433 research outputs found

    Transcriptome analysis and prediction of the metabolic state of stress-induced viable but non-culturable Bacillus subtilis cells

    Get PDF
    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

    BrevicidineB, a New Member of the Brevicidine Family, Displays an Extended Target Specificity

    Get PDF
    The group of bacterial non-ribosomally produced peptides (NRPs) has formed a rich source for drug development. Brevicidine, a bacterial non-ribosomally produced cyclic lipo-dodecapeptide, displays selective antimicrobial activity against Gram-negative pathogens. Here, we show that brevicidineB, which contains a single substitution (Tyr2 to Phe2) in the amino acid sequence of the linear part of brevicidine, has a broadened antimicrobial spectrum, showing bactericidal activity against both Gram-negative (with a MIC value of 2 to 4 mg/L) and Gram-positive (with a MIC value of 2 to 8 mg/L) pathogens. Compared with an earlier reported member of the brevicidine family, the broadened antimicrobial spectrum of brevicidineB is caused by its increased membrane disruptive capacity on Gram-positive pathogens, which was evidenced by fluorescence microscopy assays. In addition, DiSC3(5) and resazurin assays show that brevicidine and brevicidineB exert their antimicrobial activity against Gram-negative bacteria via disrupting the proton motive force of cells. Notably, as a brevicidine family member, brevicidineB also showed neither hemolytic activity nor cytotoxicity at a high concentration of 64 mg/L. This study provides a promising antibiotic candidate (brevicidineB) with a broad antimicrobial spectrum, and provides novel insights into the antimicrobial mode of action of brevicidines

    Analysis of cross-functionality within LanBTC synthetase complexes from different bacterial sources with respect to production of fully modified lanthipeptides

    Get PDF
    Lanthipeptides belong to a family of ribosomally synthesized and posttranslationally modified peptides (RiPPs) containing (methyl)lanthionine residues. Commonly, class I lanthipeptides are synthesized by a gene cluster encoding a precursor peptide (LanA), a biosynthetic machinery (LanBTC), a protease (LanP), a two-component regulatory system (LanRK), and an immunity system (LanI and LanFEG). Although nisin and subtilin are highly similar class I lanthipeptides, the cross-regulation by LanRK and the cross-immunity by LanI and LanFEG between the nisin and subtilin systems have been proven very low. Here, the possibility of the cross-functionality by LanBTC to modify and transport nisin precursor (NisA) and subtilin precursor (SpaS) was evaluated in Bacillus subtilis and Lactococcus lactis. Interestingly, we found that a promiscuous NisBC-SpaT complex is able to synthesize and export nisin precursor, as efficiently as the native nisin biosynthetic machinery NisBTC, in L. lactis, but not in B. subtilis. The assembly of the NisBC-SpaT complex at a single microdomain, close to the old cell pole, was observed by fluorescence microscopy in L. lactis. In contrast, such a complex was not formed in B. subtilis. Furthermore, the isolation of the NisBC-SpaT complex and its subcomplexes from the cytoplasmic membrane of L. lactis by pull-down assays was successfully conducted. Our work demonstrates that the association of LanBC with LanT is critical for the efficient biosynthesis and secretion of the lanthipeptide precursor with complete modifications, and suggests a cooperative mechanism between LanBC and LanT in the modification and transport processes. IMPORTANCE A multimeric synthetase LanBTC complex has been proposed for the in vivo production of class I lanthipeptides. However, it has been demonstrated that LanB, LanC, and LanT can perform their functionality in vivo and in vitro, independently of other Lan proteins. The role of protein-protein interactions, especially between the modification complex LanBC and the transport system LanT, in the biosynthesis process of lanthipeptides is still unclear. In this study, the importance of the presence of a well-installed LanBTC complex in the cell membrane for lanthipeptide biosynthesis and transport was reinforced. In L. lactis, the recruitment of SpaT from the peripheral cell membrane to the cell poles by the NisBC complex was observed, which may explain the mechanism by which secretion of premature peptide is prevented

    Nisin- and Ripcin-Derived Hybrid Lanthipeptides Display Selective Antimicrobial Activity against Staphylococcus aureus

