New! Protease-free Bacillus subtilis host
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Reference: Zeigler DR, unpublished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
B. subtilis essential gene knockdown library
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.
pLIKE vector sequences
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.
Low-Noise Fluorescent Protein Vectors
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
Easily transformable mutant of B subtilis NCIB3610
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!
Standardized, reusable B. subtilis tools
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.
Benchmarked GFP variants
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!
pDR244 for Markerless Deletions in the BKE series
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.