Transfer of 13 species of the genus Burkholderia to the genus Caballeronia and reclassification of Burkholderia jirisanensis as Paraburkholderia jirisanensis comb. nov.

A recent study of a group of Burkholderia glathei-like bacteria resulted in the description of 13 novel species of the genus Burkholderia. However, our analysis of phylogenetic positions of these species and their molecular signatures (conserved protein sequence indels) showed that they belong to the genus Caballeronia, and we propose to transfer them to this genus. The reclassified species names are proposed as Caballeroniaarationis comb. nov., Caballeroniaarvi comb. nov., Caballeroniacalidae comb. nov., Caballeroniacatudaia comb. nov., Caballeroniaconcitans comb. nov., Caballeroniafortuita comb. nov., Caballeroniaglebae comb. nov., Caballeroniahypogeia comb. nov., Caballeroniapedi comb. nov., Caballeroniaperedens comb. nov., Caballeroniaptereochthonis comb. nov., Caballeroniatemeraria comb. nov. and Caballeronia turbans comb. nov. It is also proposed to reclassify Burkholderia jirisanensis as Paraburkholderiajirisanensis comb. nov. Based on the results of the polyphasic study, B. jirisanensis had been described as a member of the A-group of the genus Burkholderiaand the most closely related to Burkholderia rhizosphaerae, Burkholderia humisilvae and Burkholderia solisilvae currently classified as belonging to the genus Paraburkholderia.

Role of homologous recombination in adaptive diversification of extraintestinal Escherichia coli.

The contribution of homologous exchange (recombination) of core genes in the adaptive evolution of bacterial pathogens is not well understood. To investigate this, we analyzed fully assembled genomes of two Escherichia coli strains from sequence type 131 (ST131), a clonal group that is both the leading cause of extraintestinal E. coli infections and the main source of fluoroquinolone-resistant E. coli. Although the sequences of each of the seven multilocus sequence typing genes were identical in the two ST131 isolates, the strains diverged from one another by homologous recombination that affected at least 9% of core genes. This was on a par with the contribution to genomic diversity of horizontal gene transfer and point gene mutation. The genomic positions of recombinant and mobile genetic regions were partially linked, suggesting their concurrent occurrence. One of the genes affected by homologous recombination was fimH, which encodes mannose-specific type 1 fimbrial adhesin, resulting in functionally distinct copies of the gene in ST131 strains. One strain, a uropathogenic isolate, had a pathoadaptive variant of fimH that was acquired by homologous replacement into the commensal strain background. Close examination of FimH structure and function in additional ST131 isolates revealed that recombination led to acquisition of several functionally distinct variants that, upon homologous exchange, were targeted by a variety of pathoadaptive mutations under strong positive selection. Different recombinant fimH strains also showed a strong clonal association with ST131 isolates that had distinct fluoroquinolone resistance profiles. Thus, homologous recombination of core genes plays a significant role in adaptive diversification of bacterial pathogens, especially at the level of clonally related groups of isolates.

Predictive diagnostics for Escherichia coli infections based on the clonal association of antimicrobial resistance and clinical outcome.

The ability to identify bacterial pathogens at the subspecies level in clinical diagnostics is currently limited. We investigated whether splitting Escherichia coli species into clonal groups (clonotypes) predicts antimicrobial susceptibility or clinical outcome. A total of 1,679 extraintestinal E. coli isolates (collected from 2010 to 2012) were collected from one German and 5 U.S. clinical microbiology laboratories. Clonotype identity was determined by fumC and fimH (CH) sequencing. The associations of clonotype with antimicrobial susceptibility and clinical variables were evaluated. CH typing divided the isolates into >200 CH clonotypes, with 93% of the isolates belonging to clonotypes with ≥ 2 isolates. Antimicrobial susceptibility varied substantially among clonotypes but was consistent across different locations. Clonotype-guided antimicrobial selection significantly reduced "drug-bug" mismatch compared to that which occurs with the use of conventional empirical therapy. With trimethoprim-sulfamethoxazole and fluoroquinolones, the drug-bug mismatch was predicted to decrease 62% and 78%, respectively. Recurrent or persistent urinary tract infection and clinical sepsis were significantly correlated with specific clonotypes, especially with CH40-30 (also known as H30), a recently described clonotype within sequence type 131 (ST131). We were able to clonotype directly from patient urine samples within 1 to 3 h of obtaining the specimen. In E. coli, subspecies-level identification by clonotyping can be used to significantly improve empirical predictions of antimicrobial susceptibility and clinical outcomes in a timely manner.

