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.

Daptomycin is the first of a new class of cyclic lipopeptide antibiotics used against multidrug-resistant, gram-positive pathogens. The proposed mechanism of action involves disruption of the functional integrity of the bacterial membrane in a Ca 2؉ -dependent manner. We have used transcriptional profiling to demonstrate that treatment of Bacillus subtilis with daptomycin strongly induces the lia operon including the autoregulatory LiaRS two-component system (homologous to Staphylococcus aureus VraSR). The lia operon protects against daptomycin, and deletion of liaH, encoding a phage-shock protein A (PspA)-like protein, leads to threefold increased susceptibility. Since daptomycin interacts with the membrane, we tested mutants with altered membrane composition for effects on susceptibility. Deletion mutations of mprF (lacking lysyl-phosphatidylglycerol) or des (lipid desaturase) increased daptomycin susceptibility, whereas overexpression of MprF decreased susceptibility. Conversely, depletion of the cell for the anionic lipid phosphatidylglycerol led to increased resistance. Fluorescently labeled daptomycin localized to the septa and in a helical pattern around the cell envelope and was delocalized upon the depletion of phosphatidylglycerol. Together, these results indicate that the daptomycin-Ca 2؉ complex interacts preferentially with regions enriched in anionic phospholipids and leads to membrane stresses that can be ameliorated by PspA family proteins.

Bacillus subtilis encodes seven extracytoplasmic function (ECF) factors that regulate partially overlapping regulons related to cell envelope homeostasis and antibiotic resistance. Here, we investigated their physiological role by constructing a mutant set of single, double, triple, and quadruple ECF factor deletions in the undomesticated B. subtilis strain NCIB3610. This mutant set was subsequently screened for defects in motility, multicellular differentiation, and sensitivity to more than 200 chemicals by using Phenotype MicroArrays. A quadruple mutant strain, harboring deletions of the sigV, sigY, sigZ, and ylaC gene, behaved indistinguishably from the wild-type strain, indicative of either regulatory redundancy or very specific functions of these four ECF factors. In contrast, a triple mutant, inactivated for the sigM, sigW, and sigX genes (but none of the corresponding double mutants), showed a biphasic growth behavior and a complete loss of multicellular differentiation, as judged by both colony formation and the inability to form a pellicle. This triple mutant also displayed a greatly increased sensitivity to detergents and several cell wall antibiotics including ␤-lactams, polymyxin B, and D-cycloserine. In several cases, these antibiotic-sensitive phenotypes are significantly enhanced in the triple mutant strain relative to strains lacking only one or two factors.

Daptomycin (DAP) is a cyclic lipopeptide that disrupts the functional integrity of the cell membranes of Gram-positive bacteria in a Ca2؉ -dependent manner. Here we present genetic, genomic, and phenotypic analyses of an evolved DAP-resistant isolate, Dap R 1, from the model bacterium Bacillus subtilis 168. Dap R 1 was obtained by serial passages with increasing DAP concentrations, is 30-fold more resistant than the parent strain, and displays cross-resistance to vancomycin, moenomycin, and bacitracin. Dap R 1 is characterized by aberrant septum placement, notably thickened peptidoglycan at the cell poles, and pleiotropic alterations at both the transcriptome and proteome levels. Genome sequencing of Dap R 1 revealed 44 point mutations, 31 of which change protein sequences. An intermediate isolate that was 20-fold more resistant to DAP than the wild type had only three of these point mutations: mutations affecting the cell shape modulator gene mreB, the stringent response gene relA, and the phosphatidylglycerol synthase gene pgsA. Genetic reconstruction studies indicated that the pgsA(A64V) allele is primarily responsible for DAP resistance. Allelic replacement with wild-type pgsA restored DAP sensitivity to wild-type levels. The additional point mutations in the evolved strain may contribute further to DAP resistance, serve to compensate for the deleterious effects of altered membrane composition, or represent neutral changes. These results suggest a resistance mechanism by which reduced levels of phosphatidylglycerol decrease the net negative charge of the membrane, thereby weakening interaction with the positively charged Ca 2؉ -DAP complex.Daptomycin (DAP) is a cyclic lipopeptide antibiotic used to treat complicated skin and skin structure infections caused by Staphylococcus aureus or enterococci. In addition, it has been approved to treat S. aureus-induced bacteremia and infective endocarditis (21), and animal model studies suggest that it may be a useful alternative for treatment of inhalational anthrax (26). The mechanism of action involves the calcium-dependent insertion of DAP into the bacterial membrane, followed by depolarization of the membrane and extrusion of potassium ions, leading to arrest of macromolecular synthesis and to cell death (49, 51).The introduction of new antibacterial compounds seems to be followed inevitably by the emergence of resistant isolates. It is estimated that over 1 million patients have been treated with DAP (J. Silverman, personal communication). According to the SENTRY Antimicrobial Surveillance Program in the United States for the years 2002 to 2010, 99.9% of methicillin-resistant S. aureus (MRSA) isolates treated with DAP had an MIC of 1.0 g/ml or lower, with only a slight increase of MIC over time (47; http://www.gp-pathogens.com/data/default.cfm).Previous studies to define mechanisms of resistance to DAP were performed on clinical isolates and by in vitro selection (22,31). After serial passages with increasing DAP concentrations, Friedman et al. characterized three...

