Molecular analysis of the acinetobacter baumannii biofilm-associated protein

Acinetobacter baumannii is a multidrug-resistant pathogen associated with hospital outbreaks of infection across the globe, particularly in the intensive care unit. The ability of A. baumannii to survive in the hospital environment for long periods is linked to antibiotic resistance and its capacity to form biofilms. Here we studied the prevalence, expression, and function of the A. baumannii biofilm-associated protein (Bap) in 24 carbapenem-resistant A. baumannii ST92 strains isolated from a single institution over a 10-year period. The bap gene was highly prevalent, with 22/24 strains being positive for bap by PCR. Partial sequencing of bap was performed on the index case strain MS1968 and revealed it to be a large and highly repetitive gene approximately 16 kb in size. Phylogenetic analysis employing a 1,948-amino-acid region corresponding to the C terminus of Bap showed that BapMS1968 clusters with Bap sequences from clonal complex 2 (CC2) strains ACICU, TCDC-AB0715, and 1656-2 and is distinct from Bap in CC1 strains. By using overlapping PCR, the bapMS1968 gene was cloned, and its expression in a recombinant Escherichia coli strain resulted in increased biofilm formation. A Bap-specific antibody was generated, and Western blot analysis showed that the majority of A. baumannii strains expressed an ∼200-kDa Bap protein. Further analysis of three Bap-positive A. baumannii strains demonstrated that Bap is expressed at the cell surface and is associated with biofilm formation. Finally, biofilm formation by these Bap-positive strains could be inhibited by affinity-purified Bap antibodies, demonstrating the direct contribution of Bap to biofilm growth by A. baumannii clinical isolates.

Molecular analysis of type 3 fimbrial genes from Escherichia coli, Klebsiella and Citrobacter species

Background Catheter-associated urinary tract infection (CAUTI) is the most common nosocomial infection in the United States and is caused by a range of uropathogens. Biofilm formation by uropathogens that cause CAUTI is often mediated by cell surface structures such as fimbriae. In this study, we characterised the genes encoding type 3 fimbriae from CAUTI strains of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Citrobacter koseri and Citrobacter freundii. Results Phylogenetic analysis of the type 3 fimbrial genes (mrkABCD) from 39 strains revealed they clustered into five distinct clades (A-E) ranging from one to twenty-three members. The majority of sequences grouped in clade A, which was represented by the mrk gene cluster from the genome sequenced K. pneumoniae MGH78578. The E. coli and K. pneumoniae mrkABCD gene sequences clustered together in two distinct clades, supporting previous evidence for the occurrence of inter-genera lateral gene transfer. All of the strains examined caused type 3 fimbriae mediated agglutination of tannic acid treated human erythrocytes despite sequence variation in the mrkD-encoding adhesin gene. Type 3 fimbriae deletion mutants were constructed in 13 representative strains and were used to demonstrate a direct role for type 3 fimbriae in biofilm formation. Conclusions The expression of functional type 3 fimbriae is common to many Gram-negative pathogens that cause CAUTI and is strongly associated with biofilm growth. Our data provides additional evidence for the spread of type 3 fimbrial genes by lateral gene transfer. Further work is now required to substantiate the clade structure reported here by examining more strains as well as other bacterial genera that make type 3 fimbriae and cause CAUTI.

Echolocation calls, wing shape, diet and phylogenetic diagnosis of the endemic Chinese bat Myotis pequinius

