Matthew D. Hitchings

Streptomyces coelicolor Dps‐like proteins: differential dual roles in response to stress during vegetative growth and in nucleoid condensation during reproductive cell division

The Dps protein, a member of the ferritin family, contributes to DNA protection during oxidative stress and plays a central role in nucleoid condensation during stationary phase in unicellular eubacteria. Genome searches revealed the presence of three Dps‐like orthologues within the genome of the Gram‐positive bacterium Streptomyces coelicolor. Disruption of the S. coelicolor dpsA, dpsB and dpsC genes resulted in irregular condensation of spore nucleoids in a gene‐specific manner. These irregularities are correlated with changes to the spacing between sporulation septa. This is the first example of these proteins playing a role in bacterial cell division. Translational fusions provided evidence for both developmental control of DpsA and DpsC expression and their localization to sporogenic compartments of aerial hyphae. In addition, various stress conditions induced expression of the Dps proteins in a stimulus‐dependent manner in vegetative hyphae, suggesting stress‐induced, protein‐specific protective functions in addition to their role during reproductive cell division. Unlike in other bacteria, the S. coelicolor Dps proteins are not induced in response to oxidative stress.

The dpsA Gene of Streptomyces coelicolor: Induction of Expression from a Single Promoter in Response to Environmental Stress or during Development

The DpsA protein plays a dual role in Streptomyces coelicolor, both as part of the stress response and contributing to nucleoid condensation during sporulation. Promoter mapping experiments indicated that dpsA is transcribed from a single, sigB-like dependent promoter. Expression studies implicate SigH and SigB as the sigma factors responsible for dpsA expression while the contribution of other SigB-like factors is indirect by means of controlling sigH expression. The promoter is massively induced in response to osmotic stress, in part due to its sensitivity to changes in DNA supercoiling. In addition, we determined that WhiB is required for dpsA expression, particularly during development. Gel retardation experiments revealed direct interaction between apoWhiB and the dpsA promoter region, providing the first evidence for a direct WhiB target in S. coelicolor.

Genomic structure and insertion sites of Helicobacter pylori prophages from various geographical origins.

Helicobacter pylori genetic diversity is known to be influenced by mobile genomic elements. Here we focused on prophages, the least characterized mobile elements of H. pylori. We present the full genomic sequences, insertion sites and phylogenetic analysis of 28 prophages found in H. pylori isolates from patients of distinct disease types, ranging from gastritis to gastric cancer, and geographic origins, covering most continents. The genome sizes of these prophages range from 22.6–33.0 Kbp, consisting of 27–39 open reading frames. A 36.6% GC was found in prophages in contrast to 39% in H. pylori genome. Remarkably a conserved integration site was found in over 50% of the cases. Nearly 40% of the prophages harbored insertion sequences (IS) previously described in H. pylori. Tandem repeats were frequently found in the intergenic region between the prophage at the 3′ end and the bacterial gene. Furthermore, prophage genomes present a robust phylogeographic pattern, revealing four distinct clusters: one African, one Asian and two European prophage populations. Evidence of recombination was detected within the genome of some prophages, resulting in genome mosaics composed by different populations, which may yield additional H. pylori phenotypes.

Draft genomes, phylogenetic reconstruction and comparative genomics of two novel cohabiting bacterial symbionts isolated from Frankliniella occidentalis.

Obligate bacterial symbionts are widespread in many invertebrates, where they are often confined to specialized host cells and are transmitted directly from mother to progeny. Increasing numbers of these bacteria are being characterized but questions remain about their population structure and evolution. Here we take a comparative genomics approach to investigate two prominent bacterial symbionts (BFo1 and BFo2) isolated from geographically separated populations of western flower thrips, Frankliniella occidentalis. Our multifaceted approach to classifying these symbionts includes concatenated multilocus sequence analysis (MLSA) phylogenies, ribosomal multilocus sequence typing (rMLST), construction of whole-genome phylogenies, and in-depth genomic comparisons. We showed that the BFo1 genome clusters more closely to species in the genus Erwinia, and is a putative close relative to Erwinia aphidicola. BFo1 is also likely to have shared a common ancestor with Erwinia pyrifoliae/Erwinia amylovora and the nonpathogenic Erwinia tasmaniensis and genetic traits similar to Erwinia billingiae. The BFo1 genome contained virulence factors found in the genus Erwinia but represented a divergent lineage. In contrast, we showed that BFo2 belongs within the Enterobacteriales but does not group closely with any currently known bacterial species. Concatenated MLSA phylogenies indicate that it may have shared a common ancestor to the Erwinia and Pantoea genera, and based on the clustering of rMLST genes, it was most closely related to Pantoea ananatis but represented a divergent lineage. We reconstructed a core genome of a putative common ancestor of Erwinia and Pantoea and compared this with the genomes of BFo bacteria. BFo2 possessed none of the virulence determinants that were omnipresent in the Erwinia and Pantoea genera. Taken together, these data are consistent with BFo2 representing a highly novel species that maybe related to known Pantoea.

