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Many bacteria adapt their physiology and enter the viable but non-culturable state to survive prolonged exposure to adverse environmental conditions. The VBNC cells maintain active metabolism, membrane integrity and gene transcription. However, they lose the ability to form colonies on a conventional culture media. Thus, standard colony counting methods cannot detect these alive but dormant cells. The Gram-positive bacterium Bacillus subtilis was found to enter the VBNC state when pre-exposed to osmotic stress and treated with a lethal dose of kanamycin. These cells reduced their metabolic activity, ceased growth and division and became kanamycin-tolerant. Interestingly, despite active metabolism, the majority of the kanamycin tolerant cells could not be revived on LB agar. In this study, we use a robust RNA-Seq technique to elucidate the differences in transcriptional profiles of B. subtilis VBNC cells. A comparative analysis of differently expressed genes and operons performed in this study indicates high similarities in transcriptional responses of VBNC and kanamycin-sensitive cells to antibiotic treatment. Moreover, this work reveals that VBNC cells strongly upregulate genes involved in proline uptake and catabolism, suggesting a putative role of proline as nutrient in VBNC cells.

Most bacteria replicate and segregate their DNA concomitantly while growing, before cell division takes place. How bacteria synchronize these different cell cycle events to ensure faithful chromosome inheritance by daughter cells is poorly understood. Here, we identify Cell Cycle Regulator protein interacting with FtsZ (CcrZ) as a conserved and essential protein in pneumococci and related Firmicutes such as Bacillus subtilis and Staphylococcus aureus. CcrZ couples cell division with DNA replication by controlling the activity of the master initiator of DNA replication, DnaA. The absence of CcrZ causes mis-timed and reduced initiation of DNA replication, which subsequently results in aberrant cell division. We show that CcrZ from Streptococcus pneumoniae interacts directly with the cytoskeleton protein FtsZ, which places CcrZ in the middle of the newborn cell where the DnaA-bound origin is positioned. This work uncovers a mechanism for control of the bacterial cell cycle in which CcrZ controls DnaA activity to ensure that the chromosome is replicated at the right time during the cell cycle., In this Article, the authors identify CcrZ as a protein that coordinates DNA replication and cell division in Streptococcus pneumoniae and other Firmicutes. CcrZ is localized to the division site by binding directly to the divisome protein FtsZ, and there it activates DnaA, the master initiator of DNA replication, through a still unknown mechanism.

The ParABS system is essential for prokaryotic chromosome segregation. After loading at parS on the genome, ParB (partition protein B) proteins rapidly redistribute to distances of ~15 kilobases from the loading site. It has remained puzzling how this large-distance spreading can occur along DNA loaded with hundreds of proteins. Using in vitro single-molecule fluorescence imaging, we show that ParB from Bacillus subtilis can load onto DNA distantly of parS, as loaded ParB molecules themselves are found to be able to recruit additional ParB proteins from bulk. Notably, this recruitment can occur in cis but also in trans, where, at low tensions within the DNA, newly recruited ParB can bypass roadblocks as it gets loaded to spatially proximal but genomically distant DNA regions. The data are supported by molecular dynamics simulations, which show that cooperative ParB-ParB recruitment can enhance spreading. ParS-independent recruitment explains how ParB can cover substantial genomic distance during chromosome segregation, which is vital for the bacterial cell cycle.

The widely conserved twin-arginine translocases (Tat) allow the transport of fully folded cofactor-containing proteins across biological membranes. In doing so, these translocases serve different biological functions ranging from energy conversion to cell division. In the Gram-positive soil bacterium Bacillus subtilis, the Tat machinery is essential for effective growth in media lacking iron or NaCl. It was previously shown that this phenomenon relates to the Tat-dependent export of the heme-containing peroxidase EfeB, which converts Fe2+ to Fe3+ at the expense of hydrogen peroxide. However, the reasons why the majority of tat mutant bacteria perish upon dilution in NaCl-deprived medium and how, after several hours, a sub-population adapts to this condition was unknown. Here we show that, upon growth in the absence of NaCl, the bacteria face two major problems, namely severe oxidative stress at the membrane and starvation leading to death. The tat mutant cells can overcome these challenges if they are fed with arginine, which implies that severe arginine depletion is a major cause of death and resumed arginine synthesis permits their survival. Altogether, our findings show that the Tat system of B. subtilis is needed to preclude severe oxidative stress and starvation upon sudden drops in the environmental Na+ concentration as caused by flooding or rain.

