Constitutive variants of the pC194 cat gene exhibit DNA alterations in the vicinity of the ribosome binding site sequence

The chloramphenicol-inducible regulation of the expression of cat genes from two Gram-positive bacteria, Staphylococcus aureus and Bacillus pumilus has been suggested to result from the presence of inverted repeat sequences that span the ribosome-binding site (RBS) for cat. In support of this hypothesis, we demonstrate that two derivatives of the pC194 cat gene which are constitutively expressed in Bacillus subtilis are deleted for all or a major portion of the inverted-repeat sequences.     

Analysis of the essential cell division gene ftsL of Bacillus subtilis by mutagenesis and heterologous complementation

The ftsL gene is required for the initiation of cell division in a broad range of bacteria. Bacillus subtilis ftsL encodes a 13-kDa protein with a membrane-spanning domain near its N terminus. The external C-terminal domain has features of an alpha-helical leucine zipper, which is likely to be involved in the heterodimerization with another division protein, DivIC. To determine what residues are important for FtsL function, we used both random and site-directed mutagenesis. Unexpectedly, all chemically induced mutations fell into two clear classes, those either weakening the ribosome-binding site or producing a stop codon. It appears that the random mutagenesis was efficient, so many missense mutations must have been generated but with no phenotypic effect. Substitutions affecting hydrophobic residues in the putative coiled-coil domain, introduced by site-directed mutagenesis, also gave no observable phenotype except for insertion of a helix-breaking proline residue, which destroyed FtsL function. ftsL homologues cloned from three diverse Bacillus species, Bacillus licheniformis, Bacillus badius, and Bacillus circulans, could complement an ftsL null mutation in B. subtilis, even though up to 66% of the amino acid residues of the predicted proteins were different from B. subtilis FtsL. However, the ftsL gene from Staphylococcus aureus (whose product has 73% of its amino acids different from those of the B. subtilis ftsL product) was not functional. We conclude that FtsL is a highly malleable protein that can accommodate a large number of sequence changes without loss of function.     

Quorum sensing by peptide pheromones and two-component signal-transduction systems in Gram-positive bacteria

Cell-density-dependent gene expression appears to be widely spread in bacteria. This quorum-sensing phenomenon has been well established in Gram-negative bacteria, where N -acyl homoserine lactones are the diffusible communication molecules that modulate cell-density-dependent phenotypes. Similarly, a variety of processes are known to be regulated in a cell-density- or growth-phase-dependent manner in Gram-positive bacteria. Examples of such quorum-sensing modes in Gram-positive bacteria are the development of genetic competence in Bacillus subtilis and Streptococcus pneumoniae , the virulence response in Staphylococcus aureus , and the production of antimicrobial peptides by several species of Gram-positive bacteria including lactic acid bacteria. Cell-density-dependent regulatory modes in these systems appear to follow a common theme, in which the signal molecule is a post-translationally processed peptide that is secreted by a dedicated ATP-binding-cassette exporter. This secreted peptide pheromone functions as the input signal for a specific sensor component of a two-component signal-transduction system. Moreover, genetic linkage of the common elements involved results in autoregulation of peptide-pheromone production.     

Measuring single-cell gene expression dynamics in bacteria using fluorescence time-lapse microscopy

Quantitative single-cell time-lapse microscopy is a powerful method for analyzing gene circuit dynamics and heterogeneous cell behavior. We describe the application of this method to imaging bacteria by using an automated microscopy system. This protocol has been used to analyze sporulation and competence differentiation in Bacillus subtilis, and to quantify gene regulation and its fluctuations in individual Escherichia coli cells. The protocol involves seeding and growing bacteria on small agarose pads and imaging the resulting microcolonies. Images are then reviewed and analyzed using our laboratory's custom MATLAB analysis code, which segments and tracks cells in a frame-to-frame method. This process yields quantitative expression data on cell lineages, which can illustrate dynamic expression profiles and facilitate mathematical models of gene circuits. With fast-growing bacteria, such as E. coli or B. subtilis, image acquisition can be completed in 1 d, with an additional 1-2 d for progressing through the analysis procedure.     

