News

Sarah Guiziou, from the Jerome Bonnet lab at the University of Montpellier, has graciously donated a large set of plasmids and strains comprising a toolkit allowing tunable gene expression in Bacillus subtilis. The amyE integration vectors in the set contain various arrangements of natural promoters, optimized RBS sequences, and protein degradation tags. By fusing the constructs to sfGFP reporters, Guiziou et al. achieved a range of expression corresponding to an average number of GFP molecules per cell varying from 15 to 270000, a span of more than five orders of magnitude. (Some of the higher expression levels result in B. subtilis constructs that look distinctly bright yellow-green under ordinary room lighting!) A complete listing of the plasmids and B. subtilis strains in the set are beyond the scope of this news item, but I encourage you to read the paper. Supplementary data file 1, an Excel spreadsheet detailing expression levels, is especially helpful. We thank Guizhiou and colleagues for these valuable tools.

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Safe simulants that closely mimic the select agent Bacillus anthracis are needed both for laboratory and field studies. B. anthracis belongs to the Bacillus cereus group (BCG) of species. Members of the BCG are nearly identical is cell and spore morphology due to highly similar genome sequence and content. They differ primarily in toxins and virulence factors, many of which are encoded by megaplasmids that differ among isolates. B. thuringiensis (Bt) likewise belongs to the BCG. Some naturally insecticidal Bt strains have been safely used in agriculture for over half a century. Non-insecticidal derivatives of standard Bt strains would seem to make ideal simulants for B. anthracis. For this reason, Alistair Bishop and colleagues at the Defence Science and Technology Laboratory, Salisbury, Wiltshire, UK, have developed plasmid-cured derivatives of Bt kurstaki strain HD1. One of them, HD-1 Cry-, has been demonstrated to be particularly useful in studies of spore aerisolization, dispersal, and decontamination. We thank Dr. Bishop for depositing B. thuringiensis HD-1 Cry- in the BGSC. It is available as BGSC 4D24.

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Some have reported recent problems with the online ordering system. When we receive an order, we always acknowledge it within 24 hours. If you do not hear back from us within that time period, please contact me (zeigler.1@osu.edu) and let me know of the problem.

Thanks!

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Bacillus subtilis is widely used as a production platform for synthesizing enzymes, pharmaceuticals, and fine chemicals (1). Unfortunately, B. subtilis 168 secretes no fewer than seven proteases during vegetative growth and stationary phase. Strains in which multiple protease genes have been inactivated have proved to be superior to wild type strains in production of foreign proteins (2, 3). I have now constructed a seven-protease deletion strain that is free from antibiotic resistance genes or integrated plasmids. This strain, B. subtilis KO7, was generated from the commonly used laboratory host, PY79, by sequentially knocking out the coding sequences. At each step, I transformed the strain with the appropriate BKE cassette for knocking out one of the loci, removing the erythromycin resistance gene with the Cre-producing plasmid pDR244, and finally heat-curing the plasmid. All seven knockouts in KO7 were confirmed by sequencing to be marker-free, in-frame deletions with a 150-bp scar replacing the coding sequence. KO7 is prototrophic and grows rapidly in standard minimal media for B. subtilis. I am placing strain KO7 in the public domain and disclaim all downstream rights to any process or product that you develop with it. It will be available from the BGSC as accession number 1A1133. Standard user fees apply. Later this summer I hope to introduce further elaborations of KO7, such as restriction-negative and asporogenous variants. Feel free to put in requests for particular features!

BGSC Accession: 1A1133

Original Code: Bacillus subtilis KO7

Reference: Zeigler DR, unpublished

Genotype: ΔnprE ΔaprE Δepr Δmpr ΔnprB Δvpr Δbpr

Description: Free of secreted proteases; marker-free deletions in PY79 genetic background; prototrophic

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The BGSC is excited to announce the availability of a new collection of Bacillus subtilis 168 mutants designed to explore the functions of 289 essential genes in this organism. The paper describing the construction of this library and its initial characterization will be released online today (26 May 2016) and will appear in the June 2 edition of Cell. The paper is a collaboration among labs at the University of California, San Francisco, Stanford University, University of California, Berkeley, and McMaster University, Hamilton, Ontario. The co-first authors are Jason M. Peters of UCSF and Alexandre Colavin and Handuo Shi of Stanford.

