Use of single molecule sequencing for comparative genomics of an environmental and a clinical isolate of Clostridium difficile ribotype 078.

BACKGROUND: How the pathogen Clostridium difficile might survive, evolve and be transferred between reservoirs within the natural environment is poorly understood. Some ribotypes are found both in clinical and environmental settings. Whether these strains are distinct from each another and evolve in the specific environments is not established. The possession of a highly mobile genome has contributed to the genetic diversity and ongoing evolution of C. difficile. Interpretations of genetic diversity have been limited by fragmented assemblies resulting from short-read length sequencing approaches and by a limited understanding of epigenetic regulation of diversity. To address this, single molecule real time (SMRT) sequencing was used in this study as it produces high quality genome sequences, with resolution of repeat regions (including those found in mobile elements) and can generate data to determine methylation modifications across the sequence (the methylome). RESULTS: Chromosomal rearrangements and ribosomal operon duplications were observed in both genomes. The rearrangements occurred at insertion sites within two mobile genetic elements (MGEs), Tn6164 and Tn6293, present only in the M120 and CD105HS27 genomes, respectively. The gene content of these two transposons differ considerably which could impact upon horizontal gene transfer; differences include CDSs encoding methylases and a conjugative prophage only in Tn6164. To investigate mechanisms which could affect MGE transfer, the methylome, restriction modification (RM)  and the CRISPR/Cas systems were characterised for each strain. Notably, the environmental isolate, CD105HS27, does not share a consensus motif for (m4)C methylation, but has one additional spacer  when compared to the clinical isolate M120. CONCLUSIONS: These findings show key differences between the two strains in terms of their genetic capacity for MGE transfer. The carriage of horizontally transferred genes appear to have genome wide effects based on two different methylation patterns. The CRISPR/Cas system appears active although perhaps slow to evolve. Data suggests that both mechanisms are functional and impact upon horizontal gene transfer and genome evolution within C. difficile

Synergistic Activity of Mobile Genetic Element Defences in Streptococcus pneumoniae.

A diverse set of mobile genetic elements (MGEs) transmit between Streptococcus pneumoniae cells, but many isolates remain uninfected. The best-characterised defences against horizontal transmission of MGEs are restriction-modification systems (RMSs), of which there are two phase-variable examples in S. pneumoniae. Additionally, the transformation machinery has been proposed to limit vertical transmission of chromosomally integrated MGEs. This work describes how these mechanisms can act in concert. Experimental data demonstrate RMS phase variation occurs at a sub-maximal rate. Simulations suggest this may be optimal if MGEs are sometimes vertically inherited, as it reduces the probability that an infected cell will switch between RMS variants while the MGE is invading the population, and thereby undermine the restriction barrier. Such vertically inherited MGEs can be deleted by transformation. The lack of between-strain transformation hotspots at known prophage att sites suggests transformation cannot remove an MGE from a strain in which it is fixed. However, simulations confirmed that transformation was nevertheless effective at preventing the spread of MGEs into a previously uninfected cell population, if a recombination barrier existed between co-colonising strains. Further simulations combining these effects of phase variable RMSs and transformation found they synergistically inhibited MGEs spreading, through limiting both vertical and horizontal transmission

Phylogenetic analysis of resistance to ceftazidime/avibactam, ceftolozane/tazobactam and carbapenems in piperacillin/tazobactam-resistant Pseudomonas aeruginosa from cystic fibrosis patients.

Pseudomonas aeruginosa is one of the most important pathogens in cystic fibrosis. This study was conducted to analyse the genetic basis and phylogenetic profile of resistance to ceftazidime/avibactam, ceftolozane/tazobactam and carbapenems in cystic fibrosis P. aeruginosa isolates. Whole genome sequence analysis was conducted of isolates resistant to piperacillin/tazobactam collected from seven hospitals in Scotland since the introduction of these two cephalosporin/β-lactamase inhibitor combinations. Ceftazidime resistance was primarily related to AmpC induction, as tested by cloxacillin inhibition assays, while high-level ceftazidime resistance not reversed by cloxacillin was associated with amino acid variations in AmpC. Only isolates resistant to both ceftazidime/avibactam and ceftolozane/tazobactam carried AmpD mutations, likely resulting in ampC overexpression. All isolates resistant to ceftazidime/avibactam and/or ceftolozane/tazobactam were resistant to carbapenems and showed inactivating mutations in the chromosomal oprD gene. None of the isolates bore class A, B, D plasmid-encoded carbapenemases. This study showed that mutational resistance emerged in phylogenetically distant lineages, which indicates the mutations occur independently without conferring a selective advantage to any phylogenetic lineage. These findings confirm the strong contribution of mutation-driven evolution to the population structure of P. aeruginosa

Identification and characterization of chromosomal relBE toxin-antitoxin locus in Streptomyces cattleya DSM46488.

