS two component regulators. The system includes a choice of vectors: a replicative plasmid, pLIKE-rep, which is used with the B. subtilis host Bsu-LIKE1; and an integrative plasmid, pLIKE-int, which is used with host Bsu-LIKE2. The replicative version offers the advantage of maximum protein production, while the integrative version allows for stable maintenance in the absence of selection. The LIKE system has the following features: a) induction with the inexpensive antibiotic, bacitracin, or with a variety of other cell wall-active agents, such as ethanol or certain detergents; b) very low background expression in un-induced cells; c) up to 1000-fold induction upon the addition of bacitracin The components of the LIKE system are listed below. We thank Dr. Mascher and colleagues for donating this interesting expression system to the BGSC.

BGSC Strains

1A1070 B. subtilis host Bsu-LIKE1

1A1071 B. subtilis host Bsu-LIKE2

ECE255 E. coli host with pLIKE-rep

ECE256 E. coli host with pLIKE-int

Both basic research and biotechnological exploitation of the moderately thermophilic species, Geobacillus, has been hampered by the lack of suitable cloning vectors. The David Leak lab (Imperial College London) has helped to fill this void with the construction of a novel Geobacillus-E. coli shuttle vector, pUCG18. This vector offers several desirable features:

~ Modest size (6331 bp), allowing good transformation frequencies

~ Blue-white screening for inserts in E. coli

~ Use of kanamycin, the most thermal-resistant of common anibiotics, for selection in Geobacillus

~ Theta-replication in Geobacillus, advantageous for plasmid structural stability.

Plasmid pUCG18 is available in a E. coli DH5α host in BGSC strain ECE231. We thank Dr. Leak for allowing us to maintain and distribute this vector.

First, from the Simon Cutting lab at Royal Holloway, University of London comes a human fecal Bacillus subtilis isolate with significant potential as a probiotic. Strain HU58 (BGSC 3A34) forms robust biofilms, sporulates rapidly, and shows complete resistance to gastric fluids (Tam 2006, Hong 2009, Permpoonpattana 2012). In mouse models, this strain persists in the GI tract while stimulating proliferation of cells in the gut-associated lymphoid system, an indication of strong immunostimulatory activity (Tam 2006, Huang 2008). Strain HU58 has also been investigated as an oral vaccine for antigen delivery (Uyen 2007).

A second novel biofilm-forming isolate, Bacillus subtilis BD4974 (=PS-216), comes to us from Ines Mandic-Mulec of University of Ljubljana, Slovenia by way of the David Dubnau lab at the Public Health Research Institute Center of the UMDNJ - New Jersey Medical School. Strain PS-216 was described in a study assessing social interactions among closely related soil bacteria in the field (Stefanic 2009). It was isolated from a sandy soil sample near River Sava, Slovenia (46°06′N, 14°28′E) in January 2006. Although it is an undomesticated environmental isolate, PS-216 achieves high levels of natural competence during stationary phase, similar to the intensively studied lab strain, 168, to which it is closely related. PS-216 provides a genetically tractable system for studying biofilm formation and other developmental processes in Bacillus subtilis.

We thank the Cutting laboratory for donating B. subtilis HU58 and the Dubnau and Mandic-Mulec labs for donating B. subtilis PS-216.

Our thanks to the Richard Losick lab at Harvard University and the Wolfgang Schuman lab at the University of Bayreuth for supplying us with four Bacillus subtilis strains containing gene knockouts affected in either spo0A or ftsH. The Losick group has contributed our new accessions 1S141 and 1S142, which contain erm and spc resistance gene knockouts of spo0A, respectively. Each strain is constructed in a PY79 background, our strain 1A747. The Spo0A protein is of course the master regulator for sporulation and other stationary phase developmental processes. The Schumann group has contributed our new accessions 1A1060 and 1A1061, which contain cat and erm knockouts of ftsH, respectively. Each strain is constructed in a 1012 background, our strain 1A982. The FtsH protein is an integral part of both cell division and general stress adaptation and plays key roles in sporulation and secretion as well. We thank these labs for their generous contributions to the BGSC collection.

