Schematic representation of the gene clusters involved in maltodextrin and maltose utilization of <i>E. faecium</i> E1162.

<p>Genes are represented by arrows (drawn to scale). Genes putatively encoding proteins involved in maltodextrin transport and/or metabolism are indicated in blue or in red. Genes predicted to be involved in the uptake and/or metabolism of maltose are indicated in green or in purple. The genes that encode putative transcriptional regulators are indicated in red or purple. The grey arrows represent the adjacent genes that are not predicted to be involved in maltodextrin or maltose utilization. Gene names, without the EfmE1162-prefix (omitted for reasons of space), are indicated in the arrows and the gene locus tags are indicated above or below the arrows. The homologs in <i>L. monocytogenes</i> EGD-e or in <i>E. faecalis</i> V583 are shown with corresponding colors above or below the gene clusters of <i>E. faecium</i>. The gene locus tags of the homologs are indicated in the arrows. Lines link the homologous genes with corresponding genes in <i>E. faecium</i> and amino acid identities are indicated.</p

Growth of <i>E. faecium</i> on starch, maltotetraose, maltose and glucose.

<p>Growth curves of <i>E. faecium</i> E1162 wild-type (black), its isogenic mutants Δ<i>mdxR</i> (red) and Δ<i>mdxABCD-pulA</i> (blue), and the <i>in trans</i> complemented strain Δ<i>mdxR+mdxR</i> (green) on starch (panel A), maltotetraose (panel B), maltose (panel C) and glucose (panel D) are shown. The growth curve of E1162 wild-type in M1 was shown in grey as a negative control. Overnight cultures were inoculated at an initial OD₆₆₀ of 0.0025 into 300 µl semi-defined minimal medium M1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072285#B9" target="_blank">9</a>], M1 supplemented with 2.5 g/l of starch, maltotetraoise, maltose or glucose as sole carbon source, respectively, and then incubated in the Bioscreen C system at 37° C with continuous shaking. The absorbance of 600nm (A₆₀₀) was recorded every 15 min for 12 hours. Growth curves are mean data of three independent experiments. Note that A₆₀₀ at the start of the experiment is higher in M1 + starch than in the other conditions, due to increased turbidity of the medium containing starch.</p

Growth curves of <i>E. faecium</i> E1162 and <i>pbp5</i> mutants in BHI with 20 µg ml⁻¹ ampicillin.

<p>Overnight cultures of wild-type <i>E. faecium</i> E1162, the insertional <i>pbp5</i> mutant (<i>pbp5</i>::pWS3), the markerless deletion mutant Δ<i>pbp5</i> and the <i>in trans</i> complemented deletion mutant (Δ<i>pbp5</i>+<i>pbp5</i>) were inoculated at an initial cell density of OD₆₆₀ 0.0025 in BHI with 20 µg ml⁻¹ ampicillin. Growth curves are mean data of three independent experiments.</p

Footprinting analysis of the transposon mutant library.

<p>(A) Schematic overview of the transposon footprinting strategy. PCR is performed using a gene specific primer and a primer corresponding to the transposon sequence. (B) Agarose gel electrophoresis of transposon footprinting on the essential gene <i>ddl</i> (lane 1), and the non-essential genes <i>nox</i> and <i>esp</i> (lane 2 and 3, respectively). Each band represents a PCR product of a different size, corresponding to a transposon insertion in a different position. The red box represents the product size range expected for transposon insertions within the essential <i>ddl</i> gene.</p

<i>E. faecium</i> genes involved in ampicillin resistance as determined by M-TraM analysis.

a<p>Indicates the gene containing the transposon insertion.</p>b<p>Indicates the fold-change derived from the ratio of the unselected control library to the ampicillin competitively selected library. <i>e.g.</i> the value 32.5 means that the relative quantity of mutants of EfmE1162_0447 in the ampicillin-selected library was 32.5-fold less than in the control library grown without selective pressure. This indicates that mutants in EfmE1162_0447 have a lower relative fitness in the presence of ampicillin than wild type cells. The value of −7.5 for EfmE1162_2487 indicates that mutants in this gene outgrow the other mutants in the ampicillin-selected library by 7.5-fold, indicating that mutants of EfmE1162_2487 have higher relative fitness in the presence of ampicillin.</p

Percentage survival of <i>E. faecium</i> cells following a lysozyme challenge.

<p>Survival of the indicated wild-type, mutant strains and <i>in trans</i> complemented strains following a 30-minute incubation in PBS containing 0.5 mg ml⁻¹ lysozyme relative to the survival of the strains after a 30-minute incubation in PBS without lysozyme. Bars represent the standard deviation of the mean of three independent experiments. Asterisks represent significant differences (<i>P</i><0.005 as determined by a two-tailed Student's <i>t</i>-test) between the indicated mutants and the wild-type strain.</p

D,D-carboxypeptidase activity in <i>E. faecium</i> membrane fractions.

<p>Membrane extracts were isolated from the indicated strains grown in BHI medium (with or without the addition 20 µg ml⁻¹ ampicillin) until an OD₆₀₀ of 0.7. The D,D-carboxypeptidase activity (nmol min⁻¹ mg⁻¹) was defined as the number of nmoles of D-Ala released from pentapeptide (7.5 mM) per min and per mg of protein in the membrane fractions. D-Ala was assayed using D-amino acid oxidase coupled to peroxidase. Bars represent the standard deviation of the mean of three independent experiments. Asterisks represent significant difference (^*<i>P</i><0.05, ^**<i>P</i><0.005 as determined by a two-tailed Student's <i>t</i>-test) between the different strains and conditions.</p

ABPHM15 EtherPad Archive

<p>An archived record of the EtherPad collaborative notes document from Applied Bioinformatics for Public Health Microbiology conference, held 6-8 May, 2015 at the Wellcome Trust Sanger Centre, Cambridge, UK. Live EtherPad at https://etherpad.mozilla.org/rxHOz9HvDr</p

Genome Sequence and Characterization of Coliphage Môr Ffagbaw

Background: The quality of coastal waters around the United Kingdom is an area of increasing concern following sewer overflow, where wastewater is discharged into the environment. Coliphages, viruses that infect coliform bacteria, are associated with water quality in aquatic systems, yet remain largely uncharacterized at the genomic level. Materials and Methods: Phage môr ffagbaw was isolated from seawater against Escherichia coli by enrichment and plaque assays. Whole genome sequencing, transmission electron microscopy, and host range analysis against the E. coli reference (ECOR) collection were used to characterize the phage. Results: The virion had a siphovirus morphology and genomic analysis placed it within the family Drexlerviridae, subfamily Tempevirinae, and forms a new species within the genus Hanrivervirus. Spot assays revealed that phage môr ffagbaw could form plaques on 6 out of 72 ECOR strains (8%). Conclusions: Môr ffagbaw represents a new species of phage within the genus Hanrivervirus, with a narrow host range

Multilocus sequence types (STs) of recovered ARE isolates and previous occurrence among other sources.

a<p><a href="http://efaecium.mlst.net/" target="_blank">http://efaecium.mlst.net/</a> queried March 2010;</p>b<p>HAI = hospital-associated isolates (i.e., clinical isolates, hospital surveillance, hospital outbreak);</p>c<p>CI = clinical isolate; CS = Community human surveillance; LS = Livestock; D = dogs; C = cats.</p