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 ( 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.

B. subtilis 168 gene knockout library available!

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!

New Catalog: The Family Paenibacillaceae

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!

Bacillus subtilis PS216, wild transformable strain

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.

New! Tools for monitoring rrn gene expression

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

Four Marine Bacillus isolates

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.

Newly Sequenced Isolate of Paenibacillus lautus

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!

Visualizing the B. subtilis replication origin

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.