Sublancin 168 is a highly potent and stable antimicrobial peptide secreted by the Gram-positive bacterium Bacillus subtilis. Production of sublancin gives B. subtilis a major competitive growth advantage over a range of other bacteria thriving in the same ecological niches, the soil and plant rhizosphere. B. subtilis protects itself against sublancin by producing the cognate immunity protein SunI. Previous studies have shown that both the sunA gene for sublancin and the sunI immunity gene are encoded by the prophage SPβ. The sunA gene is under control of several transcriptional regulators. Here we describe the mechanisms by which sunA is heterogeneously expressed within a population, while the sunI gene encoding the immunity protein is homogeneously expressed. The key determinants in heterogeneous sunA expression are the transcriptional regulators Spo0A, AbrB and Rok. Interestingly, these regulators have only a minor influence on sunI expression and they have no effect on the homogeneous expression of sunI within a population of growing cells. Altogether, our findings imply that the homogeneous expression of sunI allows even cells that are not producing sublancin to protect themselves at all times from the active sublancin produced at high levels by their isogenic neighbors. This suggests a mutualistic evolutionary strategy entertained by the SPβ prophage and its Bacillus host, ensuring both stable prophage maintenance and a maximal competitive advantage for the host at minimal costs.

Di- and tricarboxylates found as intermediates in the tricarboxylic acid cycle can be utilized by many bacteria and serve as carbon and energy source under aerobic and anaerobic conditions. A prerequisite for metabolism is that the carboxylates are transported into the cells across the cytoplasmic membrane. Bacillus subtilis is able to metabolize many di- and tricarboxylates and in this overview the available data on all known and putative di- and tricarboxylate transporters in B. subtilis is summarized. The B. subtilis transporters, that are of the secondary type, are discussed in the context of the protein families to which they belong. Available data on biochemical characterization, regulation of gene expression and the physiological function is summarized. It is concluded that in B. subtilis multiple transporters are present for tricarboxylic acid cycle intermediates.

The genome of Bacillus subtilis contains two genes that code for membrane proteins that belong to the 2-hydroxycarboxylate transporter family. Here we report the functional characterization of one of the two, yxkJ , which codes for a transporter protein named CimHbs. The gene was cloned and expressed in Escherichia coli and complemented the citrate-negative phenotype of wild-type E. coli and the malate-negative phenotype of the E. coli strain JRG4008, which is defective in malate uptake. Subsequent uptake studies in whole cells expressing CimHbs clearly demonstrated the citrate and malate transport activity of the protein. Immunoblot analysis showed that CimHbs is a 48-kDa protein that is well expressed in E. coli . Studies with right-side-out membrane vesicles demonstrated that CimHbs is an electroneutral proton-solute symporter. No indications were found for the involvement of Na + ions in the transport process. Inhibition of the uptake catalyzed by CimHbs by divalent metal ions, together with the lack of effect on transport by the chelator EDTA, showed that CimHbs translocates the free citrate and malate anions. Among a large set of substrates tested, only malate, citramalate, and citrate competitively inhibited citrate transport catalyzed by CimHbs. The transporter is strictly stereoselective, recognizing only the S enantiomers of malate and citramalate. Remarkably, though citramalate binds to the transporter, it is not translocated.

Transcription factor PrfA controls the expression of virulence genes essential forListeria monocytogenespathogenesis. To gain insight into the structure-function relationship of PrfA, we devised a positive-selection system to isolate mutations reducing or abolishing transcriptional activity. The system is based on the observation that the listerialiapgene, encoding the p60 protein, is lethal if overexpressed inBacillus subtilis. A plasmid in which theiapgene is placed under the control of the PrfA-dependenthlypromoter was constructed and introduced intoB. subtilis. This strain was rapidly killed when expression ofiapwas induced by introduction of a second plasmid carryingprfA. Two classes ofB. subtilissurvivor mutants were identified: one carried mutations iniap, and the second carried mutations inprfA. Sequence analysis of the defectiveprfAgenes identified mutations in three regions of the PrfA protein: region A, between amino acids 58 and 67 in the β-roll domain of PrfA; region B, between amino acids 169 and 193, which corresponds to the DNA-binding helix-turn-helix motif; and region C, comprising the 38 C-terminal amino acids of PrfA, which form a leucine zipper-like structure. PrfA proteins with mutations in regions B and C were unable to bind to the PrfA-binding site in the target DNA, while mutations in region A resulted in a protein still binding the target DNA but unable to form a stable complex with RNA polymerase and initiate transcription in vitro.