We describe the echolocation calls, flight morphology and diet of the endemic Chinese bat Myotis pequinius Thomas, 1908. Orientation calls are broadband, and reach low terminal frequencies. Diet comprised 80% beetles by volume. Wing shape and call design suggest that the bats fly in cluttered habitats, and the possession of moderately long ears and the dietary composition imply they forage at least sometimes by gleaning. Myotis pequinius resembles a larger Oriental version of the western Palaearctic species M. nattereri. Phylogenetic analysis based on sequences of the cytochrome b gene of mitochondrial DNA (1,140 base pairs) from a range of Palaearctic Myotis species confirmed that M. pequinius is close to the nattereri group, and is a sister-species to the eastern Palaearctic M. bombinus. One bat sequenced from China could not be identified from available species descriptions. It was smaller than M. pequinius, and also differed from it in sequence divergence by 6.7%, suggesting the existence of additional, cryptic taxonomic diversity in this group. Our phylogenetic analysis also supports the recognition of M. schaubi as a species distinct from M. nattereri in Transcaucasia and south-western Asia. Myotis nattereri tschuliensis is more closely related to M. schaubi than to M. nattereri, and is best considered either as a subspecies of M. schaubi, or possibly as a distinct species.

Comparative analysis and distribution of Omega-3 lcPUFA Biosynthesis genes in marine molluscs

Recent research has identified marine molluscs as an excellent source of omega-3 long-chain polyunsaturated fatty acids (lcPUFAs), based on their potential for endogenous synthesis of lcPUFAs. In this study we generated a representative list of fatty acyl desaturase (Fad) and elongation of very long-chain fatty acid (Elovl) genes from major orders of Phylum Mollusca, through the interrogation of transcriptome and genome sequences, and various publicly available databases. We have identified novel and uncharacterised Fad and Elovl sequences in the following species: Anadara trapezia, Nerita albicilla, Nerita melanotragus, Crassostrea gigas, Lottia gigantea, Aplysia californica, Loligo pealeii and Chlamys farreri. Based on alignments of translated protein sequences of Fad and Elovl genes, the haeme binding motif and histidine boxes of Fad proteins, and the histidine box and seventeen important amino acids in Elovl proteins, were highly conserved. Phylogenetic analysis of aligned reference sequences was used to reconstruct the evolutionary relationships for Fad and Elovl genes separately. Multiple, well resolved clades for both the Fad and Elovl sequences were observed, suggesting that repeated rounds of gene duplication best explain the distribution of Fad and Elovl proteins across the major orders of molluscs. For Elovl sequences, one clade contained the functionally characterised Elovl5 proteins, while another clade contained proteins hypothesised to have Elovl4 function. Additional well resolved clades consisted only of uncharacterised Elovl sequences. One clade from the Fad phylogeny contained only uncharacterised proteins, while the other clade contained functionally characterised delta-5 desaturase proteins. The discovery of an uncharacterised Fad clade is particularly interesting as these divergent proteins may have novel functions. Overall, this paper presents a number of novel Fad and Elovl genes suggesting that many mollusc groups possess most of the required enzymes for the synthesis of lcPUFAs.

Phylogenetic relationships of unclassified, satellitic Pasteurellaceae obtained from different species of birds as demonstrated by 16S rRNA gene sequence comparison

Avian haemophili demonstrating in vitro satellitic growth, also referred to as the V-factor or NAD requirement, have mainly been classified with Avibacterium paragallinarum (Haemophilus paragallinarum), Avibacterium avium (Pasteurella avium), Avibacterium volantium (Pasteurella volantium) and Avibacterium sp. A (Pasteurella species A). The aim of the present study was to assess the taxonomic position of 18 V-factor-requiring isolates of unclassified Haemophilus-like organisms isolated from galliforme, anseriforme, columbiforme and gruiforme birds as well as kestrels and psittacine birds including budgerigars by conventional phenotypic tests and 16S rRNA gene sequencing. All isolates shared phenotypical characteristics which allowed classification with Pasteurellaceae. Haemolysis of bovine red blood cells was negative. Haemin (X-factor) was not required for growth. Maximum-likelihood phylogenetic analysis including bootstrap analysis showed that six isolates were related to the avian 16S rRNA group and were classified as Avibacterium according to 16S rRNA sequence analysis. Surprisingly, the other 12 isolates were unrelated to Avibacterium. Two isolates were unrelated to any of the known 16S rRNA groups of Pasteurellaceae. Two isolates were related to Volucribacter of the avian 16S rRNA group. Seven isolates belonged to the Testudinis 16S rRNA group and out of these, two isolates were closely related to taxa 14 and 32 of Bisgaard, whereas four other isolates were found to form a genus-like group distantly related to taxon 40 and one isolated remained distantly related to other members of the Testudinis group. One isolate was closely related to taxon 26 (a member of Actinobacillus sensu stricto). The study documented major genetic diversity among V-factor-requiring avian isolates beyond the traditional interpretation that they only belong to Avibacterium, underlining the limited value of satellitic growth for identification of avian members of Pasteurellaceae. Our study also emphasized that these organisms will never be isolated without the use of special media satisfying the V-factor requirement.

