News

What makes Bacillus subtilis 168 such a powerful model system? Many factors: a world-wide community of investigators and a 60-year history of focused enquiry; a carefully annotated genome sequence with associated proteomic and transcriptomic data; a suite of useful curated databases making all of this information discoverable and accessible. But what sets B. subtilis apart from most other microbial model systems is a genetic toolbox of great sophistication and variety. One important set of tools are designed to introduce marker-free mutations to the B. subtilis genome. Several technologies are available, including Cre-lox marker loop-out and CRISPR methodologies. But an older technique--allele replacement by temperature-sensitive plasmids--remains highly useful. And the method is potentially extensible to any Gram-positive bacterium with an available plasmid-transformation system, whether by natural competence of by physical methods such as electroporation.

One such vector is pminiMAD2 (also called simply pminiMAD in the research literature). Originally constructed by Patrick and Kearns (2008), this shuttle vector replicates normally in E. coli with selection for ampicillin resistance and in temperature sensitive fashion in Bacillus, where selection is for erythromycin resistance. In Gram-positive hosts, plasmid replication is permissive at normal room temperature but restricted at 37°C. In practice, one simply inserts a fragment from the target chromosome, altered with either a point mutation, deletion, or insertion. The plasmid construct is introduced into the host by transformation, and then selection is maintained as the temperature is raised to 37°C. Single crossovers produce Campbell-type insertion events, where the entire vector is integrated into the chromosome at the target locus, flanked on either side by a normal and mutated copy of the insert. Transformants are next cultured at the permissive temperature in the absence of selection. Several generations of growth, usually at room temperature overnight, allow the plasmid to excise from the genome by hommologous recombination. In many cells, the plasmid will be cured spontaneously, leaving behind either a wild type or mutated form of the target locus in the chromosome. A simple screening step by PCR and sequencing can identify the desired mutant.

The PubMed Central database lists over 40 publications that use pminiMAD2 (or pminiMAD). At least 10 were published during the last two years; they are listed in the article citations below. All describe work in B. subtilis 168 or its wild type ancestor NCIB 3610 with one exception. Spacapan et al. (2018) used pminiMAD2 to introduce a marker-free deletion into an environmental isolate of B. subtilis, PS216 (BGSC accession 3A36). In principle, however, the vector could be used with any mesophilic isolate from Bacillus or related genera.

We thank the Dan Kearns laboratory at Indiana University for donating pminiMAD2 to the BGSC. It is available in an E. coli host as our catalog number ECE765.

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We are pleased to announce the availability of prophage-cured Bacillus subtilis strain Δ6 (BGSC 1A1299). This derivative of strain 168 was deleted of six prophage-like regions in the 168 chromosome, including SPβ, the defective phage PBSX, the skin element, and the prophage 1 and prophage 3 regions, together with the large polyketide synthesis operon (pks). Interestingly, mobile element ICEBs1 was later discovered to have been spontaneously cured (Reuss 2016). As a result of these deletions, the genome size of strain Δ6 has been reduced 8.1% relative to the 168 parent. This strain has demonstrated usefulness as a production platform (Commichau 2014, Juhas 2014, Van Dijl 2013) and as a host for phage studies (Willms 2016, Willms 2017). The genome sequence is publicly available at GenBank accession CP015975. We thank Jan Maarten van Dijl of the University of Groningen for donating strain Δ6 to the BGSC!

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The BGSC is pleased to announce the availability of the Bacillus BioBrick Box 2.0 (Popp et al. 2017), a collection of standardized parts for assembling modules for B. subtilis. These tools include several new plasmid vectors, which are detailed below. They also include a collection of genes encoding fluorescent proteins that as a set can span the entire visible spectrum. This parts collection extends the highly successful BioBrick Box 1.0 (Radeck et al. 2013), which is also available from the BGSC. We thank the Thorsten Mascher lab at TU Dresden for donating this exciting collection of tools!