The library uses a CRISPR interference (CRISPRi) strategy to created a tunable “knockdown” of individual essential genes. Every strain in the library has a Streptococcus pyogenes dcas9 gene integrated into the B. subtilis lacA locus, where it has been placed under control of the xylose-inducible Pxyl promoter. Each strain also has a single-guide RNA (sgRNA) targeting a specific essential gene. The sgRNA coding sequence is integrated into B. subitlis amyE, where it has been placed under the control of the strongly constitutive Pveg promoter. The dCas9 protein lacks nuclease activity. But when dCas9 is present, the sgRNA enables it to bind to the 5’ end of the target gene, where it effectively blocks transcription via steric hindrance. Basal level expression of dcas9 in the absence of xylose knocks down expression of the targeted essential gene about 3-fold. This reduction creates subtle phenotypes, such as increased sensitivity to specific antibiotics and chemical inhibitors, but allows for essentially normal growth under standard laboratory conditions. Full induction of dcas9 with xylose (1%) reduces expression of the essential gene ~150-fold, with drastic consequences for cell morphology and viability. Varying the concentration of xylose between 0.001% and 0.1% allows tunable expression of the essential gene. Peters et al. have not only reported the construction of the library, but have demonstrated its power for analyzing essential genes. They used chemical genomics, for example, to reveal the essential gene network of B. subtilis, revealing interesting connections between seemingly unrelated processes.

These strains provide an invaluable tool for a systematic study of essential genes in a bacterial model system. We thank Jason Peters, Carol Gross, and the entire consortium for donating the library to the BGSC, and we look forward to supplying strains from it to scientists from the B. subtilis research community and beyond. For a complete list of the genes targeted in the library, please see the Peters et al. publication. Summaries of what has been learned previously about most of these genes can be accessed at SubtiWiki. It will take a little while for us to update the BGSC online database to include these strains, but they are available for immediate distribution. But their naming convention is simple. The numeric portion of the gene’s locus tag is appended to the prefix “BEC” to produce the strain name. Hence the knockdown strain for the essential gene ligA, which encodes DNA ligase and carries the locus tag BSU06620, is BCE06620. The full genotype of this strain is lacA::Pxyl-dcas9 amyE::Pveg-sgRNA(ligA) trpC2, and it carries resistance markers for erythromycin and chloramphenicol. Users may request these strains by giving us the targeted gene name or locus tag. Standard user fees apply.

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Over the past two years, there has been considerable interest in the pLIKE vectors from the Thorsten Mascher lab at Ludwig-Maximilians-University (LMU) Munich. These expression vectors feature a bacitracin-inducible promoter, available both in a replicative plasmid (our ECE255) and an amyE-integration vector (ECE256). For the convenience of our users, we have uploaded maps and DNA sequences of these plasmids onto our website. You can download them here. Thanks to Prof. Mascher for providing this document.

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Some of the most useful tools for Bacillus geneticists are integrative vectors designed to fuse a promoter of interest to a coding sequence specifying a fluorescent protein. Strains bearing these vectors are among the most widely requested items in the BGSC collection, and improved versions offering some advantage over first generation vectors have regularly appeared on this page. Such is the case today, as we feature the pXFP_Star reporter system, developed and kindly donated by Stephanie Trauth and Ilka B. Bischofs in the Bischofs lab at the University of Heidelberg. These vectors, which come in green, cyan, and yellow “flavors,” feature extremely low noise. A B. subtilis strain with an empty pXFP_Star vector integrated into its chromosome has no greater background fluorescence than the host strain alone. This significant improvement was achieved by including a strong transcription terminator just upstream of the promoter-cloning site. The pXFP_Star vectors will be useful for studying all kinds of promoters, but should be especially advantageous for analyzing those that are weakly expressed. As a bonus, they also are designed to allow ligation independent cloning (LIC). The vectors are available in the following BGSC strains. Note that the NCBI sequence files may not be released for a few weeks, but in the meantime are available from the BGSC upon request.

