Genetic codes optimized as a traveling salesman problem.

The Standard Genetic Code (SGC) is robust to mutational errors such that frequently occurring mutations minimally alter the physio-chemistry of amino acids. The apparent correlation between the evolutionary distances among codons and the physio-chemical distances among their cognate amino acids suggests an early co-diversification between the codons and amino acids. Here we formulated the co-minimization of evolutionary distances between codons and physio-chemical distances between amino acids as a Traveling Salesman Problem (TSP) and solved it with a Hopfield neural network. In this unsupervised learning algorithm, macromolecules (e.g., tRNAs and aminoacyl-tRNA synthetases) associating codons with amino acids were considered biological analogs of Hopfield neurons associating "tour cities" with "tour positions". The Hopfield network efficiently yielded an abundance of genetic codes that were more error-minimizing than SGC and could thus be used to design artificial genetic codes. We further argue that as a self-optimization algorithm, the Hopfield neural network provides a model of origin of SGC and other adaptive molecular systems through evolutionary learning.

Genome Plasticity of agr-Defective Staphylococcus aureus during Clinical Infection.

Therapy for bacteremia caused by Staphylococcus aureus is often ineffective, even when treatment conditions are optimal according to experimental protocols. Adapted subclones, such as those bearing mutations that attenuate agr-mediated virulence activation, are associated with persistent infection and patient mortality. To identify additional alterations in agr-defective mutants, we sequenced and assembled the complete genomes of clone pairs from colonizing and infected sites of several patients in whom S. aureus demonstrated a within-host loss of agr function. We report that events associated with agr inactivation result in agr-defective blood and nares strain pairs that are enriched in mutations compared to pairs from wild-type controls. The random distribution of mutations between colonizing and infecting strains from the same patient, and between strains from different patients, suggests that much of the genetic complexity of agr-defective strains results from prolonged infection or therapy-induced stress. However, in one of the agr-defective infecting strains, multiple genetic changes resulted in increased virulence in a murine model of bloodstream infection, bypassing the mutation of agr and raising the possibility that some changes were selected. Expression profiling correlated the elevated virulence of this agr-defective mutant to restored expression of the agr-regulated ESAT6-like type VII secretion system, a known virulence factor. Thus, additional mutations outside the agr locus can contribute to diversification and adaptation during infection by S. aureus agr mutants associated with poor patient outcomes.

Genomic confirmation of vancomycin-resistant Enterococcus transmission from deceased donor to liver transplant recipient.

In a liver transplant recipient with vancomycin-resistant Enterococcus (VRE) surgical site and bloodstream infection, a combination of pulsed-field gel electrophoresis, multilocus sequence typing, and whole genome sequencing identified that donor and recipient VRE isolates were highly similar when compared to time-matched hospital isolates. Comparison of de novo assembled isolate genomes was highly suggestive of transplant transmission rather than hospital-acquired transmission and also identified subtle internal rearrangements between donor and recipient missed by other genomic approaches. Given the improved resolution, whole-genome assembly of pathogen genomes is likely to become an essential tool for investigation of potential organ transplant transmissions.

Isolation and Characterization of a Novel Gammaherpesvirus from a Microbat Cell Line.

While employing deep sequencing and de novo assembly to characterize the mRNA transcript profile of a cell line derived from the microbat Myotis velifer incautus, we serendipitously identified mRNAs encoding proteins with a high level of identity to herpesviruses. A majority were closely related to proteins of equine herpesvirus 2 (EHV-2), a horse gammaherpesvirus. We demonstrated by electron microscopy the presence of herpesvirus-like particles in the microbat cells. Passage of supernatants from microbat cells to Vero cells resulted in syncytium formation, and expression of viral genes and amplification of viral DNA were demonstrated by quantitative PCR. Susceptibility of human cell lines to productive infection was also demonstrated. Next-generation sequencing and de novo assembly of the viral genome from supernatants from Vero cells yielded a single contig of approximately 130 kb with at least 77 open reading frames (ORFs), predicted microRNAs (miRNAs), and a gammaherpesvirus genomic organization. Phylogenic analysis of the envelope glycoprotein (gB) and DNA polymerase (POLD1) revealed similarity to multiple gammaherpesviruses, including those from as-yet-uncultured viruses of the Rhadinovirus genus that were obtained by deep sequencing of bat tissues. Moreover, the assembled genome revealed ORFs that share little or no homology to known ORFs in EHV-2 but are similar to accessory proteins of other gammaherpesviruses. Some also have striking homology to predicted Myotis bat proteins. Cumulatively, this study provides the first isolation and characterization of a replication-competent bat gammaherpesvirus. IMPORTANCE Bats are of significant interest as reservoirs for zoonotic viral pathogens; however, tools to dissect bat-virus interactions are limited in availability. This study serendipitously identified, in an established bat cell line, a fully replication-competent gammaherpesvirus; determined the complete genome sequence of the virus; and generated a viral transcript map. This virus can replicate in select human and nonhuman primate cell lines. However, analyses of viral sequences support a bat origin for this virus; we therefore refer to the virus as bat gammaherpesvirus 8 (BGHV8). The viral genome contains unique open reading frames that likely encode modulators of bat innate and adaptive immune signaling pathways and expresses viral miRNAs. The virus and its gene products should provide a unique tool to dissect both bat and gammaherpesvirus biology.

