Construction and Analysis of Two Genome-Scale Deletion Libraries for Bacillus subtilis.

A systems-level understanding of Gram-positive bacteria is important from both an environmental and health perspective and is most easily obtained when high-quality, validated genomic resources are available. To this end, we constructed two ordered, barcoded, erythromycin-resistance- and kanamycin-resistance-marked single-gene deletion libraries of the Gram-positive model organism, Bacillus subtilis. The libraries comprise 3,968 and 3,970 genes, respectively, and overlap in all but four genes. Using these libraries, we update the set of essential genes known for this organism, provide a comprehensive compendium of B. subtilis auxotrophic genes, and identify genes required for utilizing specific carbon and nitrogen sources, as well as those required for growth at low temperature. We report the identification of enzymes catalyzing several missing steps in amino acid biosynthesis. Finally, we describe a suite of high-throughput phenotyping methodologies and apply them to provide a genome-wide analysis of competence and sporulation. Altogether, we provide versatile resources for studying gene function and pathway and network architecture in Gram-positive bacteria.

MurJ and a novel lipid II flippase are required for cell wall biogenesis in Bacillus subtilis.

Bacterial surface polysaccharides are synthesized from lipid-linked precursors at the inner surface of the cytoplasmic membrane before being translocated across the bilayer for envelope assembly. Transport of the cell wall precursor lipid II in Escherichia coli requires the broadly conserved and essential multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) exporter superfamily member MurJ. Here, we show that Bacillus subtilis cells lacking all 10 MOP superfamily members are viable with only minor morphological defects, arguing for the existence of an alternate lipid II flippase. To identify this factor, we screened for synthetic lethal partners of MOP family members using transposon sequencing. We discovered that an uncharacterized gene amj (alternate to MurJ; ydaH) and B. subtilis MurJ (murJBs; formerly ytgP) are a synthetic lethal pair. Cells defective for both Amj and MurJBs exhibit cell shape defects and lyse. Furthermore, expression of Amj or MurJBs in E. coli supports lipid II flipping and viability in the absence of E. coli MurJ. Amj is present in a subset of gram-negative and gram-positive bacteria and is the founding member of a novel family of flippases. Finally, we show that Amj is expressed under the control of the cell envelope stress-response transcription factor σ(M) and cells lacking MurJBs increase amj transcription. These findings raise the possibility that antagonists of the canonical MurJ flippase trigger expression of an alternate translocase that can resist inhibition.

Vibrio cholerae LexA coordinates CTX prophage gene expression.

The filamentous bacteriophage CTX Phi transmits the cholera toxin genes by infecting and lysogenizing its host, Vibrio cholerae. CTX Phi genes required for virion production initiate transcription from the strong P(A) promoter, which is dually repressed in lysogens by the phage-encoded repressor RstR and the host-encoded SOS repressor LexA. Here we identify the neighboring divergent rstR promoter, P(R), and show that RstR both positively and negatively autoregulates its own expression from this promoter. LexA is absolutely required for RstR-mediated activation of P(R) transcription. RstR autoactivation occurs when RstR is bound to an operator site centered 60 bp upstream of the start of transcription, and the coactivator LexA is bound to a 16-bp SOS box centered at position -23.5, within the P(R) spacer region. Our results indicate that LexA, when bound to its single site in the CTX Phi prophage, both represses transcription from P(A) and coactivates transcription from the divergent P(R). We propose that LexA coordinates P(A) and P(R) prophage transcription in a gene regulatory circuit. This circuit is predicted to display transient switch behavior upon induction of CTX Phi lysogens.

LexA represses CTXphi transcription by blocking access of the alpha C-terminal domain of RNA polymerase to promoter DNA.

CTXPhi is a Vibrio cholerae-specific temperate filamentous phage that encodes cholera toxin. CTXPhi lysogens can be induced with DNA damage-inducing agents such as UV light, leading to the release of CTXPhi virions and the rapid dissemination of cholera toxin genes to new V. cholerae hosts. This environmental regulation is directly mediated by LexA, the host-encoded global SOS transcription factor. LexA and a phage-encoded repressor, RstR, both repress transcription from P(rstA), the primary CTXPhi promoter. Because the LexA binding site is located upstream of the core P(rstA) promoter and overlaps with A-tract sequences, we speculated that LexA represses P(rstA) by occluding a promoter UP element, a binding site for the C-terminal domain of the alpha subunit of RNA polymerase (RNAP) (alphaCTD). Using in vitro transcription assays, we have shown that the LexA binding site stimulates maximal rstA transcription in the absence of any added factors. The alphaCTD of RNAP is required for this stimulation, demonstrating that the LexA site contains, or overlaps with, a promoter UP element. LexA represses rstA transcription by normal RNAP but fails to repress rstA transcription catalyzed by RNAP lacking the alphaCTD. DNase I footprint analysis mapped the alphaCTD binding site to the upstream promoter region that includes the LexA binding site. The addition of free alpha subunits blocked the binding of LexA to rstA promoter DNA, indicating that LexA and the alphaCTD directly compete for binding to their respective sites. To our knowledge, this is the first report of a repressor blocking transcription initiation by occluding a promoter UP element.

