News

The BGSC is pleased to announce the acquisition of a toolbox of standardized, reusable building blocks for Bacillus subtilis genetics. The Thorsten Mascher lab at Ludwig-Maximilians-Universität München, Germany, has constructed several vector tools following the BioBrick standard. Full details are available in the cited reference, including sequence files in the online supporting material. For convenience, I am including a brief description of these tools.

First, the Bacillus BioBrick Box includes five integration vectors. Each vector contains flanking homology regions for integration into the B. subtilis chromosome; a resistance cassette for selection of integrants; and a multiple cloning site that contains EcoRI, NotI, and XbaI upstream from an rfp cassette, and SpeI, NotI, and PstI downstream from the cassette. The rfp cassette turns the E. coli host bright red unless the cassette is removed by cloning within the multiple cloning site, making it simple to screen for recombinants. Except for pBS2E, all can be linearized by ScaI digestion prior to transformation into B. subtilis. Vector pBS2E can be linearized with BsaI or PciI digestion prior to transformation. The five vectors include:

pBS1C (BGSC accession ECE257), an empty vector that integrates into amyE with chloramphenicol selection;

pBS2E (ECE258), an empty vector that integrates into lacA with erythromycin selection;

pBS4S (ECE259), an empty vector that integrates into thrC with spectinomycin selection;

pBS1ClacZ (ECE260), for integration of lacZ reporter into amyE with chloramphenicol selection;

pBS3Clux (ECE261), for integration of luxABCDE (luciferase) reporter into sacA with chloramphenicol selection.

Second, the Box includes six promoters. Each has been cloned into the EcoRI and SpeI sites of the plasmid backbone pSB1C3 (http://parts.igem.org/Part:pSB1C3). These plasmids are chloramphenicol-resistant in E. coli and do not replicate in B. subtilis or other Gram-positives.

Four of the promoters are constitutive in B. subtilis:

Pveg (ECE262), very strong constitutive promoter;

PliaG (ECE263), constitutive promoter;

PlepA (ECE264), strong constitutive promoter;

J23101 (ECE266), very weak constitutive promoter (although strong in E. coli)

Two of the promoters are inducible in B. subtilis:

PliaI (ECE267), bacitracin-inducible promoter;

PxylA (ECE268), xylose-inducible promoter

Finally, the Box contains five commonly used epitope tags. Each epitope tag fragment has likewise been cloned into the EcoRI and SpeI sites of pSB1C3.

His10 (ECE269), for tagging proteins with 10xHis;

FLAG (ECE270), for tagging proteins with FLAG;

StrepII (ECE271), for tagging proteins with Streptactin;

HA (ECE272), for tagging proteins with HA;

cMyc (ECE273), for tagging proteins with cMyc

These tools should greatly facilitate a wide variety of genetic experiments for B. subtilis. We are grateful to the Mascher lab for sharing them with us, with special thanks to Jara Radeck for her patient work in preparing the plasmids for the BGSC and answering questions about them.

The laboratory of Jan-Willem Veening at the University of Groningen have kindly provided a collection of Green Flourescent Protein (GFP) vectors that have been benchmarked for performance in Bacillus subtilis, Streptococcus pneumoniae, and Lactococcus lactis. Seven GFP variants were selected for analysis (see below).

The seven gfp variant genes were inserted into each of three plasmid constructs. For B. subtilis, each gene was placed under the control of the IPTG-inducible Phyperspank promoter in the amyE integration vector pDR111. For S. pneumoniae, each gene was placed under the control of the Zn(++)-inducible promoter PZn in a bgaA integration vector. For L. lactis, each gene was placed under the control of a constitutive promoter in the pseudo 10 integration vector pSEUDO.