ECE295 | pGFP_Star | KJ411636

ECE296 | pYFP_Star | KJ411637

ECE297 | pCFP_Star | KJ411638

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The well-studied Gram-positive model organism, Bacillus subtilis 168, is a highly domesticated descendent of type strain of the species, B. subtilis Marburg. Forensic sequencing evidence (Zeigler 2008) suggests that the wild type strain was passaged multiple times in the laboratory, acquiring a few key mutations that eliminated swarming and motility behaviors, allowing for more uniform, regular colonies to grow on agar (Aguilar 2007). This domesticated Marburg was further adapted to life in the laboratory by selecting for variants that showed improved growth on glucose-ammonium minimal medium (Burkholder, Giles 1947). Finally, this variant was subjected to X-ray mutagenesis, resulting in a tryptophan-requiring mutant, strain 168 (Burkholder, Giles 1947). When Spizizen discovered that it could develop natural genetic competence during early stationary phase (Spizizen 1958), strain 168 quickly emerged as the organism of choice for genetic studies and eventually for biotechnology applications.

Curiously, the wild type parent for strain 168, B. subtilis NCIB 3610 (BGSC accession 3A1), is only weakly competent for transformation--about 1000-fold less than its domesticated offspring. This deficiency is unfortunate, because in recent years much attention has been focused on the wild type strain. Strain NCIB 3610 is capable of many social behaviors that require a sophisticated degree of mulicelluarity, including biofilm formation, swarming motility, and cell signaling (Kearns 2010; Shank, Kolter 2011; Romero 2013; Vlamakis 2013). A highly competent derivative of NCIB 3610 that nevertheless retain multicellarity would be a boon to research aimed at elucidating these behaviors. Such a strain has now been described. During its domestication or mutagenesis, strain 168 lost an 84-kb endogenous plasmid that is native to NCIB 3610 (see GenBank: KF365913.1 (Konkol 2013). This plasmid encodes a 30-amino acid peptide, ComI, that is largely responsible for the inhibition of competence development in strain NCIB 3610. Strain DK1042 is identical with NCIB except for a single point mutation, comI(Q12L), that inactivates the inhibitor and increases competence 100-fold (Konkol 2013).

The Dan Kearns laboratory at Indiana University, who constructed this mutant, has now kindly donated it to the BGSC. We have accessioned it as Bacillus subtilis 3A38. We anticipate that BGSC 3A38 will be of great use to those who wish to dissect the genetics of mulicelluarity in B. subtilis. We thank the Kearns lab for making it available!

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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.

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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!

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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.

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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!

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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!

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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.

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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

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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.

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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!

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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.

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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

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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.

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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.

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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.

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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.

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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.

BGSC	Original	Background	Promoter	Reporter	Monitors	Reference
1A1040	SB33	JH642	PtagC	YFP	SOS response	(2)
1A1013	AM48	JH642	PyneA	LacZ	SOS response	(1)
1A1009	SH507	JH642	PdnaA	LacZ	DnaA response	(4)
1A1036	KM81	JH642	PspoIIE	GFP	sporulation initiation	(4)
1A1034	BB827	JH642 Δsda	PspoIIE	LacZ	sporulation initiation	(6)
1A1033	BB825	JH642	PspoIIE	LacZ	sporulation initiation	(6)
1A1011	SH536	JH642	PkatA	LacZ	oxidative stress	(4)
1A1010	SH517	JH642	PkatA	GFP	oxidative stress	(4)
1A96	JH642	JH642	-	-	negative control	(3)
1A1014	AM62	JH642	-	LacZ	negative control	(5)

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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.

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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.

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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.

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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!

BGSC	Strain	Locus	Reference
1A954	GHB1	ktrAB	(2)
1A955	GHB6	ktrC	(2)
1A956	GHB12	ktrD	(2)
1A957	SMB53	mscL	(1)
1A958	SMB58	yhdY	(1)
1A959	SMB62	yfkC	(1)
1A960	SMB63	ykuT	(1)

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