You can download the plasmid sequences in a zip file here.

Below is a general description of the items in this collection.

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The following vectors replicate in E. coli with selection for ampicillin resistance. They contain an rfp gene in the multiple cloning site to facilitate screening for inserts.

General purpose shuttle vector

pBS0E, supplied in E. coi ECE732

Notes: Replicates in Bacillus from ori-1030 origin of replication with selection for MLS resistance.

Shuttle vectors with inducible promoters

pBS0EP liaI (V2) and pBS0EXylRP xylA (V2), supplied in E. coli ECE742 and ECE743

Notes: The pBS0E shuttle vector, with either the bacitracin-inducible or xylose-inducible promoters upstream from the multiple cloning site.

General purpose integration vectors

pBS1E, pBS1K supplied in E. coli ECE730 and ECE731

Notes: Integrate by double crossover events into the B. subtilis amyE locus with selection for MLS or kanamycin, respectively.

Integration vectors with inducible promoters

pBS2EP xylA (V2), pBS2EP liaI (V2), and pBS2EXylRP xylA (V2), supplied in E. coli ECE739, ECE740, and ECE741

Notes: Integrate by double crossover events into the B. subtilis lacA locus with selection for MLS; with either the bacitracin-inducible or xylose-inducible promoters upstream from the multiple cloning site.

Integration vectors with reporter genes

pBS3Klux and pBS3Elux supplied in E. coli ECE733 and ECE734

Notes: lux-reporter vectors; integrate into B. subtilis lacA locus with selection for kanamycin and MLS, respectively

pBS3Kcatlux and pBS3Ecatlux supplied in E. coli ECE735 and ECE736

Notes: lux-reporter vectors; integrate into B. subtilis lacA locus with selection for kanamycin and MLS, respectively; the promoterless cat gene, encoding chloramphenicol acetyl transferase, serves as a co-selection marker to evaluate the strength of promoters.

pBS1CαlacZ and pBS3Cαlux, supplied in E. coli ECE737 and ECE738

Notes: reporter vectors for evaluating ribosome binding sites for expression in B. subtilis; pBS1CαlacZ integrates into amyE and pBS3Cαlux integrates into sacA. Insertion of a functional RBS into the multiple cloning site, replacing the rfp gene, allows for red-blue-white color screening.

Fluorescent protein genes

The following parts are carried in E. coli plasmids with selection for resistance to chloramphenicol:

mTagBFP (codon usage for E. coli, excitation/emission 399/465) supplied in E. coli ECE744

mTagBFP_Bsu (codon optimized for B. subtilis, excitation/emission 399/465) supplied in E. coli ECE745

eCFP_Bsu (codon optimized for B. subtilis, excitation/emission 449/479) supplied in E. coli ECE746

sfGFP_Spn (codon optimized for S. pneumoniae, excitation/emission 481/511) supplied in ECE747 (RFC10) and ECE748 (RFC25)

GFPmut1 (codon usage for A. victoria, excitation/emission 483/513) supplied in E. coli ECE749

GFPmut1 (LT) (codon optimized for B. subtilis excitation/emission 483/513) supplied in E. coli ECE750

mEYFP (codon usage for E. coli, excitation/emission 500/530) supplied in E. coli ECE751

mEYFP_Bsu (codon optimized for B. subtilis, excitation/emission 500/530 supplied in E. coli ECE752

SYFP2 (codon usage for E. coli, excitation/emission 500/530) supplied in E. coli ECE753 (RFC10) and ECE754 (RFC25)

mCherry (codon usage for E. coli, excitation/emission 585/615) supplied in E. coli ECE755

mCherry_Bsu (codon optimized for B. subtilis excitation/emission 585/615) supplied in E. coli ECE756 (RFC10) and ECE757 (RFC25)

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BGSC strains appeared in at least eight peer-reviewed journal articles in June 2018. We only have space here for the briefest of mentions. Check out the references for ideas about how our strains and genetic tools might be useful in your own research!