The seven benchmarked GFP variants are as follows (with more information available in Table 2 and in the main text of the publication):

GFPmut1; optimized for original A. victoria; 35-fold brighter than wild GFP

GFP+; optimized for E. coli; 130-fold brighter than wild GFP

GFP+(htrA); optimized for E. coli; improved translation efficiency for S. pneumoniae

GFP(Sp); optimized for S. pneumoniae; functions as monomer

sfGFP(Bs); optimized for B. subtilis; superfolder

sfGFP(Sp); optimized for S. pneumoniae; superfolder

sfGFP(iGEM); optimized for B. subtilis/E. coli compromise; superfolder

Interestingly, Overkamp et al. found that the variant producing the strongest signal in B. subtilis---whether in planktonic cells or biofilms---was GFP(Sp), which had been optimized with S. pneumoniae codon usage. Conversely, the variant producing the strongest signal in S. pneumoniae and L. lactis was sfGFP(Bs), which had been optimized for B. subtilis. The entire collection of 21 plasmids is available from the BGSC in our strains ECE275 through ECE295A. Probably the most heavily used plasmids will be pDR111_GFP(Sp), found in our E. coli host ECE278; pKB01_sfGFP(Bs), found in our ECE286; and pSEUDO::Pusp45-sfGFP(Bs), found in our ECE293. They provide the strongest signals in B. subtilis, S. pneumoniae, and L. lactis, respectively. But the entire collection may find a use in future optimization experiments for related species and for other Gram-positive organisms. We thank the Veening lab for their generosity!

Last year we announced the availability of a knockout library for Bacillus subtilis 168. This BKE series includes over 4000 strains. In each of them a single gene has been deleted and replaced with an erythromycin resistance cassette. We are also pleased to offer a vector, pDR244, that makes it a simple matter to loop out the BKE cassette, leaving behind only a small scar consisting of bar code and universal primer sequences. The plasmid contains a functional cre gene that catalyzes a site-specific recombination between the lox sites that flank the BKE cassette. Because it is based on the pE194-ts replicon, pDR244 is quickly cured from its B. subtilis host by growing it at 42°C. Using a BKE strain together with pDR224 makes it possible to generate a markerless deletion in only a few days with a minimum amount of hands-on effort.

A map of pDR244 and a summary of the steps required to generate a markerless deletion can be found here. Plasmid pDR244 is available in the BGSC E. coli strain ECE274.

The BGSC is excited to announce the availability of the newly constructed gene knockout library for Bacillus subtilis 168. As many of you know, Byoung-Mo Koo and colleagues in the Carol Gross laboratory at the University of California, San Francisco assisted by members of David Rudner’s laboratory at Harvard Medical School have been constructing this and a similar library for the past couple of years. The work has been performed under a Grand Opportunity grant awarded to a consortium led by David Rudner. Dr. Koo, along with all those involved in the project, have generously consented to deposit one of these libraries in the BGSC collection prior to publication.

The library currently comprises nearly 4000 strains, each containing an erythromycin-resistance cassette inserted into one of the non-essential genes in the B. subtilis 168 genome. (The library will be augmented with a few additional strains in the near future.) Very soon we will upload data concerning these strains in our online searchable database. The strains are already available for distribution, however. The strains follow a simple naming scheme. Each begins with “BKE” (Bacillus Knockout Erythromycin), prefixed to the locus number of the knocked-out gene. Suppose, for example, one wished to obtain a spo0A::erm knockout. The spo0A gene has the locus_tag “BSU24220”. The library strain with this knockout (along with trpC2, of course) is called “BKE24220.” If you wish to request this strain, you could either request strain BKE24220 or ask for our spo0A knockout from the BKE library. Please note that the BGSC does not have the capacity to distribute the entire collection or any large subsets of it. But we are ready and able to send individual strains or smaller sets in keeping with our normal distribution policy.

We thank Byoung-Mo Koo, along with the Gross and Rudner labs for making this library available to the Bacillus subtilis research community!

The BGSC is pleased to announce the online publication of part 5 of its catalog of strains, The Family Paenibacillaceae. Our holdings of Paenibacillaceae are not large--only 45 strains at the time of pulbication. Interest in these bacteria is high, however. Included in our collection are members of the genera Paenibacillus, Brevibacillus, and Aneurinibacillus. They include isolates that have been studied for plant growth promotion, biocontrol of invertebrate pests, bacterial colony pattern formation, and bacterial taxonomy. Please take a moment to download the catalog if any of these topics is of interest to you. We welcome corrections to any mistakes you find. We also invite you to submit any well-characterized isolates of Paenibacillaceae to our collection. More new catalogs and updates to older ones will be appearing soon!

