Class IIa bacteriocin resistance in Enterococcus faecalis V583: The mannose PTS operon mediates global transcriptional responses

<p>Abstract</p> <p>Background</p> <p>The class IIa bacteriocin, pediocin PA-1, has clear potential as food preservative and in the medical field to be used against Gram negative pathogen species as <it>Enterococcus faecalis </it>and <it>Listeria monocytogenes</it>. Resistance towards class IIa bacteriocins appear in laboratory and characterization of these phenotypes is important for their application. To gain insight into bacteriocin resistance we studied mutants of <it>E. faecalis </it>V583 resistant to pediocin PA-1 by use of transcriptomic analyses.</p> <p>Results</p> <p>Mutants of <it>E. faecalis </it>V583 resistant to pediocin PA-1 were isolated, and their gene expression profiles were analyzed and compared to the wild type using whole-genome microarray. Significantly altered transcription was detected from about 200 genes; most of them encoding proteins involved in energy metabolism and transport. Glycolytic genes were down-regulated in the mutants, but most of the genes showing differential expression were up-regulated. The data indicate that the mutants were relieved from glucose repression and putative catabolic responsive elements (<it>cre</it>) could be identified in the upstream regions of 70% of the differentially expressed genes. Bacteriocin resistance was caused by reduced expression of the <it>mpt </it>operon encoding the mannose-specific phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS), and the same transcriptional changes were seen in a <it>mptD</it>-inactivated mutant. This mutant also had decreased transcription of the whole <it>mpt </it>operon, showing that the PTS is involved in its own transcriptional regulation.</p> <p>Conclusion</p> <p>Our data confirm the important role of mannose PTS in class IIa bacteriocin sensitivity and we demonstrate its importance involving global carbon catabolite control.</p

DNA binding kinetics of two response regulators, PlnC and PlnD, from the bacteriocin regulon of Lactobacillus plantarum C11

<p>Abstract</p> <p>Background</p> <p>Bacteriocin production in the lactic acid bacterium <it>Lactobacillus plantarum </it>C11 is regulated through a quorum sensing based pathway involving two highly homologous response regulators (59% identity and 76% similarity), PlnC as a transcriptional activator and PlnD as a repressor. Previous <it>in vitro </it>studies have shown that both regulators bind, as homodimers, to the same DNA regulatory repeats to exert their regulatory functions. As the genes for these two proteins are located on the same auto-regulatory operon, hence being co-expressed upon gene activation, it is plausible that their opposite functions must somehow be differentially regulated, either in terms of timing and/or binding kinetics, so that their activities do not impair each other in an uncontrolled manner. To understand the nature behind this potential differentiation, we have studied the binding kinetics of the two regulators on five target promoters (P_{<it>plnA</it>}, P_{<it>plnM</it>}, P_{<it>plnJ</it>}, P_{<it>plnE </it>}and P_{<it>plnG</it>}) from the bacteriocin regulon of <it>L. plantarum </it>C11.</p> <p>Results</p> <p>By using surface plasmon resonance spectroscopy we obtained parameters such as association rates, dissociation rates and dissociation constants, showing that the two regulators indeed differ greatly from each other in terms of cooperative binding and binding strength to the different promoters. For instance, cooperativity is very strong for PlnC binding to the promoter of the regulatory operon (P_{<it>plnA</it>}), but not to the promoter of the transport operon (P_{<it>plnG</it>}), while the opposite is seen for PlnD binding to these two promoters. The estimated affinity constants indicate that PlnC can bind to P_{<it>plnA </it>}to activate transcription of the key regulatory operon <it>plnABCD </it>without much interference from PlnD, and that the repressive function of PlnD might act through a different mechanism than repression of the regulatory operon.</p> <p>Conclusion</p> <p>We have characterised the DNA binding kinetics of the two regulators PlnC and PlnD from the bacteriocin locus in <it>L. plantarum </it>C11. Our data show that PlnC and PlnD, despite their strong homology to each other, differ greatly from each other in terms of binding affinity and cooperativity to the different promoters of the <it>pln </it>regulon.</p