Bacillus subtilis  L16601tagE contains a pMUTIN4 insertion in its tagE locus. It is impaired in major cell wall teichoic acid glucosylation. This strain should be grown in the presence of the selective agent erythromycin (0.5 µg/ml), the inducer IPTG (0.5 mM), which insures transcription of the downstream tagF gene, and MgCl2 (1 mM), which helps maintain normal cell wall morphology in the absence of the tagE product. We thank Carlos São José for donating the strain to the BGSC collection.

From the Bill Burkholder lab at Stanford comes a collection of Bacillus subtilis strains engineered to monitor several key physiological states in the cell: the SOS response and DnaA response to DNA damage; the initiation of sporulation, through the accumulation of high levels of Spo0A~P; and the oxidative stress response to the addition of peroxide or other reactive oxygen species. In each strain, a regulated promoter is fused either to lacZ or to a fluorescent reporter gene; this fusion has been integrated ectopically into the amyE locus. We are excited to offer these tools to the research community, and we thank Bill Burkholder, Allison Mo, Steve Biller, and Sharon Hoover for providing them to us.

We thank Xiao-Zhou Zhang and Y.-H. Percival Zhang of Virginia Tech for donating Bacillus subtilis SCK6, a novel strain in which the comK gene has been placed under the control of a xylose-inducible promoter. Very high levels of transformation efficiency are achieved after the addition of xylose, up to 107 cfu/µg for multimeric plasmids and 107 cfu/µg for ligated plasmid DNA. With this system it is now possible for several kinds of experiments to generate libraries directly in B. subtilis without a need for an intermediate host such as Escherichia coli. For more information, consult the reference below. B. subtilis SCK6 is available from the BGSC as 1A976.

Bacillus thuringiensis subsp. israelensis HD522 (=OVR60) was originally isolated from a raw sewage pond of Kibbutz Hulda, Israel in 1977 (1). It is toxic to dipteran larvae, including the horn fly Haematobia irritans (2). A draft genome sequence of HD522 is available NCBI AAJM01000000. We thank Donald H. Dean for supplying us with this strain, which has been accessioned into the BGSC as strain 4Q12.

Bacillus subtilis  BSF2470 is a derivative of 168 that contains a pMUTIN insertion into its liaI locus, placing the lacZ reporter gene under a cell envelope-stress inducible promoter. Antibiotics such as vancomycin, bacitracin, and nisin induce a very strong response via the LiaRS two-component sensory system, while surfactants and organic solvents induce a moderate response (2). This strain allowed Burkard and Stein to develop a microtiter plate bioassay for screening novel compounds that interfere with cell envelope synthesis or integrity (1). We thank the John D. Helmann lab at Cornell for donating this strain to the BGSC.

For a number of years the Erhard Bremer lab at the Philipps-Universität Marburg has been analyzing the stress responses of Bacillus subtilis to changing osmolarity. In nature, a soil microbe like B. subtilis must cope with sudden and unpredictable changes in water availability. To protect against sudden osmotic upshifts, B. subtilis imports potassium ions through dedicated uptake systems. To protect against sudden downshifts, the organism uses mechanosensitive channels that can quickly discharge osmoprotectants that have accumulated in the cell. The Bremer lab has donated seven mutants with knockout mutations in several genes believed to be involved in these processes (see below). We thank the Bremer lab for their generosity!

From 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!

The laboratories of John D. Helmann at Cornell University and Mohammed Marahiel at Philipps-Universität Marburg, Germany, have donated several strains that have greatly expanded the BGSC collection of B. subtilis mutants affected in alternative sigma factor genes. A list of these new strains can be found below. We thank these researchers for their generosity!

Juan C. Alonso of the Campus Universidad Autonoma de Madrid has kindly supplied a collection of knockout mutants impaired in many of the genes implicated as being involved in DNA recombination and repair in Bacillus subtilis. These mutants should prove invaluable for those who wish to study the differences--sometimes subtle and sometimes dramatic--between Gram-positive and Gram-negative model systems in this area of cell function. We thank Dr. Alonso for his kindness.