Whole-genome sequencing identifies emergence of a quinolone resistance mutation in a case of Stenotrophomonas maltophilia bacteremia.

Whole-genome sequences for Stenotrophomonas maltophilia serial isolates from a bacteremic patient before and after development of levofloxacin resistance were assembled de novo and differed by one single-nucleotide variant in smeT, a repressor for multidrug efflux operon smeDEF. Along with sequenced isolates from five contemporaneous cases, they displayed considerable diversity compared against all published complete genomes. Whole-genome sequencing and complete assembly can conclusively identify resistance mechanisms emerging in S. maltophilia strains during clinical therapy.

Draft Genome Sequence of Bacillus alcalophilus AV1934, a Classic Alkaliphile Isolated from Human Feces in 1934.

Bacillus alcalophilus AV1934, isolated from human feces, was described in 1934 before microbiome studies and recent indications of novel potassium ion coupling to motility in this extremophile. Here, we report draft sequences that will facilitate an examination of whether that coupling is part of a larger cycle of potassium ion-coupled transporters.

The rise and fall of breakpoint reuse depending on genome resolution.

BACKGROUND: During evolution, large-scale genome rearrangements of chromosomes shuffle the order of homologous genome sequences ("synteny blocks") across species. Some years ago, a controversy erupted in genome rearrangement studies over whether rearrangements recur, causing breakpoints to be reused. METHODS: We investigate this controversial issue using the synteny block's for human-mouse-rat reported by Bourque et al. and a series of synteny blocks we generated using Mauve at resolutions ranging from coarse to very fine-scale. We conducted analyses to test how resolution affects the traditional measure of the breakpoint reuse rate. RESULTS: We found that the inversion-based breakpoint reuse rate is low at fine-scale synteny block resolution and that it rises and eventually falls as synteny block resolution decreases. By analyzing the cycle structure of the breakpoint graph of human-mouse-rat synteny blocks for human-mouse and comparing with theoretically derived distributions for random genome rearrangements, we showed that the implied genome rearrangements at each level of resolution become more "random" as synteny block resolution diminishes. At highest synteny block resolutions the Hannenhalli-Pevzner inversion distance deviates from the Double Cut and Join distance, possibly due to small-scale transpositions or simply due to inclusion of erroneous synteny blocks. At synteny block resolutions as coarse as the Bourque et al. blocks, we show the breakpoint graph cycle structure has already converged to the pattern expected for a random distribution of synteny blocks. CONCLUSIONS: The inferred breakpoint reuse rate depends on synteny block resolution in human-mouse genome comparisons. At fine-scale resolution, the cycle structure for the transformation appears less random compared to that for coarse resolution. Small synteny blocks may contain critical information for accurate reconstruction of genome rearrangement history and parameters.

The landscape of C. elegans 3'UTRs.

Three-prime untranslated regions (3'UTRs) of metazoan messenger RNAs (mRNAs) contain numerous regulatory elements, yet remain largely uncharacterized. Using polyA capture, 3' rapid amplification of complementary DNA (cDNA) ends, full-length cDNAs, and RNA-seq, we defined approximately 26,000 distinct 3'UTRs in Caenorhabditis elegans for approximately 85% of the 18,328 experimentally supported protein-coding genes and revised approximately 40% of gene models. Alternative 3'UTR isoforms are frequent, often differentially expressed during development. Average 3'UTR length decreases with animal age. Surprisingly, no polyadenylation signal (PAS) was detected for 13% of polyadenylation sites, predominantly among shorter alternative isoforms. Trans-spliced (versus non-trans-spliced) mRNAs possess longer 3'UTRs and frequently contain no PAS or variant PAS. We identified conserved 3'UTR motifs, isoform-specific predicted microRNA target sites, and polyadenylation of most histone genes. Our data reveal a rich complexity of 3'UTRs, both genome-wide and throughout development.

Co-evolution of the outer surface protein C gene (ospC) and intraspecific lineages of Borrelia burgdorferi sensu stricto in the northeastern United States.