The genome of Bacillus licheniformis DSM13 consists of a single chromosome that has a size of 4,222,748 base pairs. The average G+C ratio is 46.2%. 4,286 open reading frames, 72 tRNA genes, 7 rRNA operons and 20 transposase genes were identified. The genome shows a marked co-linearity with Bacillus subtilis but contains defined inserted regions that can be identified at the sequence as well as at the functional level. B. licheniformis DSM13 has a well-conserved secretory system, no polyketide biosynthesis, but is able to form the lipopeptide lichenysin. From the further analysis of the genome sequence, we identified conserved regulatory DNA motives, the occurrence of the glyoxylate bypass and the presence of anaerobic ribonucleotide reductase explaining that B. licheniformis is able to grow on acetate and 2,3-butanediol as well as anaerobically on glucose. Many new genes of potential interest for biotechnological applications were found in B. licheniformis; candidates include proteases, pectate lyases, lipases and various polysaccharide degrading enzymes.

Genome sequences are available for increasing numbers of organisms. The proteomes (protein complement expressed by the genome) of many such organisms are being studied with two-dimensional (2D) gel electrophoresis. Here we have investigated the application of short N-terminal and C-terminal sequence tags to the identification of proteins separated on 2D gels. The theoretical N and C termini of 15, 519 proteins, representing all SWISS-PROT entries for the organisms Mycoplasma genitalium, Bacillus subtilis, Escherichia coli, Saccharomyces cerevisiae and human, were analysed. Sequence tags were found to be surprisingly specific, with N-terminal tags of four amino acid residues found to be unique for between 43% and 83% of proteins, and C-terminal tags of four amino acid residues unique for between 74% and 97% of proteins, depending on the species studied. Sequence tags of five amino acid residues were found to be even more specific. To utilise this specificity of sequence tags for protein identification, we created a world-wide web-accessible protein identification program, TagIdent (http://www.expasy.ch/www/tools.html), which matches sequence tags of up to six amino acid residues as well as estimated protein pI and mass against proteins in the SWISS-PROT database. We demonstrate the utility of this identification approach with sequence tags generated from 91 different E. coli proteins purified by 2D gel electrophoresis. Fifty-one proteins were unambiguously identified by virtue of their sequence tags and estimated pI and mass, and a further 11 proteins identified when sequence tags were combined with protein amino acid composition data. We conlcude that the TagIdent identification approach is best suited to the identification of proteins from prokaryotes whose complete genome sequences are available. The approach is less well suited to proteins from eukaryotes, as many eukaryotic proteins are not amenable to sequencing via Edman degradation, and tag protein identification cannot be unambiguous unless an organism's complete sequence is available.

The abundance of all tetra- and pentanucleotide sequences is calculated for a set of DNA sequence data comprising 767,393 nucleotides of the E. coli K-12 genome. Observed frequencies are compared to those expected from a Markov chain prediction algorithm. Systematic and extreme non-random representations are found for special sets of sequences. These are interpreted as arising from incorporation of a 2'-deoxyguanosine residue opposite thymidine during replication which, in special sequence contexts, leads to a T/G mismatch that is simultaneously substrate for two competing DNA mismatch repair systems: the mutHLS and the VSP pathway. Processing by the former leads to error correction, by the latter to mutation fixation. The significance of the latter process, as demonstrated here, makes it unlikely that VSP repair has evolved mainly as a mutation avoidance mechanism. It is proposed that in E. coli K-12, VSP repair, together with DNA cytosine methylation, constitutes a mutagenesis/recombination system capable of promoting gene-conversion-like unidirectional transfer of short stretches of DNA sequence.