1. • 1A889 (= BG339) mfd::cat trpC2 metB5 amyE sigB37 xre-1 attSPβ attICEBs1 Cm; see Ayora, S. F., et al. (1996)
2. • 1A890 (= BG775) recJ::six trpC2 metB5 amyE sigB37 xre-1 attSPβ attICEBs1 Cm; see Sanchez, H. D., et al. (2005)
3. • 1A891 (= BG281) recN::cat trpC2 metB5 amyE sigB37 xre-1 attSPβ attICEBs1 recF15 Cm; see Alonso, J.C., et al. (1993)
4. • 1A892 (= BG439) recO::cat trpC2 metB5 amyE sigB37 xre-1 attSPβ attICEBs1 Cm; see Fernández, S., et al. (1999)
5. • 1A893 (= BG705) recQ::six trpC2 metB5 amyE sigB37 xre-1 attSPβ attICEBs1; see Sanchez, H. D., et al. (2005)
6. • 1A894 (= BG425) recS::cat trpC2 metB5 amyE sigB37 xre-1 attSPβ attICEBs1 Cm; see Fernández, S., et al. (1998)
7. • 1A895 (= BG633) recU::six trpC2 metB5 amyE sigB37 xre-1 attSPβ attICEBs1; see Sanchez, H. D., et al. (2005)
8. • 1A896 (= BG703) ruvAB::six trpC2 metB5 amyE sigB37 xre-1 attSPβ attICEBs1; see Sanchez, H. D., et al. (2005)
9. • 1A897 (= BG707) recG::six trpC2 metB5 amyE sigB37 xre-1 attSPβ attICEBs1; see Sanchez, H. D., et al. (2005)
10. • 1A898 (= BG811) sbcC::cat trpC2 metB5 amyE sigB37 xre-1 attSPβ attICEBs1 Cm; Mascarenhas, J., et al. (2006)
11. • 1A899 (= BG551) helD::erm trpC2 metB5 amyE sigB37 xre-1 attSPβ attICEBs1 Em; Alonso, J. C. (unpublished)

Tsutomu Sato of the Tokyo University of Agriculture and Technology has donated to the collection a Bacillus subtilis mutant cured of the prophage-like skin element. The BGSC code for this mutant is 1A884. In B. subtilis 168, the 48-kb skin element interrupts the coding sequence for the sporulation-specific sigma factor, σK, splitting it into two genes, spoIVCB and spoIIIC, that are reassembled during sporulation by the excision of skin in the mother cell chromosome. The skin element contains 57 reading frames, many of them similar in sequence to known temperate phage genes. Also included on skin is an operon involved in the extrusion of toxic arsenical compounds. The \\\\"skinless\\\\" mutant is therefore sensitive to sensitive to arsenate (1 mM) or arsenite (0.5 mM). It sporulates normally, indicating that skin is dispensable for spore formation. We are grateful to Dr. Sato for allowing us to maintain and distribute this interesting mutant.

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:

1. • Bacillus subtilis subsp. spizizenii TU-B-10T (BGSC 2A11T), isolated from the Sahara Desert near Nefta, Tunisia;
2. • Bacillus subtilis subsp. spizizenii DV1-B-1 (= BGSC 2A12), isolated from Death Valley National Monument, California;
3. • Bacillus subtilis AUSI98 (=BGSC 3A26), isolated from a soil sample collected near Salzburg, Austria;
4. • Bacillus subtilis subsp. subtilis RO-NN-1(=BGSC 3A27), isolated from the Mojave Desert near Rosamond, California;
5. • Bacillus vallismortis DV1-F-3 (=BGSC 28A4), isolated from a sand dune with mesquite tree in Death Valley National Monument, California;
6. • Bacillus mojavensis RO-H-1 (=BGSC 28A5), isolated from the Mojave Desert near Rosamond, California

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.

From the laboratory of Prof. M. A. Marahiel at Philipps Universität Marburg come three Bacillus subtilis mutants containing knockouts in cshA and cshB. These genes encode two cold-shock helicase-like proteins. Single knockouts of cshA (strain CB30, BGSC 1A881) and cshB (strain CB40, BGSC 1A882) grow normally at 15°C under laboratory conditions, but a double knockout is lethal. A cshA knockout with a cshA-gfp fusion is also available (CB50, BGSC 1A883). The mutants are described in the paper cited below. We thank Prof. Marahiel for the gift of these strains!

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!

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:

Our thanks to Dr. Wiegert for his generous donation!

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!

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.

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.

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.

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.

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.

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

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.

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 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!

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