Comparative genomics of Enterococcus faecalis from healthy Norwegian infants

<p>Abstract</p> <p>Background</p> <p><it>Enterococcus faecalis</it>, traditionally considered a harmless commensal of the intestinal tract, is now ranked among the leading causes of nosocomial infections. In an attempt to gain insight into the genetic make-up of commensal <it>E. faecalis</it>, we have studied genomic variation in a collection of community-derived <it>E. faecalis </it>isolated from the feces of Norwegian infants.</p> <p>Results</p> <p>The <it>E. faecalis </it>isolates were first sequence typed by multilocus sequence typing (MLST) and characterized with respect to antibiotic resistance and properties associated with virulence. A subset of the isolates was compared to the vancomycin resistant strain <it>E. faecalis </it>V583 (V583) by whole genome microarray comparison (comparative genomic hybridization (CGH)). Several of the putative enterococcal virulence factors were found to be highly prevalent among the commensal baby isolates. The genomic variation as observed by CGH was less between isolates displaying the same MLST sequence type than between isolates belonging to different evolutionary lineages.</p> <p>Conclusion</p> <p>The variations in gene content observed among the investigated commensal <it>E. faecalis </it>is comparable to the genetic variation previously reported among strains of various origins thought to be representative of the major <it>E. faecalis </it>lineages. Previous MLST analysis of <it>E. faecalis </it>have identified so-called high-risk enterococcal clonal complexes (HiRECC), defined as genetically distinct subpopulations, epidemiologically associated with enterococcal infections. The observed correlation between CGH and MLST presented here, may offer a method for the identification of lineage-specific genes, and may therefore add clues on how to distinguish pathogenic from commensal <it>E. faecalis</it>. In this work, information on the core genome of <it>E. faecalis </it>is also substantially extended.</p

The Response of Enterococcus faecalis V583 to Chloramphenicol Treatment

Many Enterococcus faecalis strains display tolerance or resistance to many antibiotics, but genes that contribute to the resistance cannot be specified. The multiresistant E. faecalis V583, for which the complete genome sequence is available, survives and grows in media containing relatively high levels of chloramphenicol. No specific genes coding for chloramphenicol resistance has been recognized in V583. We used microarrays to identify genes and mechanisms behind the tolerance to chloramphenicol in V583, by comparison of cells treated with subinhibitory concentrations of chloramphenicol and untreated V583 cells. During a time course experiment, more than 600 genes were significantly differentially transcribed. Since chloramphenicol affects protein synthesis in bacteria, many genes involved in protein synthesis, for example, genes for ribosomal proteins, were induced. Genes involved in amino acid biosynthesis, for example, genes for tRNA synthetases and energy metabolism were downregulated, mainly. Among the upregulated genes were EF1732 and EF1733, which code for potential chloramphenicol transporters. Efflux of drug out of the cells may be one mechanism used by V583 to overcome the effect of chloramphenicol

Identification of proteins related to the stress response in Enterococcus faecalis V583 caused by bovine bile

<p>Abstract</p> <p>Background</p> <p><it>Enterococcus faecalis </it>is an opportunistic pathogen and one of the most important causes of hospital infections. Bile acids are a major stress factor bacteria have to cope with in order to colonize and survive in the gastro-intestinal tract. The aim of this study was to investigate the effects of bile acids on the intracellular proteome of <it>E. faecalis </it>V583.</p> <p>Results</p> <p>The proteomes of cells challenged with 1% bile were analyzed after 20 - 120 minutes exposure, using 2D gel electrophoresis and mass spectrometry. Among the approximately 500 observed proteins, 53 unique proteins were found to be regulated in response to bile and were identified with mass spectrometry. The identified proteins belonged to nine different functional classes, including fatty acid- and phospholipid-biosynthesis, energy metabolism, and transport and binding. Proteins involved in fatty acid and phospholipid biosynthesis pathways were clearly overrepresented among the identified proteins and all were down-regulated upon exposure to bile. The proteome data correlated reasonably well with data from previous transcriptome experiments done under the same conditions, but several differences were observed.</p> <p>Conclusion</p> <p>The results provide an overview of potentially important proteins that <it>E. faecalis </it>V583 needs to regulate in order to survive and adapt to a bile-rich environment, among which are several proteins involved in fatty acid and phospholipid biosynthesis pathways. In addition, this study reveals several hypothetical proteins, which are both abundant and clearly regulated and thus stand out as targets for future studies on bile stress.</p