Distinct Phylogenetic Clusters Research Articles

Bacterial community composition and fhs profiles of low- and high-ammonia biogas digesters reveal novel syntrophic acetate-oxidising bacteria.

BackgroundSyntrophic acetate oxidation (SAO) is the predominant pathway for methane production in high ammonia anaerobic digestion processes. The bacteria (SAOB) occupying this niche and the metabolic pathway are poorly understood. Phylogenetic diversity and strict cultivation requirements hinder comprehensive research and discovery of novel SAOB. Most SAOB characterised to date are affiliated to the physiological group of acetogens. Formyltetrahydrofolate synthetase is a key enzyme of both acetogenic and SAO metabolism. The encoding fhs gene has therefore been identified as a suitable functional marker, using a newly designed primer pair. In this comparative study, we used a combination of terminal restriction fragment length polymorphism profiling, clone-based comparison, qPCR and Illumina amplicon sequencing to assess the bacterial community and acetogenic sub-community prevailing in high- and low-ammonia laboratory-scale digesters in order to delineate potential SAOB communities. Potential candidates identified were further tracked in a number of low-ammonia and high-ammonia laboratory-scale and large-scale digesters in order to reveal a potential function in SAO.ResultsAll methodical approaches revealed significant changes in the bacterial community composition concurrently with increasing ammonia and predominance of SAO. The acetogenic community under high ammonia conditions was revealed to be generally heterogeneous, but formed distinct phylogenetic clusters. The clusters differed clearly from those found under low-ammonia conditions and represented an acetogenic assemblage unique for biogas processes and recurring in a number of high-ammonia processes, indicating potential involvement in SAO.ConclusionsThe phylogenetic affiliation and population dynamics observed point to a key community, belonging mainly to the Clostridia class, in particular to the orders Clostridiales and Thermoanaerobacterales, which appear to specialise in SAO rather than being metabolically versatile. Overall, the results reported here provide evidence of functional importance of the bacterial families identified in high-ammonia systems and extend existing knowledge of bacterial and acetogenic assemblages at low and high ammonia levels. This information will be of help in monitoring and assessing the impacts on the SAOB community in order to identify characteristics of robust and productive high ammonia biogas processes.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0454-9) contains supplementary material, which is available to authorized users.

A novel homolog of protein tyrosine kinase Fyn identified in Lampetra japonica with roles in the immune response

The non-receptor protein tyrosine kinase (nrPTK) Fyn, a member of the avian sarcoma virus transforming gene (Src) kinase family, plays a very significant role in cell growth, survival, apoptosis, tumor formation and immune response. In this study, a homolog of nrPTK Fyn was identified for the first time in the lamprey, Lampetra japonica and was named “Lja-Fyn”. The cDNA fragment of lamprey lja-fyn contains a 1611-bp open reading frame, which encodes a protein of 537 amino acids. Multiple sequence alignment analysis showed that it shares four conserved domains (Src homology (SH) 4, SH3, SH2 and protein kinases catalytic domains) and a variable unique domain with vertebrates Fyn molecules. Though Lja-Fyn has high sequence similarity with typical Fyn and Yes molecules of jawed vertebrates, the identities among Lja-Fyn and typical Fyn molecules in unique domain are relatively higher than that among Lja-Fyn and typical Yes molecules. The result indicates that Lja-Fyn is a homolog of Fyn rather than Yes. The phylogenetic analysis showed that Fyn, Yes and Src molecules are grouped into three distinct phylogenetic clusters, and Lja-Fyn is grouped as a single branch in Fyn cluster. The real-time quantitative PCR assay revealed the wide distribution of the lja-fyn mRNA in lamprey immune related tissues. After stimulation with mixed antigens, the levels of lja-fyn mRNA were obviously up-regulated in the gill and lymphocyte-like cells, and the similar results were got by western blot analysis of Lja-Fyn protein expression. These results indicated that nrPTK Lja-Fyn was likely to be involved in immune response. Furthermore, our present findings also provide the necessary information for understanding the distinction between lamprey Lja-Fyn and other members of jawed vertebrates in Src family.