Local genes for local bacteria: Evidence of allopatry in the genomes of transatlantic Campylobacter populations

The genetic structure of bacterial populations can be related to geographical locations of isolation. In some species, there is a strong correlation between geographical distance and genetic distance, which can be caused by different evolutionary mechanisms. Patterns of ancient admixture in Helicobacter pylori can be reconstructed in concordance with past human migration, whereas in Mycobacterium tuberculosis it is the lack of recombination that causes allopatric clusters. In Campylobacter, analyses of genomic data and molecular typing have been successful in determining the reservoir host species, but not geographical origin. We investigated biogeographical variation in highly recombining genes to determine the extent of clustering between genomes from geographically distinct Campylobacter populations. Whole‐genome sequences from 294 Campylobacter isolates from North America and the UK were analysed. Isolates from within the same country shared more recently recombined DNA than isolates from different countries. Using 15 UK/American closely matched pairs of isolates that shared ancestors, we identify regions that have frequently and recently recombined to test their correlation with geographical origin. The seven genes that demonstrated the greatest clustering by geography were used in an attribution model to infer geographical origin which was tested using a further 383 UK clinical isolates to detect signatures of recent foreign travel. Patient records indicated that in 46 cases, travel abroad had occurred <2 weeks prior to sampling, and genomic analysis identified that 34 (74%) of these isolates were of a non‐UK origin. Identification of biogeographical markers in Campylobacter genomes will contribute to improved source attribution of clinical Campylobacter infection and inform intervention strategies to reduce campylobacteriosis.

Recombination-Mediated Host Adaptation by Avian Staphylococcus aureus

Staphylococcus aureus are globally disseminated among farmed chickens causing skeletal muscle infections, dermatitis, and septicaemia. The emergence of poultry-associated lineages has involved zoonotic transmission from humans to chickens but questions remain about the specific adaptations that promote proliferation of chicken pathogens. We characterized genetic variation in a population of genome-sequenced S. aureus isolates of poultry and human origin. Genealogical analysis identified a dominant poultry-associated sequence cluster within the CC5 clonal complex. Poultry and human CC5 isolates were significantly distinct from each other and more recombination events were detected in the poultry isolates. We identified 44 recombination events in 33 genes along the branch extending to the poultry-specific CC5 cluster, and 47 genes were found more often in CC5 poultry isolates compared with those from humans. Many of these gene sequences were common in chicken isolates from other clonal complexes suggesting horizontal gene transfer among poultry associated lineages. Consistent with functional predictions for putative poultry-associated genes, poultry isolates showed enhanced growth at 42 °C and greater erythrocyte lysis on chicken blood agar in comparison with human isolates. By combining phenotype information with evolutionary analyses of staphylococcal genomes, we provide evidence of adaptation, following a human-to-poultry host transition. This has important implications for the emergence and dissemination of new pathogenic clones associated with modern agriculture.

A tale of tails: deciphering the contribution of terminal tails to the biochemical properties of two Dps proteins from Streptomyces coelicolor

Dps proteins are members of an extensive family of proteins that oxidise and deposit iron in the form of ferric oxide, and are also able to bind DNA. Ferroxidation centres are formed at the interface of anti-parallel dimers, which further assemble into dodecameric nanocages with a hollow core where ferric oxide is deposited. Streptomyces coelicolor encodes three Dps-like proteins (DpsA, B and C). Despite sharing the conserved four-helix bundle organisation observed in members of the Dps family, they display significant differences in the length of terminal extensions, or tails. DpsA possess both N- and C-terminal tails of different lengths, and their removal affects quaternary structure assembly to varying degrees. DpsC quaternary structure, on the other hand, is heavily dependent on its N-terminal tail as its removal abolishes correct protein folding. Analysis of the crystal structure of dodecamers from both proteins revealed remarkable differences in the position of tails and interface surface area; and provides insight to explain the differences in biochemical behaviour observed while comparing DpsA and DpsC.

