Insights into secondary metabolism from a global analysis of prokaryotic biosynthetic gene clusters.

Although biosynthetic gene clusters (BGCs) have been discovered for hundreds of bacterial metabolites, our knowledge of their diversity remains limited. Here, we used a novel algorithm to systematically identify BGCs in the extensive extant microbial sequencing data. Network analysis of the predicted BGCs revealed large gene cluster families, the vast majority uncharacterized. We experimentally characterized the most prominent family, consisting of two subfamilies of hundreds of BGCs distributed throughout the Proteobacteria; their products are aryl polyenes, lipids with an aryl head group conjugated to a polyene tail. We identified a distant relationship to a third subfamily of aryl polyene BGCs, and together the three subfamilies represent the largest known family of biosynthetic gene clusters, with more than 1,000 members. Although these clusters are widely divergent in sequence, their small molecule products are remarkably conserved, indicating for the first time the important roles these compounds play in Gram-negative cell biology.

Whole-genome comparison of urinary pathogenic Escherichia coli and faecal isolates of UTI patients and healthy controls.

The faecal flora is a common reservoir for urinary tract infection (UTI), and Escherichia coli (E. coli) is frequently found in this reservoir without causing extraintestinal infection. We investigated these E. coli reservoirs by whole-genome sequencing a large collection of E. coli from healthy controls (faecal), who had never previously had UTI, and from UTI patients (faecal and urinary) sampled from the same geographical area. We compared MLST types, phylogenetic relationship, accessory genome content and FimH type between patient and control faecal isolates as well as between UTI and faecal-only isolates, respectively. Comparison of the accessory genome of UTI isolates to faecal isolates revealed 35 gene families which were significantly more prevalent in the UTI isolates compared to the faecal isolates, although none of these were unique to one of the two groups. Of these 35, 22 belonged to a genomic island and three putatively belonged to a type VI secretion system (T6SS). MLST types and SNP phylogeny indicated no clustering of the UTI or faecal E. coli from patients distinct from the control faecal isolates, although there was an overrepresentation of UTI isolates belonging to clonal lineages CC73 and CC12. One combination of mutations in FimH, N70S/S78N, was significantly associated to UTI, while phylogenetic analysis of FimH and fimH identified no signs of distinct adaptation of UTI isolates compared to faecal-only isolates not causing UTI. In summary, the results showed that (i) healthy women who had never previously had UTI carried faecal E. coli which were overall closely related to UTI and faecal isolates from UTI patients; (ii) UTI isolates do not cluster separately from faecal-only isolates based on SNP analysis; and (iii) 22 gene families of a genomic island, putative T6SS proteins as well as specific metabolism and virulence associated proteins were significantly more common in UTI isolates compared to faecal-only isolates and (iv) evolution of fimH for these isolates was not linked to the clinical source of the isolates, apart from the mutation combination N70S/S78N, which was correlated to UTI isolates of phylogroup B2. Combined, these findings illustrate that faecal and UTI isolates, as well as faecal-only and faecal-UTI isolates, are closely related and can only be distinguished, if at all, by their accessory genome.

Adaptation of Escherichia coli traversing from the faecal environment to the urinary tract.

The majority of extraintestinal pathogenic Escherichia coli (ExPEC) causing urinary tract infections (UTI) are found in the patient's own gut flora, but only limited knowledge is available on the potential adaptation that may occur in the bacteria in order to traverse the perineum and successfully infect the urinary tract. Here, matching pairs of faecal and UTI isolates from 42 patients were compared pairwise using in-depth whole-genome sequencing to investigate whether genetic changes were evident for successful colonization in these two different environments. The identified non-synonymous mutations (0-12 substitutions in each pair) were primarily associated to genes encoding virulence factors and nutrient metabolism; and indications of parallel evolution were observed in genes encoding the major phase-variable protein antigen 43, a toxin/antitoxin locus and haemolysin B. No differences in virulence potential were observed in a mouse UTI model for five matching faecal and UTI isolates with or without mutations in antigen 43 and haemolysin B. Variations in plasmid content were observed in only four of the 42 pairs. Although, we observed mutations in known UTI virulence genes for a few pairs, the majority showed no detectable differences with respect to mutations or mobilome when compared to their faecal counterpart. The results show that UPECs are successful in colonizing both the bladder and gut without adaptation.

Molecular tracing of the emergence, adaptation, and transmission of hospital-associated methicillin-resistant Staphylococcus aureus.