Convergent molecular evolution of genomic cores in Salmonella enterica and Escherichia coli.

One of the strongest signals of adaptive molecular evolution of proteins is the occurrence of convergent hot spot mutations: repeated changes in the same amino acid positions. We performed a comparative genome-wide analysis of mutation-driven evolution of core (omnipresent) genes in 17 strains of Salmonella enterica subspecies I and 22 strains of Escherichia coli. More than 20% of core genes in both Salmonella and E. coli accumulated hot spot mutations, with a predominance of identical changes having recent evolutionary origin. There is a significant overlap in the functional categories of the adaptively evolving genes in both species, although mostly via separate molecular mechanisms. As a strong evidence of the link between adaptive mutations and virulence in Salmonella, two human-restricted serovars, Typhi and Paratyphi A, shared the highest number of genes with serovar-specific hot spot mutations. Many of the core genes affected by Typhi/Paratyphi A-specific mutations have known virulence functions. For each species, a list of nonrecombinant core genes (and the hot spot mutations therein) under positive selection is provided.

Wash functions downstream of Rho and links linear and branched actin nucleation factors.

Wiskott-Aldrich Syndrome (WAS) family proteins are Arp2/3 activators that mediate the branched-actin network formation required for cytoskeletal remodeling, intracellular transport and cell locomotion. Wasp and Scar/WAVE, the two founding members of the family, are regulated by the GTPases Cdc42 and Rac, respectively. By contrast, linear actin nucleators, such as Spire and formins, are regulated by the GTPase Rho. We recently identified a third WAS family member, called Wash, with Arp2/3-mediated actin nucleation activity. We show that Drosophila Wash interacts genetically with Arp2/3, and also functions downstream of Rho1 with Spire and the formin Cappuccino to control actin and microtubule dynamics during Drosophila oogenesis. Wash bundles and crosslinks F-actin and microtubules, is regulated by Rho1, Spire and Arp2/3, and is essential for actin cytoskeleton organization in the egg chamber. Our results establish Wash and Rho as regulators of both linear- and branched-actin networks, and suggest an Arp2/3-mediated mechanism for how cells might coordinately regulate these structures.

Formins in development: orchestrating body plan origami.

Formins, proteins defined by the presence of an FH2 domain and their ability to nucleate linear F-actin de novo, play a key role in the regulation of the cytoskeleton. Initially thought to primarily regulate actin, recent studies have highlighted a role for formins in the regulation of microtubule dynamics, and most recently have uncovered the ability of some formins to coordinate the organization of both the microtubule and actin cytoskeletons. While biochemical analyses of this family of proteins have yielded many insights into how formins regulate diverse cytoskeletal reorganizations, we are only beginning to appreciate how and when these functional properties are relevant to biological processes in a developmental or organismal context. Developmental genetic studies in fungi, Dictyostelium, vertebrates, plants and other model organisms have revealed conserved roles for formins in cell polarity, actin cable assembly and cytokinesis. However, roles have also been discovered for formins that are specific to particular organisms. Thus, formins perform both global and specific functions, with some of these roles concurring with previous biochemical data and others exposing new properties of formins. While not all family members have been examined across all organisms, the analyses to date highlight the significance of the flexibility within the formin family to regulate a broad spectrum of diverse cytoskeletal processes during development.

Human subtelomeres are hot spots of interchromosomal recombination and segmental duplication.