Refined Global Analysis of Bemisia tabaci (Hemiptera: Sternorrhyncha: Aleyrodoidea: Aleyrodidae) Mitochondrial Cytochrome Oxidase 1 to Identify Species Level Genetic Boundaries.

Identifying species boundaries within morphologically indistinguishable cryptic species complexes is often contentious. For the whitefly Bemisia tabaci (Gennadius) (Hemiptera: Sternorrhyncha: Aleyrodoidea: Aleyrodidae), the lack of a clear understanding about the genetic limits of the numerous genetic groups and biotypes so far identified has resulted in a lack of consistency in the application of the terms, the approaches use to apply them and in our understanding of what genetic structure within B. tabaci means. Our response has been to use mitochondrial gene cytochrome oxidase one to consider how to clearly and consistently define genetic separation. Using Bayesian phylogenetic analysis and analysis of sequence pairwise divergence we found a considerably higher to number of genetic groups than had been previously determined with two breaks in the distribution, one at 11% and another at 3.5%. At >11% divergence, 11 distinct groups were resolved, whereas at >3.5% divergence 24 groups were identified. Consensus sequences for each of these groups were determined and were shown to be useful in the correct assignment of sequences of unknown origin. The 3.5% divergence bound is consistent with species level separations in other insect taxa and Suggests that B. tabaci is it cryptic species composed of at least 24 distinct species. We further show that the placement of Bemesia atriplex (Froggatt) within the B. tabaci in, group adds further weight to the argument for species level separation within B. tabaci. This new analysis, which constructs consensus sequences and uses these its a standard against which unknown sequences call be compared, provides for the first time it consistent means of identifying the genetic hounds of each species with it high degree of certainty.

An inordinate fondness for Fusarium: Phylogenetic diversity of fusaria cultivated by ambrosia beetles in the genus Euwallacea on avocado and other plant hosts

Ambrosia beetle fungiculture represents one of the most ecologically and evolutionarily successful symbioses, as evidenced by the 11 independent origins and 3500 species of ambrosia beetles. Here we document the evolution of a clade within Fusarium associated with ambrosia beetles in the genus Euwallacea (Coleoptera: Scolytinae). Ambrosia Fusarium Clade (AFC) symbionts are unusual in that some are plant pathogens that cause significant damage in naive natural and cultivated ecosystems, and currently threaten avocado production in the United States, Israel and Australia. Most AFC fusaria produce unusual clavate macroconidia that serve as a putative food source for their insect mutualists. AFC symbionts were abundant in the heads of four Euwallacea spp., which suggests that they are transported within and from the natal gallery in mandibular mycangia. In a four-locus phylogenetic analysis, the AFC was resolved in a strongly supported monophyletic group within the previously described Cade 3 of the Fusarium solani species complex (FSSC). Divergence-time estimates place the origin of the AFC in the early Miocene similar to 21.2 Mya, which coincides with the hypothesized adaptive radiation of the Xyleborini. Two strongly supported clades within the AFC (Clades A and B) were identified that include nine species lineages associated with ambrosia beetles, eight with Euwallacea spp. and one reportedly with Xyleborus ferrugineus, and two lineages with no known beetle association. More derived lineages within the AFC showed fixation of the clavate (club-shaped) macroconidial trait, while basal lineages showed a mix of clavate and more typical fusiform macroconidia. AFC lineages consisted mostly of genetically identical individuals associated with specific insect hosts in defined geographic locations, with at least three interspecific hybridization events inferred based on discordant placement in individual gene genealogies and detection of recombinant loci. Overall, these data are consistent with a strong evolutionary trend toward obligate symbiosis coupled with secondary contact and interspecific hybridization. (C) 2013 Elsevier Inc. All rights reserved.