    Get PDF
    [Image: see text] Lanthipeptides are (methyl)lanthionine ring-containing ribosomally synthesized and post-translationally modified peptides (RiPPs). Many lanthipeptides show strong antimicrobial activity against bacterial pathogens, including antibiotic-resistant bacterial pathogens. The group of disulfide-bond-containing antimicrobial peptides (AMPs) is well-known in nature and forms a rich source of templates for the production of novel peptides with corresponding (methyl)lanthionine analogues instead of disulfides. Here, we show that novel macrocyclic lanthipeptides (termed thanacin and ripcin) can be synthesized using the known antimicrobials thanatin and rip-thanatin as templates. Notably, the synthesized nisin(1–20)–ripcin hybrid lanthipeptides (ripcin B–G) showed selective antimicrobial activity against S. aureus, including an antibiotic-resistant MRSA strain. Interestingly, ripcin B–G, which are hybrid peptides of nisin(1–20) and ripcin that are each inactive against Gram-negative pathogens, showed substantial antimicrobial activity against the tested Gram-negative pathogens. Moreover, ripcin B–G was highly resistant against the nisin resistance protein (NSR; a peptidase that removes the C-terminal 6 amino acids of nisin and strongly reduces its antimicrobial activity), opposed to nisin itself. This study provides an example of converting disulfide-bond-based AMPs into (methyl)lanthionine-based macrocyclic hybrid lanthipeptides and can yield antimicrobial peptides with selective antimicrobial activity against S. aureus

    NADH-Mediated Gene Expression in Streptococcus pneumoniae and Role of Rex as a Transcriptional Repressor of the Rex-Regulon

    Get PDF
    Nicotinamide adenine dinucleotides (NAD(H)) play a vital role in various biological processes, including keeping the cellular redox balance. In this study, we investigate the regulatory responses of Streptococcus pneumoniae D39 to NADH and characterize the role of the Rex protein as a transcriptional repressor of the gapN, fba, pncB, adhB2, gap, and adhE genes. Transcriptomic analysis was used to observe the response of S. pneumoniae D39 to NADH. Our microarray studies revealed elevated expression of various genes/operons involved in transport and biosynthesis of niacin (gapN, fba, pncB, adhB2, gap, and adhE). Promoter lacZ-fusion assays and microarray studies with the rex mutant revealed the role of Rex as a transcriptional repressor of gapN, fba, pncB, adhB2, gap, and adhE involved in niacin uptake and biosynthesis, in the presence of NADH. We predict the operator site (5 ' TTGTKAWAAWWTTCACAA-3 ' of Rex in the regulatory regions of Rex-regulated genes that was subsequently validated by promoter mutational experiments

    Ascorbic acid-dependent gene expression in Streptococcus pneumoniae and the activator function of the transcriptional regulator UlaR2

    Get PDF
    In this study, we have explored the impact of ascorbic acid on the transcriptome of Streptococcus pneumoniae D39. The expression of several genes and operons, including the ula operon (which has been previously shown to be involved in ascorbic acid utilization), the AdcR regulon (which has been previously shown to be involved in zinc transport and virulence) and a PTS operon (which we denote here as ula2 operon) were altered in the presence of ascorbic acid. The ula2 operon consists of five genes, including the transcriptional activator ulaR2. Our β-galactosidase assay data and transcriptome comparison of the ulaR2 mutant with the wild-type demonstrated that the transcriptional activator UlaR2 in the presence of ascorbic acid activates the expression of the ula2 operon. We further predict a 16-bp regulatory site (5'-ATATTGTGCTCAAATA-3') for UlaR2 in the Pula2. Furthermore, we have explored the effect of ascorbic acid on the expression of the AdcR regulon. Our ICP-MS analysis showed that addition of ascorbic acid to the medium causes zinc starvation in the cell which leads to the activation of the AdcR regulon

    Controlled overproduction of proteins by lactic acid bacteria

    Get PDF
    Lactic acid bacteria are widely used in industrial food fermentations, contributing to flavour, texture and preservation of the fermented products. Here we describe recent advances in the development of controlled gene expression systems, which allow the regulated overproduction of any desirable protein by lactic acid bacteria. Some systems benefit from the fact that the expression vectors, marker genes and inducing factors can be used directly in food applications since they are all derived from food-grade lactic acid bacteria. These systems have also been employed for the development of autolytic bacteria, suitable for various industrial applications.

    Mimicry of a Non-ribosomally Produced Antimicrobial, Brevicidine, by Ribosomal Synthesis and Post-translational Modification

    Get PDF
    Zhao et al. describe a strategy to synthesize mimics of the recently discovered antimicrobial non-ribosomal peptide, brevicidine. The engineered mimics show antimicrobial activities against pathogens susceptible to brevicidine, which demonstrate that conversion of NRPs to RiPPs is feasible and offer great opportunities for engineering a wide range of effective antibiotics
    corecore