The relBE family of Type II toxin-antitoxin (TA) systems have been widely reported in bacteria but none in Streptomyces. With the conserved domain searches for TA pairs in the sequenced Streptomyces genomes, we identified two putative relBE loci, relBE1sca and relBE2sca, on the chromosome of Streptomyces cattleya DSM 46488. Overexpression of the S. cattleya toxin RelE2sca caused severe growth inhibition of E. coli and S. lividans, but RelE1sca had no toxic effect. The toxicity of RelE2sca could be abolished by the co-expression of its cognate RelB2sca antitoxin. Moreover, the RelBE2sca complex, or the antitoxin RelB2sca alone, specifically interacted with the relBE2sca operon and repressed its transcription. The relBE2sca operon transcription was induced under osmotic stress, along with the ClpP proteinase genes. The subsequent in vivo analysis showed that the antitoxin was degraded by ClpP. Interestingly, the E. coli antitoxin RelBeco was able to alleviate the toxicity of S. cattleya RelE2sca while the mutant RelB2sca(N61V&M68L) but not the wild type could alleviate the toxicity of E. coli RelEeco as well. The experimental demonstration of the relBEsca locus might be helpful to investigate the key roles of type II TA systems in Streptomyces physiology and environmental stress responses

Growth of mutants in the EIIB^Man and EIIA^Man domains of the CelR beta-glucoside regulator.

<p>Wild type strain (blue) and a its isogenic mutants in the regulator EIIB domain (panels A, B and C) and EIIA domain (panels D, E and F) were collected from agar plates, resuspended to OD₅₉₀ of 0.2 and inoculated in CAT medium containing one of the following carbohydrates: glucose 0.3% (panels A and D), gentiobiose 0.3% (panels B and E) and cellobiose 0.3% (panels C and F). Pneumococcal strains were DP1004 (blue, all panels), EIIB domain knockout (green, panel A, B and C), EIIB_C414A mutant (red, panel A, B and C), EIIB_C414D mutant (violet panel A, B and C), EIIA domain knockout (green, panels D, E and F) and EIIA_H577A mutant (violet, in panels D, E and F). Panel G through I report growth in 1%, 0.3% and 0.1% cellobiose respectively of the <i>wt</i> (blue), the EIIB_C414A mutant (red as in panels A-D), the EIIBCA^Glc spr0505 mutant (violet), and the EIIB_C414A and EIIBCA^Glc spr0505 double mutant (green). Panel J shows the wt (blue), the EIIBCA^Glc spr0505 insertion mutant (violet as in panels G-I) and the spr0505_EIIA in frame deletion mutant (black) grown in 0.1% cellobiose. Panel K shows growth of our wt strain in gentibiose (continuous blue), gentibiose plus methyl-beta-glucoside (dashed blue), cellobiose (red), cellobiose plus methyl-beta-glucoside (dashed red), methyl-beta-glucoside only (black dashed), and without sugar (black). All sugar concentrations in panel K are 0.2%.</p

Schematic drawing of the CelR regulator and proposed model for regulation.

<p>Panel (A) shows the CelR regulator composed of a helix-turn-helix (HTH) domain, a M trans-acting positive regulator (Mga) domain, two PTS-regulatory domain (PRD), and phosphotransferase system EIIB^Gat-like and EIIA^Mtl domains <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047393#pone.0047393-Finn1" target="_blank">[26]</a>. The phosphorylable conserved residues, five histidine and one cysteine, are shown <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047393#pone.0047393-Zeng1" target="_blank">[8]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047393#pone.0047393-Joyet1" target="_blank">[17]</a>. Panel (B) shows the proposed regulatory circuit in presence of cellobiose and absence of glucose. In such a situation in <i>S. mutans</i> HPr protein does not phosphorylate the glucose transporter (pneumococcal orthologue spr0259-60-61), but phosphorylates the cellobiose PTS (pneumococcal orthologue spr0278-80-82) and the CelR regulator <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047393#pone.0047393-Zeng1" target="_blank">[8]</a>. In addition the cellobiose PTS does also dephosphorylate CelR <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047393#pone.0047393-Zeng1" target="_blank">[8]</a>. Phosphorylation in presence of cellobiose of the second beta-glucoside PTS <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047393#pone.0047393-Bidossi1" target="_blank">[5]</a> is in accordance with gene expression data (Safeeq and Kuipers, personal communication). Phosphorylation of the CelR EIIB domain phosphorylable cysteine by the CelR EIIA domain is deduced from our growth phenotypes. The putative interaction of the spr0505 EIIBA domains with the PRD domains of CelR is shown as dashed line.</p

Schematic representation of the dynamic model.

<p>The glucose and beta-glucoside substrates are supplemented initially at a certain concentration. The enzymes spr0259-60-61 and spr0278-80-82 are induced by glucose and beta-glucoside substrates respectively through transcriptional regulation. The <i>S. pneumoniae</i> growth process is sustained by glucose and beta-glucoside through enzymes spr0259-60-61 and spr0278-80-82.</p

Parameters of the model and their biological meaning.

<p>Parameters of the model and their biological meaning.</p

Strains and isogenic mutants in the EIIB and EIIA domains used in this study.

<p>Strains and isogenic mutants in the EIIB and EIIA domains used in this study.</p

Parameters estimation with cellobiose concentration = 3 g/l.

<p>Parameters estimation with cellobiose concentration  = 3 g/l.</p