CTXphi and Vibrio cholerae: exploring a newly recognized type of phage-host cell relationship.

The genes encoding cholera toxin, one of the principal virulence factors of the diarrhoeal pathogen Vibrio cholerae, are part of the genome of CTXphi, a filamentous bacteriophage. Thus, CTXphi has played a critical role in the evolution of the pathogenicity of V. cholerae. Unlike the well-studied F pilus-specific filamentous coliphages, CTXphi integrates site-specifically into its host chromosome and forms stable lysogens. Here we focus on the CTXphi life cycle and, in particular, on recent studies of the mechanism of CTXphi integration and the factors that govern lysogeny. These and other processes illustrate the remarkable dependence of CTXphi on host-encoded factors.

LexA cleavage is required for CTX prophage induction.

The physiologic conditions and molecular interactions that control phage production have been studied in few temperate phages. We investigated the mechanisms that regulate production of CTXphi, a temperate filamentous phage that infects Vibrio cholerae and encodes cholera toxin. In CTXphi lysogens, the activity of P(rstA), the only CTXphi promoter required for CTX prophage development, is repressed by RstR, the CTXvphi repressor. We found that the V. cholerae SOS response regulates CTXvphi production. The molecular mechanism by which this cellular response to DNA damage controls CTXphi production differs from that by which the E. coli SOS response controls induction of many prophages. UV-stimulated CTXphi production required RecA-dependent autocleavage of LexA, a repressor that controls expression of numerous host DNA repair genes. LexA and RstR both bind to and repress P(rstA). Thus, CTXphi production is controlled by a cellular repressor whose activity is regulated by the cell's response to DNA damage.

The CTXphi repressor RstR binds DNA cooperatively to form tetrameric repressor-operator complexes.

CTX is a filamentous bacteriophage that encodes cholera toxin and integrates into the Vibrio cholerae genome to form stable lysogens. In CTX lysogens, gene expression originating from the rstA phage promoter is repressed by the phage-encoded repressor RstR. The N-terminal region of RstR contains a helix-turn-helix DNA-binding element similar to the helix-turn-helix of the cI/Cro family of phage repressors, whereas the short C-terminal region is unrelated to the oligomerization domain of cI repressor. Purified His-tagged RstR bound to three extended 50-bp operator sites in the rstA promoter region. Each of the RstR footprints exhibited a characteristic staggered pattern of DNase I-accessible regions that suggested RstR binds DNA as a dimer-of-dimers. In gel permeation chromatography and cross-linking experiments, RstR oligomerized to form dimers and tetramers. RstR was shown to be tetrameric when bound to operator DNA by performing mobility shift experiments with mixtures of RstR and a lengthened active variant of RstR. Binding of RstR to the high affinity O1 site could be fit to a cooperative model of operator binding in which two RstR dimers associate to form tetrameric RstR-operator complexes. The binding of RstR dimers to the left or right halves of O1 operator DNA was not observed in mobility shift assays. These observations support a model in which protein-protein contacts between neighboring RstR dimers contribute to strong operator binding.

A satellite phage-encoded antirepressor induces repressor aggregation and cholera toxin gene transfer.

CTXphi is a filamentous bacteriophage whose genome encodes cholera toxin, the principal virulence factor of Vibrio cholerae. We have found that the CTXphi-related element RS1 is a satellite phage whose transmission depends upon proteins produced from a CTX prophage (its helper phage). However, unlike other satellite phages and satellite animal viruses, RS1 can aid the CTX prophage as well as exploit it, due to the RS1-encoded protein RstC. RstC, whose function previously was unknown, is an antirepressor that counteracts the activity of the phage repressor RstR. RstC promotes transcription of genes required for phage production and thereby promotes transmission of both RS1 and CTXphi. Antirepression by RstC also induces expression of the cholera toxin genes, ctxAB, and thus may contribute to the virulence of V.cholerae. In vitro, RstC binds directly to RstR, producing unusual, insoluble aggregates containing both proteins. In vivo, RstC and RstR are both found at the cell pole, where they again appear to form stable complexes. The sequestration/inactivation process induced by RstC resembles those induced by mutant polyglutamine-containing proteins implicated in human neurodegenerative disorders.