Thiol-disulphide oxidoreductase modules in the low-GC Gram-positive bacteria

Disulphide bond formation catalysed by thiol-disulphide oxidoreductases (TDORs) is a universally conserved mechanism for stabilizing extracytoplasmic proteins. In Escherichia coli, disulphide bond formation requires a concerted action of distinct TDORs in thiol oxidation and subsequent quinone reduction. TDOR function in other bacteria has remained largely unexplored. Here we focus on TDORs of low-GC Gram-positive bacteria, in particular DsbA of Staphylococcus aureus and BdbA-D of Bacillus subtilis. Phylogenetic analyses reveal that the homologues DsbA and BdbD cluster in distinct groups typical for Staphylococcus and Bacillus species respectively. To compare the function of these TDORs, DsbA was produced in various bdb mutants of B. subtilis. Next, we assessed the ability of DsbA to sustain different TDOR-dependent processes, including heterologous secretion of E. coli PhoA, competence development and bacteriocin (sublancin 168) production. The results show that DsbA can function in all three processes. While BdbD needs a quinone oxidoreductase for activity, DsbA activity appears to depend on redox-active medium components. Unexpectedly, both quinone oxidoreductases of B. subtilis are sufficient to sustain production of sublancin. Moreover, DsbA can functionally replace these quinone oxidoreductases in sublancin production. Taken together, our unprecedented findings imply that TDOR systems of low-GC Gram-positive bacteria have a modular composition.     

Cloning of lysozyme and lysostaphin genes of Staphylococcus aureus and their expression in Bacillus subtilis cells

The gene of microbial lysozyme (lyz) of S. aureus 118 and the gene of lysostaphin (lzf) of S. aureus RN 3239 were cloned and their expression in B. subtilis cells was shown. Lysozyme production in B. subtilis recombinant clone pLF14-Lyz, obtained as the result of cloning, was 2.5-fold greater than lysozyme production in S. aureus wild strain 118. Lysostaphin production in B. subtilis recombinant strain pLF14-Lzf which had inherited the cloned genes was approximately equal to lysostaphin production observed in S. aureus initial strain RN 3239. The production of lysozyme and lysostaphin in the cells of B. subtilis recombinant strains was observed at 30 degrees C and pH 5.5, while in S. aureus initial strains 118 and RN 3239 bacteria produced lysozyme and lysostaphin at 37 degrees C and pH 7.5 respectively.     

The toxin-antitoxin system of the streptococcal plasmid pSM19035

pSM19035 of the pathogenic bacterium Streptococcus pyogenes is a low-copy-number plasmid carrying erythromycin resistance, stably maintained in a broad range of gram-positive bacteria. We show here that the omega-epsilon-zeta operon of this plasmid constitutes a novel proteic plasmid addiction system in which the epsilon and zeta genes encode an antitoxin and toxin, respectively, while omega plays an autoregulatory function. Expression of toxin Zeta is bactericidal for the gram-positive Bacillus subtilis and bacteriostatic for the gram-negative Escherichia coli. The toxic effects of zeta gene expression in both bacterial species are counteracted by proper expression of epsilon. The epsilon-zeta toxin-antitoxin cassette stabilizes plasmids in E. coli less efficiently than in B. subtilis.     

Control of key metabolic intersections in Bacillus subtilis

The remarkable ability of bacteria to adapt efficiently to a wide range of nutritional environments reflects their use of overlapping regulatory systems that link gene expression to intracellular pools of a small number of key metabolites. By integrating the activities of global regulators, such as CcpA, CodY and TnrA, Bacillus subtilis manages traffic through two metabolic intersections that determine the flow of carbon and nitrogen to and from crucial metabolites, such as pyruvate, 2-oxoglutarate and glutamate. Here, the latest knowledge on the control of these key intersections in B. subtilis is reviewed.     