Characteristics of <i>Acidibacillus</i> Spp.: A Novel Genus of Acidophilic Iron-Oxidising <i>Firmicutes</i>

Strains of acidophilic bacteria, isolated from different global locations, were shown to be representatives of a novel genus within theFirmicutesphylum. All isolates oxidised and reduced iron and catalysed the oxidative dissolution of pyrite. Together they formed two distinct phylogenetic clusters, one of which included mesophilic strains that did not oxidise sulfur, and the other thermotolerant/moderately thermophilic strains that could oxidise sulfur. The novel genusAcidibacillusis proposed for these new isolates, to includeA. ferrooxidans(mesophilic iron-oxidising strains) andA.sulfooxidans(moderately thermophilic iron-and sulfur-oxidising strains) as candidate species.

Whole-Genome Sequencing of Kaposi's Sarcoma-Associated Herpesvirus from Zambian Kaposi's Sarcoma Biopsy Specimens Reveals Unique Viral Diversity

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent for Kaposi's sarcoma (KS). Both KSHV and KS are endemic in sub-Saharan Africa where approximately 84% of global KS cases occur. Nevertheless, whole-genome sequencing of KSHV has only been completed using isolates from Western countries-where KS is not endemic. The lack of whole-genome KSHV sequence data from the most clinically important geographical region, sub-Saharan Africa, represents an important gap since it remains unclear whether genomic diversity has a role on KSHV pathogenesis. We hypothesized that distinct KSHV genotypes might be present in sub-Saharan Africa compared to Western countries. Using a KSHV-targeted enrichment protocol followed by Illumina deep-sequencing, we generated and analyzed 16 unique Zambian, KS-derived, KSHV genomes. We enriched KSHV DNA over cellular DNA 1,851 to 18,235-fold. Enrichment provided coverage levels up to 24,740-fold; therefore, supporting highly confident polymorphism analysis. Multiple alignment of the 16 newly sequenced KSHV genomes showed low level variability across the entire central conserved region. This variability resulted in distinct phylogenetic clustering between Zambian KSHV genomic sequences and those derived from Western countries. Importantly, the phylogenetic segregation of Zambian from Western sequences occurred irrespective of inclusion of the highly variable genes K1 and K15. We also show that four genes within the more conserved region of the KSHV genome contained polymorphisms that partially, but not fully, contributed to the unique Zambian KSHV whole-genome phylogenetic structure. Taken together, our data suggest that the whole KSHV genome should be taken into consideration for accurate viral characterization. Our results represent the largest number of KSHV whole-genomic sequences published to date and the first time that multiple genomes have been sequenced from sub-Saharan Africa, a geographic area where KS is highly endemic. Based on our new sequence data, it is apparent that whole-genome KSHV diversity is greater than previously appreciated and differential phylogenetic clustering exists between viral genomes of Zambia and Western countries. Furthermore, individual genes may be insufficient for KSHV genetic characterization. Continued investigation of the KSHV genetic landscape is necessary in order to effectively understand the role of viral evolution and sequence diversity on KSHV gene functions and pathogenesis.

Analysis of conserved NCS2 motifs in the Escherichia coli xanthine permease XanQ.

The xanthine permease XanQ of Escherichia coli is a paradigm for transporters of the evolutionarily broad family nucleobase-cation symporter-2 (NCS2) that transport key metabolites or anti-metabolite analogs. Most functionally known members are xanthine/uric acid transporters related to XanQ and belong to a distinct phylogenetic cluster of the family. Here, we present a comprehensive mutagenesis of XanQ based on the identification and Cys-scanning analysis of conserved sequence motifs in this cluster. Results are interpreted in relation to homology modeling on the structurally known template of UraA and previous data on critical binding-site residues in transmembrane segments (TMs) 3, 8 and 10. The current analysis, of motifs distant to the binding site, revealed a set of functionally important residues in TMs 2, 5, 12 and 13, including seven irreplaceable ones, of which six are Gly residues in the gate domain (159, 369, 370, 383, 409) and in TM2 (Gly-71), and one is polar (Gln-75). Gln-75 (TM2) is probably crucial in a network of hydrogen-bonding interactions in the middle of the core domain involving another essential residue, Asp-304 (TM9). Although the two residues are irreplaceable individually, combinatorial replacement of Gln-75 with Asn and of Asp-304 with Glu rescues significant transport activity.