Peter Burby (University of Michigan) updated his detailed, very useful protocol for performing CRISPR/Cas9 genome editing in Bacillus subtilis using vectors pPB41 (BGSC No. ECE389) and pPB105 (BGSC ECE390).

Kim Harris (Yale University) used one of our inducible expression vectors to study an OLE (ornate, large, extremophilic) RNA in the moderate halophile Bacillus halodurans. This noncoding RNA and its two accessory proteins are required if the organism is to be tolerant to low temperatures or to short-chain alcohols in the growth medium.

Several articles explore the use of Bacillus and Paenibacillus as biocontrol organisms. Raida Zribi Zghal (University of Sfax) and colleagues investigated the potential of a local B. thuringiensis isolate to control mosquitoes. They used B. thuringiensis servor israelensis wild type (4Q2) and crystal-minus mutant (4Q7) strains from the BGSC for comparison studies.

Other researchers investigated antifungal Bacillus strains. Lamia Abdellaziz, along with colleagues at institutions in Algeria, France, and Belgium, characterized 16 antifungal isolates for their lipopeptide production. They used genome sequence data from two B. thuringiensis strains (BGSC 4BA1 and 4CC1) to assist them in designing screening primers. Ricardo Salvatierra-Martinez, together with colleagues at institutions in Chile and Mexico, likewise studied local antifungal isolates capable of colonizing roots. The used the proven biocontrol agent B. velezensis FZB42 (BGSC 10A6) and two of its mutants (BGSC 10A9 and 10A16) as comparison strains. Ambrin Sarwar and colleagues in Pakistan and Austria also used FZB42 and B. subtilis 168 (BGSC 1A1) in a mass spectrometry analysis of antifungal lipopeptides.

Paenibacillus polymyxa is a plant-growth promoting rhizobacterium. Elizabeth Finch (Queen\'s University Belfast) used BGSC 25A2 to demonstrate that P. polymyxa soil inoculation shifts the nematode population from plant-pathogenic species to predatory species, contributing to plant growth.

Finally, Patricia Calero and Pablo I. Nikel (Technical University of Denmark) reviewed the concept of the “bacterial chassis,” which they define as “the physical, metabolic and regulatory containment for plugging‐in and plugging‐out dedicated genetic circuits and regulatory devices” for the purpose of metabolic engineering. They focus on B. subtilis as a production platform and discuss the BGSC as a source of strains and genetic tools.

We congratulate these teams on their accomplishments and are happy that the BGSC could play a part!

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In recent years, there has been increasing interest in using the Bacillus subtlis endospore as a platform for immobilizing and displaying foreign proteins. The endospore coat is proteinaceus, and the outer layer self-assembles without requiring any transport across membranes. In theory, a very wide range of proteins could be anchored to the spore surface, including enzymes for biotechnology purposes or antigens for developing diagnostic tools. Now researchers in the Thorsten Mascher laboratory at the Technical University of Dresden have developed a set of vectors that greatly facilitate spore surface display [1]. Each of the vectors can be manipulated in E. coli and then integrated into the B. subtilis amyE locus with selection for chloramphenicol resistance. The 12 vectors allow either N- or C-terminal fusions to be constructed with any of the six spore crust proteins, which include CotV, CotW, CotX, CotY, CotZ, and CgeA. Fusions are expressed under a strong sporulation promoter, PcotXY. We are grateful to Julia Bartels and her colleagues in the Mascher lab for donating this set of vectors to the BGSC, and we are excited to make them available to our user community. These vectors are accessioned in the collection under BGSC numbers ECE363-ECE374, inclusive. For more details, please consult the reference below.

