pminiMAD, a tool for markerless allele replacement

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.

Prophage-cured Bacillus subtilis strain Δ6

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!

The Bacillus BioBrick Box 2.0

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.

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)

BGSC Journal Club: June 2018

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!