Molecular epidemiological characterization of human immunodeficiency virus-1 CRF01_AE strains in China

Objective To understand the molecular epidemiological characterization of human immunodeficiency virus (HIV)-1 CRF01_AE strains in China. Methods Data were extracted by a systematic search in the databases combined with literature review. Data were then grouped according to the sites and risk groups for a Meta-analysis. Sequences of CRF01_AE pol genes from China were downloaded from Los Alamos database to build phylogenetic trees by means of FastTree2.1. The Bayes factor test was calculated by BEAST V1.6.2 package and Spread to explore spatial transmission links. Results Meta-analysis demonstrated that CRF01_AE strains accounted for more than 45.0% of all subtypes among men who have sex with men (MSM) in six areas of China. Among heterosexuals in eastern, northern, southwestern and south-central China, the proportions of CRF01_AE strains exceeded 30.0%. The strains were also prevalent among intravenous drug users in south-central regions, accounting for 57.3% (95%CI: 35.1%-79.6%), and were only detected among blood donors in south-central China (10.6%, 95%CI: 6.2%-14.9%). Seven distinct phylogenetic clusters of CRF01_AE strains which were transmitted independently were identified. Clusters 1, 3 and 5 were prevalent among heterosexuals, while clusters 2 and 6 were found primarily among MSM, and clusters 4 was detected chiefly among sexual contact people. Geographically, clusters 1, 2, 3, 4 and 5 were prevalent in southwestern areas. Clusters 1 and 7 were circulating in south-central areas. Clusters 2 and 6 were prevalent in northern areas, while clusters 2 and 4 were dominant in eastern regions and cluster 6 was prevalent in northeastern China. The Bayes factor test reveals the complexity of transmission links among eastern, south-central, southwestern, northern, and northeastern provinces. Conclusion CRF01_AE strains are prevalent in most high risk groups in multiple regions of China and the transmission between different regions is complicated. Key words: CRF01_AE strains; Epidemiology, molecular; Meta-analysis; Phylogenetic analysis; Bayes theorem

A genomic and transcriptomic approach to investigate the blue pigment phenotype in Pseudomonas fluorescens

Pseudomonas fluorescens is a well-known food spoiler, able to cause serious economic losses in the food industry due to its ability to produce many extracellular, and often thermostable, compounds. The most outstanding spoilage events involving P. fluorescens were blue discoloration of several food stuffs, mainly dairy products. The bacteria involved in such high-profile cases have been identified as belonging to a clearly distinct phylogenetic cluster of the P. fluorescens group. Although the blue pigment has recently been investigated in several studies, the biosynthetic pathway leading to the pigment formation, as well as its chemical nature, remain challenging and unsolved points.In the present paper, genomic and transcriptomic data of 4 P. fluorescens strains (2 blue-pigmenting strains and 2 non-pigmenting strains) were analyzed to evaluate the presence and the expression of blue strain-specific genes. In particular, the pangenome analysis showed the presence in the blue-pigmenting strains of two copies of genes involved in the tryptophan biosynthesis pathway (including trpABCDF). The global expression profiling of blue-pigmenting strains versus non-pigmenting strains showed a general up-regulation of genes involved in iron uptake and a down-regulation of genes involved in primary metabolism. Chromogenic reaction of the blue-pigmenting bacterial cells with Kovac's reagent indicated an indole-derivative as the precursor of the blue pigment. Finally, solubility tests and MALDI-TOF mass spectrometry analysis of the isolated pigment suggested that its molecular structure is very probably a hydrophobic indigo analog.