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As discovered by John Spizizen in 1958 [1], Bacillus subtilis 168 can become transiently competent to take up DNA from its environment during early stationary phase. Competence is achieved by a minority population within a culture as a consequence of a developmental change known as the K-state, marked by growth cessation, arrest of septum formation, synthesis of DNA-uptake machinery, and activation of recombination-repair systems [2]. Entry of cells into the K-state is under control of the master regulatory protein ComK [3]. Over-expression of ComK leads to the phenomenon of super-competence, in which essentially every cell in a culture stops growing and takes up any DNA in its immediate environment. Removal of the inducer allows growth to resume.

The BGSC has two super-competent lines of B. subtilis. In the first, strain SCK6, the comK gene has been placed under the control of the xylose-inducible promoter PxylA [4]. Addition of xylose to 1% (w/v) to B. subtilis cultures in LB allowed for plasmid DNA transformation frequencies of up to 10^7 with multimeric plasmid preps or 10^4 with ligated plasmid DNA. In the second, strain REG19, comK has been placed under the control of the mannitol-inducible promoter PmtlA, together with a second competence gene, comS [5]. Transformation frequencies were slightly lower than those reported for SCK6, but still much higher than observed with the parental culture under an ordinary competence protocol.

These super-competent lines essentially eliminate technical difficulties associated with standard competence protocols, which generally require closely-monitored growth curves and extended incubation of cultures in two different minimal media. Their high levels of competence allow for simple alteration of chromosomal sequences via amplification fragments, for example by Gibson assembly [5]. Some care must be taken not to allow non-competent mutants to take over stock cultures; super-competent cell lines tend to form smaller colonies on plates (Zhang, personal communication). But their availability should make complicated strain construction projects much simpler for many applications. Strain SCK6 is available from the BGSC under accession number 1A976. Strain REG19 is available under accession number 1A1276.

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Over the weekend we completed a bulk update of our online database. Included are each strain in the BKE knockout library, the CRISPRi knockdown library, the Bacillus subtilis gene expression toolbox, and many more. You should have a greatly increased ability to locate strains and plasmids using our online search engine.

You can enter string of three or more characters into the search box and find strains by their BGSC Code, Original Code, genotype, published reference, and in some cases a GenBank accession ID (we are working to update that last field).

For example: suppose you need a knockout of the B. subtilis xpaC gene. Simply enter xpaC in the search box, press enter, and you will discover that a knockout is available in strain BKE00250. Or suppose you are looking at a 1999 publication by Levin et al., Identification and characterization of a negative regulator of FtsZ ring formation in Bacillus subtilis. You could enter a phrase from the title (FtsZ ring formation) or the PMID for the article (10449747) and discover that we have two of the strains used in this research. Or suppose a BLAST search turns up a sequenced strain with GenBank accession number CP002905. Enter that number in the box, and you will discover we have the strain. Do we have the common lab strain PY79? Enter that name in the box, press enter, and you\'ll find that the answer is yes!

Of course we are always happy to answer inquiries about our holdings or to brainstorm with you about strains or plasmids that might work for your project. Write or call anytime!

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We at the BGSC view supporting STEM education as one of our most important roles. For this reason I want to take a moment to congratulate two gold medalists at the 2016 iGEM competition. If you are not familiar with iGEM (International Genetically Engineered Machine), you should be! This year, over 5000 students in 42 nations participated at the high school, undergraduate, and overgraduate levels. The competition culminated in a jamboree held October 27-31 in Boson, Massachusetts, where over 3000 gathered to share and celebrate their achievements. The BGSC is proud to have supplied strains and advice to two gold medalists. (Please let me know if I am forgetting anyone!) Team Freiburg explored the use of Bacillus subtilis spore display for the targeted delivery of therapeutic drugs, with a test case of immune suppression therapy for ulcerative colitis. For more on their work, see their project website. Team UC Davis asked whether B. subtilis could be engineered to produce natural food colorants. Their proof of concept experiments suggested that cyanobacterial protein pigments could potentially replace Blue dye #1, or Brilliant Blue. For details, see the project website. A shout out to both teams! There are still plenty of Bacillus-related project for future IGEM competitions, and the BGSC is here to help.

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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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