The Staphylococcus aureus CsoR regulates both chromosomal and plasmid-encoded copper resistance mechanisms

Copper is an essential metal which is used as a cofactor in several enzymes and is required for numerous essential biochemical reactions. However, free copper ions can be toxic to cellular systems if the intracellular concentration is not tightly regulated. In this study we show that Staphylococcus aureus copper resistance is not the same in every staphylococcal isolate, but in fact varies considerably between clinical strains. Hyper-copper-resistance was shown to be due to the carriage of an additional plasmid-encoded copper homeostasis mechanism, copBmco. This plasmid can be transferred into the copper-sensitive S. aureus Newman to confer a hyper-copper-resistant phenotype, showing that copper resistance has the potential to spread to other S. aureus strains. This is the first time that plasmid-encoded copper resistance has been reported and shown to be transferable between pathogenic bacteria isolated from humans. A homologue of the Bacillus subtilis and Mycobacterium tuberculosis CsoR regulators was identified in S. aureus. The S. aureus csoR gene is conserved in all sequenced S. aureus genomes and was found to be copper-induced and transcribed along with two downstream genes: a putative copper chaperone (csoZ) and a hypothetical gene. Mutational and complementation studies showed that unlike other homologues, the S. aureus CsoR negatively regulates both chromosomal and plasmid-encoded copper homeostasis mechanisms in response to excess-copper conditions.     

Regulated Antisense RNA Eliminates Alpha-Toxin Virulence in Staphylococcus aureus Infection

The ability to selectively disrupt gene function remains a critical element in elucidating information regarding gene essentiality for bacterial growth and/or pathogenesis. In this study, we adapted a tet regulatory expression system for use in Staphylococcus aureus, with the goal of downregulating gene expression via induction of antisense RNA. We demonstrate that this system exhibits a 50- to 100-fold dose-dependent level of induction in bacterial cells grown in culture (i.e., in vitro) and also functions in mice (i.e., in vivo) following oral administration of inducer. To determine whether induced antisense RNA could interfere with chromosomally derived gene expression, we cloned a fragment of the S. aureus alpha-toxin gene (hla) in antisense orientation downstream of the tet promoter system and introduced the construct into S. aureus. Induced antisense hla RNA downregulated chromosomally derived hla gene expression in vitro approximately 14-fold. Similarly, induction of hla antisense RNA in vivo dramatically reduced alpha-toxin expression in two different murine models of S. aureus infection. Most importantly, this reduction completely eliminated the lethality of the infection. These results indicate that the tet regulatory system functions efficiently in S. aureus and induced antisense RNA can effectively downregulate chromosomal gene expression both in vitro and in vivo.     

Identification of a novel essential two-component signal transduction system, YhcSR, in Staphylococcus aureus

Two-component signal transduction systems play an important role in the ability of bacteria to adapt to various environments by sensing changes in their habitat and by altering gene expression. In this study, we report a novel two-component system, YhcSR, in Staphylococcus aureus which is required for bacterial growth in vitro. We found that the down-regulation of yhcSR expression by induced yhcS antisense RNA can inhibit and terminate bacterial growth. Moreover, without complementary yhcS or yhcR, no viable yhcS or yhcR gene replacement mutant was recoverable. Collectively, these results demonstrated that the YhcSR regulatory system is indispensable for S. aureus growth in culture. Moreover, induced yhcS antisense RNA selectively increased bacterial susceptibility to phosphomycin. These data suggest that YhcSR probably modulates the expression of genes critical for bacterial survival and may be a potential target for the development of novel antibacterial agents.     