Hospital-associated infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are a global health burden dominated by a small number of bacterial clones. The pandemic EMRSA-16 clone (ST36-II) has been widespread in UK hospitals for 20 y, but its evolutionary origin and the molecular basis for its hospital association are unclear. We carried out a Bayesian phylogenetic reconstruction on the basis of the genome sequences of 87 S. aureus isolates including 60 EMRSA-16 and 27 additional clonal complex 30 (CC30) isolates, collected from patients in three continents over a 53-y period. The three major pandemic clones to originate from the CC30 lineage, including phage type 80/81, Southwest Pacific, and EMRSA-16, shared a most recent common ancestor that existed over 100 y ago, whereas the hospital-associated EMRSA-16 clone is estimated to have emerged about 35 y ago. Our CC30 genome-wide analysis revealed striking molecular correlates of hospital- or community-associated pandemics represented by mobile genetic elements and nonsynonymous mutations affecting antibiotic resistance and virulence. Importantly, phylogeographic analysis indicates that EMRSA-16 spread within the United Kingdom by transmission from hospitals in large population centers in London and Glasgow to regional health-care settings, implicating patient referrals as an important cause of nationwide transmission. Taken together, the high-resolution phylogenomic approach used resulted in a unique understanding of the emergence and transmission of a major MRSA clone and provided molecular correlates of its hospital adaptation. Similar approaches for hospital-associated clones of other bacterial pathogens may inform appropriate measures for controlling their intra- and interhospital spread.

Comparative and functional genomics of Rhodococcus opacus PD630 for biofuels development.

The Actinomycetales bacteria Rhodococcus opacus PD630 and Rhodococcus jostii RHA1 bioconvert a diverse range of organic substrates through lipid biosynthesis into large quantities of energy-rich triacylglycerols (TAGs). To describe the genetic basis of the Rhodococcus oleaginous metabolism, we sequenced and performed comparative analysis of the 9.27 Mb R. opacus PD630 genome. Metabolic-reconstruction assigned 2017 enzymatic reactions to the 8632 R. opacus PD630 genes we identified. Of these, 261 genes were implicated in the R. opacus PD630 TAGs cycle by metabolic reconstruction and gene family analysis. Rhodococcus synthesizes uncommon straight-chain odd-carbon fatty acids in high abundance and stores them as TAGs. We have identified these to be pentadecanoic, heptadecanoic, and cis-heptadecenoic acids. To identify bioconversion pathways, we screened R. opacus PD630, R. jostii RHA1, Ralstonia eutropha H16, and C. glutamicum 13032 for growth on 190 compounds. The results of the catabolic screen, phylogenetic analysis of the TAGs cycle enzymes, and metabolic product characterizations were integrated into a working model of prokaryotic oleaginy.

Candidate targets of balancing selection in the genome of Staphylococcus aureus.

Signatures of balancing selection can highlight polymorphisms and functions that are important to the long-term fitness of a species. We performed a first genome-wide scan for balancing selection in a bacterial species, Staphylococcus aureus, which is a common cause of serious antimicrobial-resistant infections of humans. Using a sliding window approach, the genomes of 16 strains of S. aureus, including 5 new genome sequences presented here, and 1 outgroup strain of S. epidermidis were scanned for signatures of balancing selection. A total of 195 short windows were investigated based on their extreme values of both Tajima's D (>2.03) and π/K ratios (>0.12) relative to the rest of the genome. To test the unusualness of these windows, an Approximate Bayesian Computation framework was used to select a null demographic model that better accounted for the observed data than did the standard neutral model. A total of 186 windows were demonstrated to be unusual under the null model and, thus, represented candidate loci under balancing selection. These 186 candidate windows were located within 99 candidate genes that were spread across 62 different loci. Nearly all the signal (97.2%) was located within coding sequences; balancing selection on gene regulation apparently occurs through the targeting of global regulators such as agr and gra/aps. The agr locus had some of the strongest signatures of balancing selection, which provides new insight into the causes of diversity at this locus. The list of candidate genes included multiple virulence-associated genes and was significantly enriched for functions in amino acid and inorganic ion transport and metabolism and in defense mechanisms against innate immunity and antimicrobials, highlighting these particular functions as important to the fitness of this pathogen.

Draft genome sequences of Staphylococcus aureus sequence type 34 (ST34) and ST42 hybrids.

Staphylococcus aureus is a major cause of antimicrobial-resistant infections of humans. Hybrids of S. aureus, which originate from large-scale chromosomal recombinations between parents of distinct genetic backgrounds, are of interest from clinical and evolutionary perspectives. We present draft genome sequences of two S. aureus hybrids of sequence type 34 (ST34) and ST42.

Dynamics of Pseudomonas aeruginosa genome evolution.

One of the hallmarks of the Gram-negative bacterium Pseudomonas aeruginosa is its ability to thrive in diverse environments that includes humans with a variety of debilitating diseases or immune deficiencies. Here we report the complete sequence and comparative analysis of the genomes of two representative P. aeruginosa strains isolated from cystic fibrosis (CF) patients whose genetic disorder predisposes them to infections by this pathogen. The comparison of the genomes of the two CF strains with those of other P. aeruginosa presents a picture of a mosaic genome, consisting of a conserved core component, interrupted in each strain by combinations of specific blocks of genes. These strain-specific segments of the genome are found in limited chromosomal locations, referred to as regions of genomic plasticity. The ability of P. aeruginosa to shape its genomic composition to favor survival in the widest range of environmental reservoirs, with corresponding enhancement of its metabolic capacity is supported by the identification of a genomic island in one of the sequenced CF isolates, encoding enzymes capable of degrading terpenoids produced by trees. This work suggests that niche adaptation is a major evolutionary force influencing the composition of bacterial genomes. Unlike genome reduction seen in host-adapted bacterial pathogens, the genetic capacity of P. aeruginosa is determined by the ability of individual strains to acquire or discard genomic segments, giving rise to strains with customized genomic repertoires. Consequently, this organism can survive in a wide range of environmental reservoirs that can serve as sources of the infecting organisms.