One of the more interesting developments in Bacillus subtilis genetics over the past decade is the application of lessons learned with the model organism, strain 168 (=BGSC 1A1), to wild relatives, especially strain NCIB 3610 (=BGSC 3A1), a direct descendant of the 168 parental strain. Strain 3610 has a variety of important phenotypes that have been lost in the domesticated strain 168, including swarming, biofilm formation, and development of complex colony architecture. Unfortunately, strain 3610 is poorly competent, primarily due to a plasmid-encoded competence antagonist, ComI (Konkol 2013), making genetic analysis of these wild phenotypes much more difficult. A novel environmental isolate, B. subtilis PS216 (=BGSC 3A36), offers a way to circumvent this problem. This strain, isolated from sandy soil sample near River Sava, Slovenia (46°06′N, 14°28′E) in January 2006, shows wild phenotypes comparable to strain 3610 yet acquires natural competence at levels similar to strain 168 (Stefanic 2009). A draft genome sequence of strain PS216 is now available (Durrett 2013). Strain PS216 lacks the large plasmid that bears the comI gene in strain 3610; it also lacks the prophage SPβ and the mobile element ICEBs1. Otherwise, there are only 140 SNPs distinguishing PS216 from strain 3610. In four important regulatory genes that have been altered during the domestication of 168--oppD, comP, degQ, and sigH--strain PS216 shows an identical sequence to that of 3610. Strain PS216 should offer a useful comparison for strain 3610. We are pleased to offer this strain (our BGSC 3A36) and thank Drs. Dubnau and Mandic-Mulec for donating it to our collection.

Alex Rosenberg of the Ben-Yehuda laboratory at The Hebrew University of Jerusalem has deposited a set of Bacillus subtilis strains in which operons of the translational machinery can be monitored at a single cell level by GFP fusions. This set includes transcriptional fusions to promoters for seven rRNA operons and one ribosomal protein gene, as well as a translational fusion to ribosomal protein L1 (see below). All are constructed in a PY79 (= BGSC 1A747) genetic background. Using these tools, Rosenberg et al. (2012) were able to monitor the expression of each of the major rRNA operons and the synthesis of ribosomal proteins during the B. subtilis life cycle in a variety of media, even visualizing synthesis in the developing forespore. We are pleased to offer this set of strains to the Bacillus genetics community, and we thank Rosenberg and colleagues for sharing them with us.

BGSC | Original Code | Genotype

1A1088 | B. subtilis AR5 | rplA-gfpmut2-spc Sp

1A1089 | B. subtilis AR13 | amyE:PrrnA-gfpmut2-spc Sp

1A1090 | B. subtilis AR14 | amyE:PrrnB-gfpmut2-spc Sp

1A1091 | B. subtilis AR15 | amyE:PrrnD-gfpmut2-spc Sp

1A1092 | B. subtilis AR16 | amyE:PrrnE-gfpmut2-spc Sp

1A1093 | B. subtilis AR17 | amyE:PrrnO-gfpmut2-spc Sp

1A1094 | B. subtilis AR18 | amyE:PrrnI-gfpmut2-spc Sp

1A1095 | B. subtilis AR19 | amyE:PrrnJ-gfpmut2-spc Sp

1A1096 | B. subtilis AR25 | amyE:PrplA-gfpmut2-cm Cm

We announce the addition of four marine Bacillus isolates to the BGSC collection. Actually, one of the strains, B. vietnamensis 15-1T (our 63A1T), was isolated from a traditional Vietnamese fish sauce, nuoc mam, but it is closely related to other strains isolated from marine waters (Noguchi 2004). The remaining strains were isolated directly from seawater: B. aquimaris TF-12T (64A1T) and B. marisflavii TF11T (65A1T) from a tidal flat in the Yellow Sea, Korea (Yoon 2003); and Bacillus sp. NRRL B-14850 (64A2) from the Gulf of Mexico (Siefert 2000). Each of these isolates forms pigmented colonies, ranging from light yellow (64A1T and 65A1T) to deep orange (63A1T and 64A2). This type of pigmentation, caused by carotenoids, has been associated with UV resistance in related environmental isolates (Khaneja 2010). Some of these strains are moderately halotolerant (63A1T, 64A1T, and 65A1T) or alkalitolerant (63A1T), while others are not (64A2). Marine Bacillus isolates are less studied than their terrestrial counterparts, but their role in their environment and their largely untapped biotechnological potential make them attractive candidates for further research.

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!