Paenibacillus oenotherae sp. nov. and Paenibacillus hemerocallicola sp. nov., isolated from the roots of herbaceous plants.

Two Gram-staining-positive, aerobic, endospore-forming, motile bacteria, strains DT7-4T and DLE-12T, were isolated from roots of evening primrose (Oenothera biennis) and day lily (Hemerocallis fulva), respectively, and subjected to taxonomic characterization. Analysis of 16S rRNA gene sequences indicated that the two strains fell into two distinct phylogenetic clusters belonging to the genus Paenibacillus. Strain DT7-4T was most closely related to Paenibacillus phyllosphaerae PALXIL04T and Paenibacillus taihuensis THMBG22T, with 96.3% 16S rRNA gene sequence similarity to each, and strain DLE-12T was most closely related to Paenibacillus ginsengarvi Gsoil 139T and Paenibacillus hodogayensis SGT, with 96.6 and 93.3% sequence similarity, respectively. Both isolates contained anteiso-C15 : 0 as the dominant fatty acid, meso-diaminopimelic acid as the diagnostic diamino acid in the cell-wall peptidoglycan and MK-7 as the respiratory menaquinone. The cellular polar lipids were composed of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and unidentified polar lipids. The DNA G+C contents of strains DT7-4T and DLE-12T were 50.1 ± 0.7 and 55.2 ± 0.5 mol%, respectively. The chemotaxonomic properties of both isolates were typical of members of the genus Paenibacillus. However, our biochemical and phylogenetic analyses distinguished each isolate from related species. Based on our polyphasic taxonomic analysis, strains DT7-4T and DLE-12T should be recognized as representatives of novel species of Paenibacillus, for which the names Paenibacillus oenotherae sp. nov. (type strain DT7-4T = KCTC 33186T = JCM 19573T) and Paenibacillus hemerocallicola sp. nov. (type strain DLE-12T = KCTC 33185T = JCM 19572T) are proposed.

A duplex PCR assay for the detection of Ralstonia solanacearum phylotype II strains in Musa spp.

Banana wilt outbreaks that are attributable to Moko disease-causing strains of the pathogen Ralstonia solanacearum (Rs) remain a social and economic burden for both multinational corporations and subsistence farmers. All known Moko strains belong to the phylotype II lineage, which has been previously recognized for its broad genetic basis. Moko strains are paraphyletic and are distributed among seven related but distinct phylogenetic clusters (sequevars) that are potentially major threats to Musaceae, Solanaceae, and ornamental crops in many countries. Although clustered within the Moko IIB-4 sequevar, strains of the epidemiologically variant IIB-4NPB do not cause wilt on Cavendish or plantain bananas; instead, they establish a latent infection in the vascular tissues of plantains and demonstrate an expanded host range and high aggressiveness toward Solanaceae and Cucurbitaceae. Although most molecular diagnostic methods focus on strains that wilt Solanaceae (particularly potato), no relevant protocol has been described that universally detects strains of the Musaceae-infecting Rs phylotype II. Thus, a duplex PCR assay targeting Moko and IIB-4NPB variant strains was developed, and its performance was assessed using an extensive collection of 111 strains representing the known diversity of Rs Moko-related strains and IIB-4NPB variant strains along with certain related strains and families. The proposed diagnostic protocol demonstrated both high accuracy (inclusivity and exclusivity) and high repeatability, detected targets on either pure culture or spiked plant extracts. Although they did not belong to the Moko clusters described at the time of the study, recently discovered banana-infecting strains from Brazil were also detected. According to our comprehensive evaluation, this duplex PCR assay appears suitable for both research and diagnostic laboratories and provides reliable detection of phylotype II Rs strains that infect Musaceae.