Validation of genomics-based prognostic tests in malignant pleural mesothelioma.

PURPOSE: Malignant pleural mesothelioma (MPM) is a highly lethal neoplasm with limited pretreatment prognostication strategies. In this report, we examine the accuracy of a previously proposed prognostic test in an independent cohort of MPM patients. This test uses simple ratios of gene expression levels to provide a novel prognostication scheme. EXPERIMENTAL DESIGN: Gene expression data using high-density oligonucleotide microarrays (approximately 22,000 genes) were obtained for a new cohort of human MPM tumors from patients undergoing similar treatments (n = 39). The relative expression levels for specific genes were also determined using real-time quantitative reverse transcription-PCR. We also used a subset of these tumors associated with widely divergent patient survival (n = 23) as a training set to identify new treatment-specific candidate prognostic molecular markers and gene ratio-based prognostic tests. The predictive nature of these newly discovered markers and gene ratio-based prognostic tests were then examined in an independent group of tumors (n = 52) using microarray data and quantitative reverse transcription-PCR. RESULTS: Previously described MPM prognostic genes and gene ratio-based prognostic tests predicted clinical outcome in 39 independent MPM tumor specimens in a statistically significant manner. Newly discovered treatment-specific prognostic genes and gene ratio-based prognostic tests were highly accurate and statistically significant when examined in an independent group of 52 tumors from patients undergoing similar treatment. CONCLUSIONS: The data support the use of gene ratios in translating gene expression data into easily reproducible, statistically validated clinical tests for the prediction of outcome in MPM.

Selection of unique Escherichia coli clones by random amplified polymorphic DNA (RAPD): Evaluation by whole genome sequencing.

Identifying and characterizing clonal diversity are important when analysing fecal flora. We evaluated random amplified polymorphic DNA (RAPD) PCR, applied for selection of Escherichia coli isolates, by whole genome sequencing. RAPD was fast, and reproducible as screening method for selection of distinct E. coli clones in fecal swabs.

The human olfactory receptor gene family.

Humans perceive an immense variety of chemicals as having distinct odors. Odor perception initiates in the nose, where odorants are detected by a large family of olfactory receptors (ORs). ORs have diverse protein sequences but can be assigned to subfamilies on the basis of sequence relationships. Members of the same subfamily have related sequences and are likely to recognize structurally related odorants. To gain insight into the mechanisms underlying odor perception, we analyzed the human OR gene family. By searching the human genome database, we identified 339 intact OR genes and 297 OR pseudogenes. Determination of their genomic locations showed that OR genes are unevenly distributed among 51 different loci on 21 human chromosomes. Sequence comparisons showed that the human OR family is composed of 172 subfamilies. Types of odorant structures that may be recognized by some subfamilies were predicted by identifying subfamilies that contain ORs with known odor ligands or human homologs of such ORs. Analysis of the chromosomal locations of members of each OR subfamily revealed that most subfamilies are encoded by a single chromosomal locus. Moreover, many loci encode only one or a few subfamilies, suggesting that different parts of the genome may, to some extent, be involved in the detection of different types of odorant structural motifs.

The mouse olfactory receptor gene family.

In mammals, odor detection in the nose is mediated by a diverse family of olfactory receptors (ORs), which are used combinatorially to detect different odorants and encode their identities. The OR family can be divided into subfamilies whose members are highly related and are likely to recognize structurally related odorants. To gain further insight into the mechanisms underlying odor detection, we analyzed the mouse OR gene family. Exhaustive searches of a mouse genome database identified 913 intact OR genes and 296 OR pseudogenes. These genes were localized to 51 different loci on 17 chromosomes. Sequence comparisons showed that the mouse OR family contains 241 subfamilies. Subfamily sizes vary extensively, suggesting that some classes of odorants may be more easily detected or discriminated than others. Determination of subfamilies that contain ORs with identified ligands allowed tentative functional predictions for 19 subfamilies. Analysis of the chromosomal locations of members of each subfamily showed that many OR gene loci encode only one or a few subfamilies. Furthermore, most subfamilies are encoded by a single locus, suggesting that different loci may encode receptors for different types of odorant structural features. Comparison of human and mouse OR subfamilies showed that the two species have many, but not all, subfamilies in common. However, mouse subfamilies are usually larger than their human counterparts. This finding suggests that humans and mice recognize many of the same odorant structural motifs, but mice may be superior in odor sensitivity and discrimination.