DataSheet1.xlsx

<p>Staphylococcus epidermidis is a prominent commensal member of human skin microbiome and an emerging nosocomial pathogen, making it a good model organism to provide genomic insights, correlating its transition between commensalism and pathogenicity. While there are numerous studies to understand differences in commensal and pathogenic isolates, systematic efforts to understand variation and evolutionary pattern in multiple strains isolated from healthy individuals are lacking. In the present study, using whole genome sequencing and analysis, we report presence of diverse lineages of S. epidermidis isolates in healthy individuals from two geographically diverse locations of India and North America. Further, there is distinct pattern in the distribution of candidate gene(s) for pathogenicity and commensalism. The pattern is not only reflected in lineages but is also based on geographic origin of the isolates. This is evident by the fact that North American isolates under this study are more genomically dynamic and harbor pathogenicity markers in higher frequency. On the other hand, isolates of Indian origin are less genomically dynamic, harbor less pathogenicity marker genes and possess two unique antimicrobial peptide gene clusters. This study provides a basis to understand the nature of selection pressure in a key human skin commensal bacterium with implications in its management as an opportunistic pathogen.</p

Identification, Purification and Characterization of Laterosporulin, a Novel Bacteriocin Produced by <em>Brevibacillus</em> sp. Strain GI-9

<div><h3>Background</h3><p>Bacteriocins are antimicrobial peptides that are produced by bacteria as a defense mechanism in complex environments. Identification and characterization of novel bacteriocins in novel strains of bacteria is one of the important fields in bacteriology.</p> <h3>Methodology/Findings</h3><p>The strain GI-9 was identified as <em>Brevibacillus</em> sp. by 16 S rRNA gene sequence analysis. The bacteriocin produced by strain GI-9, namely, laterosporulin was purified from supernatant of the culture grown under optimal conditions using hydrophobic interaction chromatography and reverse-phase HPLC. The bacteriocin was active against a wide range of Gram-positive and Gram-negative bacteria. MALDI-TOF experiments determined the precise molecular mass of the peptide to be of 5.6 kDa and N-terminal sequencing of the thermo-stable peptide revealed low similarity with existing antimicrobial peptides. The putative open reading frame (ORF) encoding laterosporulin and its surrounding genomic region was fished out from the draft genome sequence of GI-9. Sequence analysis of the putative bacteriocin gene did not show significant similarity to any reported bacteriocin producing genes in database.</p> <h3>Conclusions</h3><p>We have identified a bacteriocin producing strain GI-9, belonging to the genus <em>Brevibacillus</em> sp. Biochemical and genomic characterization of laterosporulin suggests it as a novel bacteriocin with broad spectrum antibacterial activity.</p> </div

Genetic organization of 4 kb region of the genome encoding laterosporulin (A) and nucleotide sequence of the ORF (B).

<p>The putative ORF encoding laterosporulin structural gene is shown by filled arrow and flanking ORFs as shown by empty arrows (A). Panel B shows the nucleotide sequence of the laterosporulin gene (encoding the indicated amino acids) with putative start codan, stop codons and ribosome binding site (RBS) shown in bold.</p

Comparison of bacteriocin production at different phases of growth curve.

<p>The growth curve analysis of strain <i>Brevibacillus</i> sp. GI-9 indicates that the production of bacteriocin initiated at late logarithmic phase. Growth measured as absorption at 600 nm is indicated by squares (left y axes), while the bacteriocin activity by triangles (right y axes).</p

Bactericidal effect of laterosporulin on <i>E. coli</i>.

<p>The scanning electron micrographs of <i>E. coli</i> cells without laterosporulin treatment (A) and after laterosporulin treatment (500 µg/ml) for 4 h (B). Note that the treatment of <i>E. coli</i> displaying roughening of cell surface with cell debris as a result of treatment.</p

MALDI-TOF mass spectrometry analysis of laterosporulin.

<p>The purified bacteriocin from <i>Brevibacillus</i> sp. GI-9 shows the mass (m/z) of 5.6 kDa.</p

Gel filtration elution profile of laterosporulin (A) and analysis (B).

<p>(A) The major peak (highlighted fraction) in gel filtration chromatography shows antimicrobial activity and the calibration curve obtained from protein standards indicates mass of the peptide in the range of 5.3 to 10.1 kDa. (B) Tricine-SDS-PAGE analysis of the major peak showed a single band; lane 1, molecular weight markers; lane 2, purified laterosporulin.</p

Putative pathogenicity elements identified in the genome of <i>Xam</i> CIO151.

1<p>Number indicates CDS identified by key word searches in iANT.</p

Phylogeny of conserved effectors in the genus <i>Xanthomonas</i>.

<p>Phylogenetic tree of concatenated conserved effector protein sequences of AvrBs2, XopK, XopL, XopN, XopQ XopR families and the Hpa1 protein, obtained with a Bayesian approach. Numbers on branches indicate Bayesian support values. Length of branches indicates the number of amino acid substitutions per site.</p

Circular representation of the genome sequence of <i>Xam</i> CIO151.

<p>From outside to inside: first circle in blue indicates CDS predicted in the positive strands for the scaffolds classified as probable chromosomal regions. Second circle in red indicates the CDS predicted in the negative strand. Red spots in the black third circle indicate the region identified with atypical nucleotide composition. The fourth circle indicates the deviation pattern from the average G+C content. Inner circle shows GC skew values, positive values are shown in purple and negative values are shown in orange. Numbers correspond to scaffold IDs.</p