Temporal dynamics and subpopulation analysis of Theileria orientalis genotypes in cattle

In Australia, outbreaks of clinical theileriosis caused by Theileria orientalis have been largely associated with the Ikeda genotype which can occur as a sole infection, or more commonly, as a mixture of genotypes. The most prevalent genotype, Chitose, frequently co-occurs with type Ikeda, however the role of this genotype in clinical disease has not been clearly established. Furthermore, the dynamics of individual genotypes in field infection of cattle have not been examined. In this study we developed quantitative PCR (qPCR) and genotyping methods to examine the role of the Chitose genotype in clinical disease and to investigate the temporal dynamics of T. orientalis Ikeda, Chitose and Buffeli genotypes in naïve animals introduced to a T. orientalis-endemic area. Analysis of the major piroplasm surface protein (MPSP) genes of Chitose isolates revealed the presence of two distinct phylogenetic clusters, Chitose A and Chitose B. A genotyping assay aimed at determining Chitose A/B allele frequency revealed that the Chitose A phylogenetic cluster is strongly associated with clinical disease but nearly always co-occurs with the Ikeda genotype. qPCR revealed that the Chitose genotype (particularly Chitose A), undergoes temporal switching in conjunction with the Ikeda genotype and contributes substantially to the overall parasite burden. The benign Buffeli genotype can also undergo temporal switching but levels of this genotype appear to remain low relative to the Ikeda and Chitose types. Interplay between vector and host immunological factors is presumed to be critical to the population dynamics observed in this study. Genotypic switching likely contributes to the persistence of T. orientalis in the host.

DNA barcoding of Japanese click beetles (Coleoptera, Elateridae).

Click beetles (Coleoptera: Elateridae) represent one of the largest groups of beetle insects. Some click beetles in larval form, known as wireworms, are destructive agricultural pests. Morphological identification of click beetles is generally difficult and requires taxonomic expertise. This study reports on the DNA barcoding of Japanese click beetles to enable their rapid and accurate identification. We collected and assembled 762 cytochrome oxidase subunit I barcode sequences from 275 species, which cover approximately 75% of the common species found on the Japanese main island, Honshu. This barcode library also contains 20 out of the 21 potential pest species recorded in Japan. Our analysis shows that most morphologically identified species form distinct phylogenetic clusters separated from each other by large molecular distances. This supports the general usefulness of the DNA barcoding approach for quick and reliable identification of Japanese elaterid species for environmental impact assessment, agricultural pest control, and biodiversity analysis. On the other hand, the taxonomic boundary in dozens of species did not agree with the boundary of barcode index numbers (a criterion for sequence-based species delimitation). These findings urge taxonomic reinvestigation of these mismatched taxa.

Oligoflexia, the Newest Class of the Phylum Proteobacteria, Consisting of only One Cultured Species and Uncultured Bacterial Phylotypes from Diverse Habitats

The phylum Proteobacteria has recently gained a new taxon Oligoflexia that represents the seventh or eighth (if yet-to-be-validated “Zetaproteobacteria” is included) class, described by the only cultured species (as of December 2014), Oligoflexus tunisiensis, as the type species. This bacterium exhibits cellular polymorphism and presence of the uncommon fatty acid C16: 1ω5c as high as >65% of total fatty acids, besides its unique 16S rRNA gene sequence. The class Oligoflexia is characterized by the distinct phylogenetic cluster within the greater proteobacterial cluster, and certain environmentally-derived 16S rRNA gene sequences, a.k.a. environmental clones or phylotypes, of uncultured bacteria are now grouped into the Oligoflexia cluster; however, the content and extent of the cluster has not been clearly depicted. This mini-review illustrates that the Oligoflexia cluster hosts a variety of environmental clones from diverse sources. Currently 20 phylotypes (or clones) are affiliated with the Oligoflexia cluster, and the sources were ranging from soils to cyanobacterial mat, bio-filter, human skin, ant colony, desert, glacial ice, earthworm intestine, and seawater. However, their frequencies in respective clone libraries were generally as low as 85% similarities (class-level affiliation) to the 16S rRNA gene sequence of O. tunisiensis, which accounts for merely 0.04% of those registered in Meta-Metagenomic Data Base (MetaMeta DB). On the other hand, >97%-similarities sequences were found in rhizosphere, and <95%-similar sequences were found from hydrocarbon-rich habitats such as petroleum reservoir. Thus, it is suggested that members of Oligoflexia may display cosmopolite distribution in general as well as endemism in certain geochemical settings.