Analysis of Puumala hantavirus in a bank vole population in northern Finland: evidence for co-circulation of two genetic lineages and frequent reassortment between strains

"In this study, for the first time, two distinct genetic lineages of Puumala virus (PUUV) were found within a small sampling area and within a single host genetic lineage (Ural mtDNA) at Pallasjarvi, northern Finland. Lung tissue samples of 171 bank voles (Myodes glareolus) trapped in September 1998 were screened for the presence of PUUV nucleocapsid antigen and 25 were found to be positive. Partial sequences of the PUUV small (S), medium (M) and large (L) genome segments were recovered from these samples using RT-PCR. Phylogenetic analysis revealed two genetic groups of PUUV sequences that belonged to the Finnish and north Scandinavian lineages. This presented a unique opportunity to study inter-lineage reassortment in PUUV; indeed, 32% of the studied bank voles appeared to carry reassortant virus genomes. Thus, the frequency of inter-lineage reassortment in PUUV was comparable to that of intra-lineage reassortment observed previously (Razzauti, M., Plyusnina, A., Henttonen, H. & Plyusnin, A. (2008). J Gen Virol 89, 1649-1660). Of six possible reassortant S/M/L combinations, only two were found at Pallasjarvi and, notably, in all reassortants, both S and L segments originated from the same genetic lineage, suggesting a non-random pattern for the reassortment. These findings are discussed in connection to PUUV evolution in Fermoscandia."

Comparative Nucleotide and Amino Acid Sequence Analysis of the Sequence-Specific RNA-Binding Rotavirus Nonstructural Protein NSP3

NSP3, an acidic nonstructural protein, encoded by gene 7 has been implicated as the key player in the assembly of the 11 viral plus-strand RNAs into the early replication intermediates during rotavirus morphogenesis. To date, the sequence or NSP3 from only three animal rotaviruses (SA11, SA114F, and bovine UK) has been determined and that from a human strain has not been reported. To determine the genetic diversity among gene 7 alleles from group A rotaviruses, the nucleotide sequence of the NSP3 gene from 13 strains belonging to nine different G serotypes, from both humans and animals, has been determined. Based on the amino acid sequence identity as well as phylogenetic analysis, NSP3 from group A rotaviruses falls into three evolutionarily related groups, i.e., the SA11 group, the Wa group, and the S2 group. The SA 11/SA114F gene appears to have a distant ancestral origin from that of the others and codes for a polypeptide of 315 amino acids (aa) in length. NSP3 from all other group A rotaviruses is only 313 aa in length because of a 2-amino-acid deletion near the carboxy-terminus, While the SA114F gene has the longest 3' untranslated region (UTR) of 132 nucleotides, that from other strains suffered deletions of varying lengths at two positions downstream of the translational termination codon. In spite of the divergence of the nucleotide (nt) sequence in the protein coding region, a stretch of about 80 nt in the 3' UTR is highly conserved in the NSP3 gene from all the strains. This conserved sequence in the 3' UTR might play an important role in the regulation of expression of the NSP3 gene. (C) 1995 Academic Press, Inc.