Human subtelomeres are polymorphic patchworks of interchromosomal segmental duplications at the ends of chromosomes. Here we provide evidence that these patchworks arose recently through repeated translocations between chromosome ends. We assess the relative contribution of the principal mechanisms of ectopic DNA repair to the formation of subtelomeric duplications and find that non-homologous end-joining predominates. Once subtelomeric duplications arise, they are prone to homology-based sequence transfers as shown by the incongruent phylogenetic relationships of neighbouring sections. Interchromosomal recombination of subtelomeres is a potent force for recent change. Cytogenetic and sequence analyses reveal that pieces of the subtelomeric patchwork have changed location and copy number with unprecedented frequency during primate evolution. Half of the known subtelomeric sequence has formed recently, through human-specific sequence transfers and duplications. Subtelomeric dynamics result in a gene duplication rate significantly higher than the genome average and could have both advantageous and pathological consequences in human biology. More generally, our analyses suggest an evolutionary cycle between segmental polymorphisms and genome rearrangements.

Human subtelomeric WASH genes encode a new subclass of the WASP family.

Subtelomeres are duplication-rich, structurally variable regions of the human genome situated just proximal of telomeres. We report here that the most terminally located human subtelomeric genes encode a previously unrecognized third subclass of the Wiskott-Aldrich Syndrome Protein family, whose known members reorganize the actin cytoskeleton in response to extracellular stimuli. This new subclass, which we call WASH, is evolutionarily conserved in species as diverged as Entamoeba. We demonstrate that WASH is essential in Drosophila. WASH is widely expressed in human tissues, and human WASH protein colocalizes with actin in filopodia and lamellipodia. The VCA domain of human WASH promotes actin polymerization by the Arp2/3 complex in vitro. WASH duplicated to multiple chromosomal ends during primate evolution, with highest copy number reached in humans, whose WASH repertoires vary. Thus, human subtelomeres are not genetic junkyards, and WASH's location in these dynamic regions could have advantageous as well as pathologic consequences.

Elevated rates of sister chromatid exchange at chromosome ends.

Chromosome ends are known hotspots of meiotic recombination and double-strand breaks. We monitored mitotic sister chromatid exchange (SCE) in telomeres and subtelomeres and found that 17% of all SCE occurs in the terminal 0.1% of the chromosome. Telomeres and subtelomeres are significantly enriched for SCEs, exhibiting rates of SCE per basepair that are at least 1,600 and 160 times greater, respectively, than elsewhere in the genome.

A sequence-based survey of the complex structural organization of tumor genomes.

BACKGROUND: The genomes of many epithelial tumors exhibit extensive chromosomal rearrangements. All classes of genome rearrangements can be identified using end sequencing profiling, which relies on paired-end sequencing of cloned tumor genomes. RESULTS: In the present study brain, breast, ovary, and prostate tumors, along with three breast cancer cell lines, were surveyed using end sequencing profiling, yielding the largest available collection of sequence-ready tumor genome breakpoints and providing evidence that some rearrangements may be recurrent. Sequencing and fluorescence in situ hybridization confirmed translocations and complex tumor genome structures that include co-amplification and packaging of disparate genomic loci with associated molecular heterogeneity. Comparison of the tumor genomes suggests recurrent rearrangements. Some are likely to be novel structural polymorphisms, whereas others may be bona fide somatic rearrangements. A recurrent fusion transcript in breast tumors and a constitutional fusion transcript resulting from a segmental duplication were identified. Analysis of end sequences for single nucleotide polymorphisms revealed candidate somatic mutations and an elevated rate of novel single nucleotide polymorphisms in an ovarian tumor. CONCLUSION: These results suggest that the genomes of many epithelial tumors may be far more dynamic and complex than was previously appreciated and that genomic fusions, including fusion transcripts and proteins, may be common, possibly yielding tumor-specific biomarkers and therapeutic targets.

Interchromosomal segmental duplications explain the unusual structure of PRSS3, the gene for an inhibitor-resistant trypsinogen.

