SUMMARY 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 3968 and 3970 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.

SummaryCTX is a filamentous phage that encodes cholera toxin, one of the principal virulence factors of Vibrio cholerae. CTX is unusual among filamentous phages because it can either replicate as a plasmid or integrate into the V. cholerae chromosome at a specific site. The CTX genome has two regions, the 'core' and RS2. Integrated CTX is frequently flanked by an element known as RS1 which is related to RS2. The nucleotide sequences of RS2 and RS1 were determined. These related elements contain three nearly identical open reading frames (ORFs), which in RS2 were designated rstR, rstA2 and rstB2. RS1 contains an additional ORF designated rstC. Functional analyses indicate that rstA2 is required for CTX replication and rstB2 is required for CTX integration. The amino terminus of RstR is similar to the amino termini of other phageencoded repressors, and RstR represses the expression of rstA2. Although genes with related functions are clustered in the genome of CTX in a way similar to those for other filamentous phages, the CTX RS2-encoded gene products mediating replication, integration and repression appear to be novel.

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 (murJ Bs ; formerly ytgP) are a synthetic lethal pair. Cells defective for both Amj and MurJ Bs exhibit cell shape defects and lyse. Furthermore, expression of Amj or MurJ Bs 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 MurJ Bs 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.T he bacterial cell wall or peptidoglycan (PG) is composed of glycan strands cross-linked together by short peptides. This 3D meshwork protects the cell from osmotic lysis and determines shape, and its assembly is the target of some of our most successful antibiotics. Cell wall synthesis begins in the cytoplasm, where a set of highly conserved enzymes catalyze the formation of the lipid-linked precursor lipid II, which is composed of undecaprenyl-pyrophosphate (UndPP) linked to N-acetylglucosamine-N-acetylmuramic acid pentapeptide. Lipid II is synthesized on the inner face of the cytoplasmic membrane (1). The molecule is then translocated to the outer face of the membrane, where the disaccharide-peptide monomer is incorporated into the existing PG by cell wall synthetic machineries composed of penicillin-binding proteins and additional factors (2). The enzymes that transport lipid II across the membrane have been the subject of extensive research and speculation (3-6). Recent work in Escherichia coli has provided strong evidence that the polytopic membrane protein MurJ is required for lipid II transport across the membrane, and is likely to be a lipid II flippase (6). MurJ is a member of the multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) exporter superfamily (7). It is broadly conserved among Eubacteria and essential for viability in many organisms. Intriguingly, work in the model gram-positive bacterium Bacillus subtilis indicates that cells lacking four MOP superfamily members similar to MurJ are viable and have...

SummaryIn Escherichia coli, precursor proteins are targeted to the membrane-bound translocase by the cytosolic chaperone SecB. SecB binds to the extreme carboxyterminus of the SecA ATPase translocase subunit, and this interaction is promoted by preproteins. The mutant SecB proteins, L75Q and E77K, which interfere with preprotein translocation in vivo, are unable to stimulate in vitro translocation. Both mutants bind proOmpA but fail to support the SecA-dependent membrane binding of proOmpA because of a marked reduction in their binding affinities for SecA. The stimulatory effect of preproteins on the interaction between SecB and SecA exclusively involves the signal sequence domain of the preprotein, as it can be mimicked by a synthetic signal peptide and is not observed with a mutant preprotein (⌬8proOmpA) bearing a non-functional signal sequence. ⌬8proOmpA is not translocated across wild-type membranes, but the translocation defect is suppressed in inner membrane vesicles derived from a prlA4 strain. SecB reduces the translocation of ⌬8proOmpA into these vesicles and almost completely prevents translocation when, in addition, the SecB binding site on SecA is removed. These data demonstrate that efficient targeting of preproteins by SecB requires both a functional signal sequence and a SecB binding domain on SecA. It is concluded that the SecB-SecA interaction is needed to dissociate the mature preprotein domain from SecB and that binding of the signal sequence domain to SecA is required to ensure efficient transfer of the preprotein to the translocase.