Unencapsulated Streptococcus pneumoniae from conjunctivitis encode variant traits and belong to a distinct phylogenetic cluster.

Streptococcus pneumoniae, an inhabitant of the upper respiratory mucosa, causes respiratory and invasive infections as well as conjunctivitis. Strains that lack the capsule, a main virulence factor and the target of current vaccines, are often isolated from conjunctivitis cases. Here we perform a comparative genomic analysis of 271 strains of conjunctivitis-causing S. pneumoniae from 72 postal codes in the US. We find that the vast majority of conjunctivitis strains are members of a distinct cluster of closely related unencapsulated strains. These strains possess divergent forms of pneumococcal virulence factors (such as CbpA and neuraminidases) that are not shared with other unencapsulated nasopharyngeal S. pneumoniae. They also possess putative adhesins that have not been described in encapsulated pneumococci. These findings suggest that the unencapsulated strains capable of causing conjunctivitis utilize a pathogenesis strategy substantially different from that described for S. pneumoniae at other infection sites.

Genetic variability of Babesia parasites in Haemaphysalis spp. and Ixodes persulcatus ticks in the Baikal region and Far East of Russia

To study Babesia diversity in Ixodid ticks in Russia, Ixodes persulcatus, Haemaphysalis japonica, Haemaphysalisconcinna, Dermacentor silvarum, and Dermacentor nuttalli ticks collected in the Far East and Baikal region were assayed for the presence of Babesia spp. using nested PCR. In total, Babesia DNA was detected in 30 of the 1125 (2.7%) I. persulcatus, 17 of the 573 (3.0%) H. concinna, and 12 of the 543 (2.2%) H. japonica but was undetectable in any of the 294 analyzed Dermacentor spp. Partial 18S rRNA gene sequences were determined for all of the positive samples. Among the positive ticks, nine I. persulcatus were infected by Babesia microti ‘US’-type, five I. persulcatus were infected by Babesia divergens-like parasites, and 11 I. persulcatus were infected by Babesia venatorum. For all three of these species, the determined 18S rRNA gene sequences were identical to those of the Babesia genetic variants found previously in I. persulcatus in Russia. In addition, five I. persulcatus from the Baikal region and all of the positive Haemaphysalis spp. ticks carried 13 different sequence variants of Babesia sensu stricto belonging to distinct phylogenetic clusters. Babesia spp. from 29 ticks of different species collected in distinct locations belonged to the cluster of cattle and ovine parasites (Babesia crassa, Babesiamajor, Babesiamotasi, Babesiabigemina, etc.). Babesia spp. from four H. japonica ticks in the Far East belonged to the cluster formed by parasites of carnivores. One more Babesia sequence variant detected in an I. persulcatus tick from the Baikal region belonged to the cluster formed by parasites of cattle and wild cervids (B. divergens, Babesiacapreoli, B. venatorum, Babesiaodocoilei, etc.).