The Evolution of an Osmotically Inducible dps in the Genus Streptomyces

Dps proteins are found almost ubiquitously in bacterial genomes and there is now an appreciation of their multifaceted roles in various stress responses. Previous studies have shown that this family of proteins assemble into dodecamers and their quaternary structure is entirely critical to their function. Moreover, the numbers of dps genes per bacterial genome is variable; even amongst closely related species - however, for many genera this enigma is yet to be satisfactorily explained. We reconstruct the most probable evolutionary history of Dps in Streptomyces genomes. Typically, these bacteria encode for more than one Dps protein. We offer the explanation that variation in the number of dps per genome among closely related Streptomyces can be explained by gene duplication or lateral acquisition, and the former preceded a subsequent shift in expression patterns for one of the resultant paralogs. We show that the genome of S. coelicolor encodes for three Dps proteins including a tailless Dps. Our in vivo observations show that the tailless protein, unlike the other two Dps in S. coelicolor, does not readily oligomerise. Phylogenetic and bioinformatic analyses combined with expression studies indicate that in several Streptomyces species at least one Dps is significantly over-expressed during osmotic shock, but the identity of the ortholog varies. In silico analysis of dps promoter regions coupled with gene expression studies of duplicated dps genes shows that paralogous gene pairs are expressed differentially and this correlates with the presence of a sigB promoter. Lastly, we identify a rare novel clade of Dps and show that a representative of these proteins in S. coelicolor possesses a dodecameric quaternary structure of high stability.

Comparative Genomics Study of Staphylococcus epidermidis Isolates from Orthopedic-Device-Related Infections Correlated with Patient Outcome

ABSTRACT Staphylococcus epidermidis has emerged as an important opportunistic pathogen causing orthopedic-device-related infections (ODRI). This study investigated the association of genome variation and phenotypic features of the infecting S. epidermidis isolate with the clinical outcome for the infected patient. S. epidermidis isolates were collected from 104 patients with ODRI. Their clinical outcomes were evaluated, after an average of 26 months, as either “cured” or “not cured.” The isolates were tested for antibiotic susceptibility and biofilm formation. Whole-genome sequencing was performed on all isolates, and genomic variation was related to features associated with “cured” and “not cured.” Strong biofilm formation and aminoglycoside resistance were associated with a “not-cured” outcome (P = 0.031 and P < 0.001, respectively). Based on gene-by-gene analysis, some accessory genes were more prevalent in isolates from the “not-cured” group. These included the biofilm-associated bhp gene, the antiseptic resistance qacA gene, the cassette chromosome recombinase-encoding genes ccrA and ccrB, and the IS256-like transposase gene. This study identifies biofilm formation and antibiotic resistance as associated with poor outcome in S. epidermidis ODRI. Whole-genome sequencing identified specific genes associated with a “not-cured” outcome that should be validated in future studies. (The study has been registered at ClinicalTrials.gov with identifier NCT02640937.)

Complete genome sequence of a psychotrophic Arthrobacter strain A3 (CGMCC 1.8987), a novel long-chain hydrocarbons producer

Arthrobacter strain A3, a psychotrophic bacterium isolated from the Tian Shan Mountain of China, can degrade the cellulose and synthesis the long-chain hydrocarbons efficiently in low temperature. Here we report the complete genome sequence of this bacterium. The complete genome sequence of Arthrobacter strain A3, consisting of a cycle chromosome with a size of 4.26 Mbp and a cycle plasmid with a size of 194kbp. In this genome, a hydrocarbon biosynthesis gene cluster (oleA, oleB/oleC and oleD) was identified. To resistant the extreme environment, this strain contains a unique mycothiol-biosynthetic pathway (mshA-D), which has not been found in other Arthrobacter species before. The availability of this genome sequence allows us to investigate the genetic basis of adaptation to growth in a nutrient-poor permafrost environment and to evaluate of the biofuel-synthetic potential of this species.