Homo sapiens possess several trypsinogen or trypsinogen-like genes of which three (PRSS1, PRSS2, and PRSS3) produce functional trypsins in the digestive tract. PRSS1 and PRSS2 are located on chromosome 7q35, while PRSS3 is found on chromosome 9p13. Here, we report a variation of the theme of new gene creation by duplication: the PRSS3 gene was formed by segmental duplications originating from chromosomes 7q35 and 11q24. As a result, PRSS3 transcripts display two variants of exon 1. The PRSS3 transcript whose gene organization most resembles PRSS1 and PRSS2 encodes a functional protein originally named mesotrypsinogen. The other variant is a fusion transcript, called trypsinogen IV. We show that the first exon of trypsinogen IV is derived from the noncoding first exon of LOC120224, a chromosome 11 gene. LOC120224 codes for a widely conserved transmembrane protein of unknown function. Comparative analyses suggest that these interchromosomal duplications occurred after the divergence of Old World monkeys and hominids. PRSS3 transcripts consist of a mixed population of mRNAs, some expressed in the pancreas and encoding an apparently functional trypsinogen and others of unknown function expressed in brain and a variety of other tissues. Analysis of the selection pressures acting on the trypsinogen gene family shows that, while the apparently functional genes are under mild to strong purifying selection overall, a few residues appear under positive selection. These residues could be involved in interactions with inhibitors.

Gene content and function of the ancestral chromosome fusion site in human chromosome 2q13-2q14.1 and paralogous regions.

Various portions of the region surrounding the site where two ancestral chromosomes fused to form human chromosome 2 are duplicated elsewhere in the human genome, primarily in subtelomeric and pericentromeric locations. At least 24 potentially functional genes and 16 pseudogenes reside in the 614-kb of sequence surrounding the fusion site and paralogous segments on other chromosomes. By comparing the sequences of genomic copies and transcripts, we show that at least 18 of the genes in these paralogous regions are transcriptionally active. Among these genes are new members of the cobalamin synthetase W domain (CBWD) and forkhead domain FOXD4 gene families. Copies of RPL23A and SNRPA1 on chromosome 2 are retrotransposed-processed pseudogenes that were included in segmental duplications; we find 53 RPL23A pseudogenes in the human genome and map the functional copy of SNRPA1 to 15qter. The draft sequence of the human genome also provides new information on the location and intron-exon structure of functional copies of other 2q-fusion genes (PGM5, retina-specific F379, helicase CHLR1, and acrosin). This study illustrates that the duplication and rearrangement of subtelomeric and pericentromeric regions have functional relevance to human biology; these processes can change gene dosage and/or generate genes with new functions.

Genomic structure and evolution of the ancestral chromosome fusion site in 2q13-2q14.1 and paralogous regions on other human chromosomes.

Human chromosome 2 was formed by the head-to-head fusion of two ancestral chromosomes that remained separate in other primates. Sequences that once resided near the ends of the ancestral chromosomes are now interstitially located in 2q13-2q14.1. Portions of these sequences had duplicated to other locations prior to the fusion. Here we present analyses of the genomic structure and evolutionary history of >600 kb surrounding the fusion site and closely related sequences on other human chromosomes. Sequence blocks that closely flank the inverted arrays of degenerate telomere repeats marking the fusion site are duplicated at many, primarily subtelomeric, locations. In addition, large portions of a 168-kb centromere-proximal block are duplicated at 9pter, 9p11.2, and 9q13, with 98%-99% average sequence identity. A 67-kb block on the distal side of the fusion site is highly homologous to sequences at 22qter. A third ~100-kb segment is 96% identical to a region in 2q11.2. By integrating data on the extent and similarity of these paralogous blocks, including the presence of phylogenetically informative repetitive elements, with observations of their chromosomal distribution in nonhuman primates, we infer the order of the duplications that led to their current arrangement. Several of these duplicated blocks may be associated with breakpoints of inversions that occurred during primate evolution and of recurrent chromosome rearrangements in humans.