Molecular and biological evidence for a severe seedling yellows strain of Citrus tristeza virus spreading in southern Italy

Citrus tristeza virus (CTV) outbreaks have been reported in the main citrus-growing regions of Italy in the past 10 years. In some areas where eradication efforts failed to suppress spread, high CTV incidence is now observed. Recently, potentially severe CTV strains were detected in Calabria (southern Italy), one of the major citrus-growing area. As a result, investigations of the virulence and molecular features of CTV populations spreading in this region were undertaken. Virus was detected by enzyme-linked immunosorbent assay (ELISA) using a broad spectrum polyclonal antiserum, and was differentiated into potential virulent categories with the severe-strain discriminating monoclonal antibody MCA13. Isolate genotyping was conducted using reverse-transcription polymerase chain reaction (RT-PCR) with multiple molecular markers (MMM), single-strand conformation polymorphism (SSCP) analysis of the amplicons from the genes coding for the coat protein (CP) p25 and the non-structural p20 protein as well as sequence analysis. Based on the serological reactivity, the isolates were differentiated in two distinct serogroups: MCA13-reactive and MCA13 non-reactive. Similarly, based on the molecular profile, the isolates were grouped in two genetically distinct phylogenetic clusters, and associated either with a T30-like or with a T3-like genotype. These data were related to the results of biological indexing on standard indicator plants, which distinguished isolates causing mild or severe seedling yellow reactions. The study has demonstrated the presence of MCA13-reactive isolates associated with a T3-like genotype and causing severe seedling yellows in sour orange, grapefruit and lemon seedlings, and stem pitting in Mexican lime.

Among the three striatin family members SG2NA was first to arise during evolution

Striatin, SG2NA, and zinedin constitute a three-member subfamily of WD-40 repeat proteins. They are found only in metazoans and are likely to have scaffolding functions. Apart from WD-40 repeats, they also have a caveolin-binding motif, a coiled-coil structure, and a calmodulin-binding domain. This paper focuses on the analysis of their evolution as a paradigm of understanding the metazoan scaffolds. Each member of the family forms distinct phylogenetic clusters, wherein striatins, SG2NAs, and zinedins have 13, 10, and 9 conserved motifs, respectively. Furthermore, two of those motifs each in striatin and in zinedin and three in SG2NA are exclusive for the respective subfamily. Of those exclusive motifs for SG2NA, two encompass the caveolin-binding and coiled-coiled domains. Collectively, they show the presence of 11 conserved motifs, suggestive of convergence of individual motifs and creation of patterns. A prokaryotic WD-40 repeat motif pCM-I was found only in the corresponding domain of SG2NA but not in other family members. It is thus hypothesized that striatin family members have evolved from bacteria, and SG2NA was the first member to arise.

Identification and Environmental Distribution ofdcpA, Which Encodes the Reductive Dehalogenase Catalyzing the Dichloroelimination of 1,2-Dichloropropane to Propene in Organohalide-Respiring Chloroflexi

Dehalococcoides mccartyi strains KS and RC grow with 1,2-dichloropropane (1,2-D) as an electron acceptor in enrichment cultures derived from hydrocarbon-contaminated and pristine river sediments, respectively. Transcription, expression, enzymatic, and PCR analyses implicated the reductive dehalogenase gene dcpA in 1,2-D dichloroelimination to propene and inorganic chloride. Quantitative real-time PCR (qPCR) analyses demonstrated a D. mccartyi cell increase during growth with 1,2-D and suggested that both D. mccartyi strains carried a single dcpA gene copy per genome. D. mccartyi strain RC and strain KS produced 1.8 × 10(7) ± 0.1 × 10(7) and 1.4 × 10(7) ± 0.5 × 10(7) cells per μmol of propene formed, respectively. The dcpA gene was identified in 1,2-D-to-propene-dechlorinating microcosms established with sediment samples collected from different geographical locations in Europe and North and South America. Clone library analysis revealed two distinct dcpA phylogenetic clusters, both of which were captured by the dcpA gene-targeted qPCR assay, suggesting that the qPCR assay is useful for site assessment and bioremediation monitoring at 1,2-D-contaminated sites.