Limitations in the use of Drosophila melanogaster as a model host for gram-positive bacterial infection

AIMS: To examine sensitivities of various Drosophila melanogaster strains towards human pathogenic and nonpathogenic gram-positive bacteria. METHODS AND RESULTS: The D. melanogaster Oregon R strain was infected by injecting the thorax with a needle containing Escherichia coli (negative control), Listeria monocytogenes, Staphylococcus aureus (both food-borne pathogens), Listeria innocua, Bacillus subtilis, Carnobacterium maltaromaticum, Lactobacillus plantarum or Pediococcus acidilactici (all nonpathogenic bacteria). Listeria monocytogenes and S. aureus killed the host rapidly compared with the negative control. Infection with L. innocua, B. subtilis or C. maltaromaticum also resulted in a high fly mortality, whereas Lact. plantarum and P. acidilactici resulted in a slightly increased mortality. Four additional D. melanogaster lines, three of which had been selected for heat, cold and desiccation resistance respectively, were subjected to infection by L. monocytogenes, S. aureus and E. coli. Mortality rates were comparable with that of the Oregon R strain. CONCLUSIONS: Use of the injection method shows the limitation of D. melanogaster as a model host for gram-positive bacteria as opportunistic infection by nonpathogenic gram-positive bacteria results in partial or high mortality. In addition, lines of fruit flies resistant to various stress exposures did not show an increased resistance to infection by gram-positive pathogens under the conditions tested. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the inadequacy of D. melanogaster infected by the injection method in order to distinguish between virulent and nonvirulent gram-positive bacteria.     

Lesions in teichoic acid biosynthesis in Staphylococcus aureus lead to a lethal gain of function in the otherwise dispensable pathway

An extensive study of teichoic acid biosynthesis in the model organism Bacillus subtilis has established teichoic acid polymers as essential components of the gram-positive cell wall. However, similar studies pertaining to therapeutically relevant organisms, such as Staphylococcus aureus, are scarce. In this study we have carried out a meticulous examination of the dispensability of teichoic acid biosynthetic enzymes in S. aureus. By use of an allelic replacement methodology, we examined all facets of teichoic acid assembly, including intracellular polymer production and export. Using this approach we confirmed that the first-acting enzyme (TarO) was dispensable for growth, in contrast to dispensability studies in B. subtilis. Upon further characterization, we demonstrated that later-acting gene products (TarB, TarD, TarF, TarIJ, and TarH) responsible for polymer formation and export were essential for viability. We resolved this paradox by demonstrating that all of the apparently indispensable genes became dispensable in a tarO null genetic background. This work suggests a lethal gain-of-function mechanism where lesions beyond the initial step in wall teichoic acid biosynthesis render S. aureus nonviable. This discovery poses questions regarding the conventional understanding of essential gene sets, garnered through single-gene knockout experiments in bacteria and higher organisms, and points to a novel drug development strategy targeting late steps in teichoic acid synthesis for the infectious pathogen S. aureus.     

金黄色葡萄球菌hmp基因缺失突变株的构建及抗一氧化氮能力分析

摘要：【目的】:构建金黄色葡萄球菌RN6390黄素血红蛋白(flavohaemoglobin, HMP)基因缺失突变株,研究其抗一氧化氮(Nitric Oxide, NO) 能力及其在细菌生物被膜形成中的作用。【方法】：根据同源重组技术的原理,利用PCR扩增RN6390的hmp基因上下游同源臂,经过抗生素和温度交替培养筛选hmp基因缺失突变株,利用基因组PCR、定量PCR对突变菌株进行鉴定。以硝普钠（SNP）为NO供体,检测了hmp基因缺失菌株的抗NO能力,并初步研究了hmp基因在生物被膜形成中的作用。【结果】：成功构建了RN6390的hmp基因缺失突变株,外源NO能够诱导菌株hmp基因的表达,hmp基因缺失菌株抗NO能力明显下降,但其生物被膜形成能力有明显提高。【结论】：获得了RN6390的hmp基因缺失突变株,该突变株的获得为进一步研究hmp基因的生物功能,以及细菌内源性NO的作用奠定了良好的技术平台。