The transglycosylation step of cell wall synthesis is a prime antibiotic target because it is essential and specific to bacteria. Two antibiotics, ramoplanin and moenomycin, target this step by binding to the substrate lipid II and the transglycosylase enzyme, respectively. Here, we compare the ramoplanin and moenomycin stimulons in the Gram-positive model organism Bacillus subtilis. Ramoplanin strongly induces the LiaRS two-component regulatory system, while moenomycin almost exclusively induces genes that are part of the regulon of the extracytoplasmic function (ECF) factor M . Ramoplanin additionally induces the ytrABCDEF and ywoBCD operons, which are not part of a previously characterized antibiotic-responsive regulon. Cluster analysis reveals that these two operons are selectively induced by a subset of cell wall antibiotics that inhibit lipid II function or recycling. Repression of both operons requires YtrA, which recognizes an inverted repeat in front of its own operon and in front of ywoB. These results suggest that YtrA is an additional regulator of cell envelope stress responses.The bacterial cell wall is a unique and vital molecular sieve. It provides the cell with structural strength and protects it from lysis due to high turgor pressures. This makes the cell wall an important target for many antimicrobial compounds (43). The major component of the cell wall is peptidoglycan (PG), an alternating polymer of the amino sugars N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc). GlcNAc and MurNAc are synthesized inside the cell and incorporated, together with a pentapeptide side chain, into the lipid-linked PG precursor designated lipid II. After lipid II is translocated to the outside cell, the GlcNAc-MurNAc-pentapepide portion is inserted and cross-linked to the existing PG by the transglycosylase (TG) and transpeptidase (TP) activities of high-molecular-weight penicillin binding proteins (HMW PBPs).The TG and TP reactions are both excellent drug targets, because they are essential, are easily accessible, and have no equivalent in eukaryotic cells. The TP reaction is specifically targeted by ␤-lactam antibiotics, which covalently modify the enzyme active site. The TG reaction is inhibited by several classes of antibiotics targeting either the substrate or the enzyme. Moenomycin (MOE) is a glycolipid that targets the active site of the TG enzyme (23). Among the TG inhibitors that bind lipid II, the glycopeptides vancomycin, teicoplanin, and ristocetin bind to the terminal D-Ala-D-Ala of the pentapeptide side chain (21, 30). The glycolipodepsipeptide ramoplanin sequesters lipid II at the interface between the extracellular environment and the bacterial membrane and binds the reducing end of the nascent glycan chain (9, 12, 13). The lantibiotic nisin recognizes the diphospho-sugar portion of lipid II (17). Bacitracin, a cyclic dodecylpeptide antibiotic, binds to the undecaprenyl pyrophosphate released after the TG reaction and thereby inhibits the recycling of the key undecaprenyl phosphate li...

A Tn7 donor plasmid, pTn7SX, was constructed for use with the model gram-positive bacterium Bacillus subtilis. This new mini-Tn7, mTn7SX, contains a spectinomycin resistance cassette and an outward-facing, xylose-inducible promoter, thereby allowing for the regulated expression of genes downstream of the transposon. We demonstrate that mTn7SX inserts are obtained at a high frequency and occur randomly throughout the B. subtilis genome. The utility of this system was demonstrated by the selection of mutants with increased resistance to the antibiotic fosfomycin or duramycin.

The ability to rapidly and accurately determine viral infectivity can help improve the speed of vaccine product development and manufacturing. Current methods to determine infectious viral titers, such as the end-point dilution (50% tissue culture infective dose, TCID50) and plaque assays are slow, labor intensive, and often subjective. In order to accelerate virus quantification, Laser Force Cytology (LFC) was used to monitor vesicular stomatitis virus (VSV) infection in Vero (African green monkey kidney) cells. LFC uses a combination of optical and fluidic forces to interrogate single cells without the use of labels or antibodies. Using a combination of variables measured by the Radiance™ LFC instrument (LumaCyte), an infection metric was developed that correlates well with the viral titer as measured by TCID50 and shortens the timeframe from infection to titer determination from 3 days to 16 h (a 4.5 fold reduction). A correlation was also developed between in-process cellular measurements and the viral titer of collected supernatant, demonstrating the potential for real-time infectivity measurements. Overall, these results demonstrate the utility of LFC as a tool for rapid infectivity measurements throughout the vaccine development process.

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