Local phylogenetic analysis identifies distinct trends in transmitted HIV drug resistance: implications for public health interventions

BackgroundHIV transmitted drug resistance (TDR) surveillance is usually conducted by sampling from a large population. However, overall TDR prevalence results may be inaccurate for many individual clinical setting. We analyzed HIV genotypes at a tertiary care setting in Ottawa, Ontario in order to evaluate local TDR patterns among sub-populations.MethodGenotyping reports were digitized from ART naïve patients followed at the Immunodeficiency Clinic at the Ottawa Hospital, between 2008 and 2010. Quality controlled, digitized sequence data were assessed for TDR using the Stanford HIV Database. Patient characteristics were analyzed according to TDR patterns. Finally, a phylogenetic tree was constructed to elucidate the observed pattern of HIV TDR.ResultsAmong the 155 clinic patients there was no statistically significantly difference in demographics as compared to the Ontario provincial HIV population. The clinic prevalence of TDR was 12.3%; however, in contrast to the data from Ontario, TDR patterns were inverted with a 21% prevalence among MSM and 5.5% among IDU. Furthermore, nearly 80% of the observed TDR was a D67N/K219Q pattern with 87% of these infections arising from a distinct phylogenetic cluster.ConclusionsLocal patterns of TDR were distinct to what had been observed provincially. Phylogenetic analysis uncovered a cluster of related infections among MSM that appeared more likely to be recent infections. Results support a paradigm of routine local TDR surveillance to identify the sub-populations under care. Furthermore, the routine application of phylogenetic analysis in the TDR surveillance context provides insights into how best to target prevention strategies; and how to correctly measure outcomes.

Genomics of an emerging clone of Salmonella serovar Typhimurium ST313 from Nigeria and the Democratic Republic of Congo

Salmonella enterica serovar Typhimurium ST313 is an invasive and phylogenetically distinct lineage present in sub-Saharan Africa. We report the presence of S. Typhimurium ST313 from patients in the Democratic Republic of Congo and Nigeria. Eighteen S. Typhimurium ST313 isolates were characterized by antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST). Additionally, six of the isolates were characterized by whole genome sequence typing (WGST). The presence of a putative virulence determinant was examined in 177 Salmonella isolates belonging to 57 different serovars. All S. Typhimurium ST313 isolates harbored resistant genes encoded by blaTEM1b, catA1, strA/B, sul1, and dfrA1. Additionally, aac(6')1aa gene was detected. Phylogenetic analyses revealed close genetic relationships among Congolese and Nigerian isolates from both blood and stool. Comparative genomic analyses identified a putative virulence fragment (ST313-TD) unique to S. Typhimurium ST313 and S. Dublin. We showed in a limited number of isolates that S. Typhimurium ST313 is a prevalent sequence-type causing gastrointestinal diseases and septicemia in patients from Nigeria and DRC. We found three distinct phylogenetic clusters based on the origin of isolation suggesting some spatial evolution. Comparative genomics showed an interesting putative virulence fragment (ST313-TD) unique to S. Typhimurium ST313 and invasive S. Dublin.

Genome and Transcriptome Adaptation Accompanying Emergence of the Definitive Type 2 Host-Restricted Salmonella enterica Serovar Typhimurium Pathovar

ABSTRACTSalmonella enterica serovar Typhimurium definitive type 2 (DT2) is host restricted to Columba livia (rock or feral pigeon) but is also closely related to S. Typhimurium isolates that circulate in livestock and cause a zoonosis characterized by gastroenteritis in humans. DT2 isolates formed a distinct phylogenetic cluster within S. Typhimurium based on whole-genome-sequence polymorphisms. Comparative genome analysis of DT2 94-213 and S. Typhimurium SL1344, DT104, and D23580 identified few differences in gene content with the exception of variations within prophages. However, DT2 94-213 harbored 22 pseudogenes that were intact in other closely related S. Typhimurium strains. We report a novel in silico approach to identify single amino acid substitutions in proteins that have a high probability of a functional impact. One polymorphism identified using this method, a single-residue deletion in the Tar protein, abrogated chemotaxis to aspartate in vitro. DT2 94-213 also exhibited an altered transcriptional profile in response to culture at 42°C compared to that of SL1344. Such differentially regulated genes included a number involved in flagellum biosynthesis and motility.