Clinical and tick isolates of Borrelia burgdorferi sensu stricto, the bacterial agent of Lyme disease, from the northeastern United States were sequenced at 12 loci located on the main chromosome and 7 plasmids (lp54, cp26, cp9, lp17, lp25, lp28-2, and lp38). The outer surface protein C gene (ospC) showed the highest number (12) of major alleles (defined as alleles differing by 5% or more in nucleotide sequence) while other ORFs had only two to four major alleles. A non-recombining chromosomal marker, the rrs-rrlA ribosomal RNA spacer, was used to infer the intraspecific phylogeny among these B. burgdorferi isolates. We were thus able to analyze the multilocus genotypes in the context of a B. burgdorferi intraspecific phylogeny. Except for ospC, sequence variations at plasmid-borne loci showed broad inconsistency with the intraspecific phylogeny, supporting DNA exchanges mediated by plasmid transfers. The multilocus linkage frequently observed in B. burgdorferi populations is due more likely to a "founder effect" than to a lack of recombination. The exceptional phylogenetic consistency of ospC, in conjunction with its selectively maintained high intraspecific diversity, suggested a dominant role ospC plays in the initiation and maintenance of adaptive differentiation in B. burgdorferi.

Composition-modified matrices improve identification of homologs of saccharomyces cerevisiae low-complexity glycoproteins.

Yeast glycoproteins are representative of low-complexity sequences, those sequences rich in a few types of amino acids. Low-complexity protein sequences comprise more than 10% of the proteome but are poorly aligned by existing methods. Under default conditions, BLAST and FASTA use the scoring matrix BLOSUM62, which is optimized for sequences with diverse amino acid compositions. Because low-complexity sequences are rich in a few amino acids, these tools tend to align the most common residues in nonhomologous positions, thereby generating anomalously high scores, deviations from the expected extreme value distribution, and small e values. This anomalous scoring prevents BLOSUM62-based BLAST and FASTA from identifying correct homologs for proteins with low-complexity sequences, including Saccharomyces cerevisiae wall proteins. We have devised and empirically tested scoring matrices that compensate for the overrepresentation of some amino acids in any query sequence in different ways. These matrices were tested for sensitivity in finding true homologs, discrimination against nonhomologous and random sequences, conformance to the extreme value distribution, and accuracy of e values. Of the tested matrices, the two best matrices (called E and gtQ) gave reliable alignments in BLAST and FASTA searches, identified a consistent set of paralogs of the yeast cell wall test set proteins, and improved the consistency of secondary structure predictions for cell wall proteins.

Efficient sorting of genomic permutations by translocation, inversion and block interchange.

MOTIVATION: Finding genomic distance based on gene order is a classic problem in genome rearrangements. Efficient exact algorithms for genomic distances based on inversions and/or translocations have been found but are complicated by special cases, rare in simulations and empirical data. We seek a universal operation underlying a more inclusive set of evolutionary operations and yielding a tractable genomic distance with simple mathematical form. RESULTS: We study a universal double-cut-and-join operation that accounts for inversions, translocations, fissions and fusions, but also produces circular intermediates which can be reabsorbed. The genomic distance, computable in linear time, is given by the number of breakpoints minus the number of cycles (b-c) in the comparison graph of the two genomes; the number of hurdles does not enter into it. Without changing the formula, we can replace generation and re-absorption of a circular intermediate by a generalized transposition, equivalent to a block interchange, with weight two. Our simple algorithm converts one multi-linear chromosome genome to another in the minimum distance.

Genetic exchange and plasmid transfers in Borrelia burgdorferi sensu stricto revealed by three-way genome comparisons and multilocus sequence typing.

Comparative genomics of closely related bacterial isolates is a powerful method for uncovering virulence and other important genome elements. We determined draft sequences (8-fold coverage) of the genomes of strains JD1 and N40 of Borrelia burgdorferi sensu stricto, the causative agent of Lyme disease, and we compared the predicted genes from the two genomes with those from the previously sequenced B31 genome. The three genomes are closely related and are evolutionarily approximately equidistant ( approximately 0.5% pairwise nucleotide differences on the main chromosome). We used a Poisson model of nucleotide substitution to screen for genes with elevated levels of nucleotide polymorphisms. The three-way genome comparison allowed distinction between polymorphisms introduced by mutations and those introduced by recombination using the method of phylogenetic partitioning. Tests for recombination suggested that patches of high-density nucleotide polymorphisms on the chromosome and plasmids arise by DNA exchange. The role of recombination as the main mechanism driving B. burgdorferi diversification was confirmed by multilocus sequence typing of 18 clinical isolates at 18 polymorphic loci. A strong linkage between the multilocus sequence genotypes and the major alleles of outer-surface protein C (ospC) suggested that balancing selection at ospC is a dominant force maintaining B. burgdorferi diversity in local populations. We conclude that B. burgdorferi undergoes genome-wide genetic exchange, including plasmid transfers, and previous reports of its clonality are artifacts from the use of geographically and ecological isolated samples. Frequent recombination implies a potential for rapid adaptive evolution and a possible polygenic basis of B. burgdorferi pathogenicity.