A comprehensive analysis of the chorion locus in silkmoth

Despite more than 40 years of intense study, essential features of the silkmoth chorion (eggshell) are still not fully understood. To determine the precise structure of the chorion locus, we performed extensive EST analysis, constructed a bacterial artificial chromosome (BAC) contig, and obtained a continuous genomic sequence of 871,711 base pairs. We annotated 127 chorion genes in two segments interrupted by a 164 kb region with 5 non-chorion genes, orthologs of which were on chorion bearing scaffolds in 4 ditrysian families. Detailed transcriptome analysis revealed expression throughout choriogenesis of most chorion genes originally categorized as ``middle'', and evidence for diverse regulatory mechanisms including cis-elements, alternative splicing and promoter utilization, and antisense RNA. Phylogenetic analysis revealed multigene family associations and faster evolution of early chorion genes and transcriptionally active pseudogenes. Proteomics analysis identified 99 chorion proteins in the eggshell and micropyle localization of 1 early and 6 Hc chorion proteins.

Phylogenetic relationships among two species of golden monkey and three species of leaf monkey inferred from rDNA variation

Restriction maps of rDNA repeats of five species of Colobinae and three outgroup taxa, Hylobates leucogenys, Macaca mulatta, and Macaca irus, were constructed using 15 restriction endonucleases and cloned 18S and 28S rRNA gene probes. The site variation between Rhinopithecus roxellana and Rhinopithecus bieti is comparable to that between Presbytis francoisi and Presbytis phayrei, implying that R. bieti is a valid species rather than a subspecies of R. roxellana. Phylogenetic analysis on the 47 informative sites supports the case for Rhinopithecus being an independent genus and closely related to Presbytis. Furthermore, branch lengths of the tree seem to support the hypothesis that the leaf monkeys share some ancestral traits as well as some automorphic characters.

Phylogenetic relationships in genus Niviventer (Rodentia : Muridae) in China inferred from complete mitochondrial cytochrome b gene

Chinese species of the genus Niviventer, predominantly distributed in the southeastern Tibetan Plateau and in Taiwan, are a diverse group and have not yet received a thorough molecular phylogenetic analysis. Here, we reconstructed the phylogenetic relatio

Molecular and morphological analysis of endangered species Gymnodiptychus integrigymnatus (Teleostei: Cyprinidae)

Gymnodiptychus integrigymnatus is a critically endangered species endemic to the Gaoligongshan Mountains. It was thought to be only distributed in several headwater-streams of the Longchuanjiang River (west slope of the Gaoligongshan Mountains, belonging to the Irrawaddy River drainage). In recent years, dozens of G. integrigymnatus specimens have been collected in some streams on the east slope of the Gaoligongshan Mountains (the Salween drainage). We performed a morphological and genetic analyses (based on cytochrome b and D-loop) of the newly discovered populations of G. integrigymnatus to determine whether the degree of separation of these populations warrants species status. Our analysis from the cytochrome b gene revealed that nine individuals from the Irrawaddy drainage area and seven individuals from the Salween drainage area each have only one unique haplotype. The genetic distance between the two haplotypes is 1.97%. Our phylogenetic analysis revealed that G. integrigymnatus is closely related to highly specialized schizothoracine fishes. Analysis from the mitochondrial control region revealed that G. integrigymnatus has relatively high genetic diversity (pi was 0.00891 and h was 0.8714), and individuals from different river drainages do not share the same haplotypes. The AMOVA results indicated 87.27% genetic variability between the Salween and Irrawaddy populations. Phylogenetic trees show two major geographic groups corresponding to the river systems. We recommend that G. integrigymnatus should be considered as a high priority for protected species status in the Gaoligongshan Mountains National Nature Reserve, and that the area of the Gaoligongshan Mountains National Nature Reserve should be expanded to cover the entire distribution of G. integrigymnatus. Populations of G. integrigymnatus from different river systems should be treated as evolutionarily significant units.

Morphological characteristics and phylogenetic relationship of Anabaena species from Lakes Dianchi and Erhai, China

Although Anabaena is one of the most prevalent planktonic freshwater genus in China, there are few taxonomic reports of Anabaena strains by morphology and genetics. In this study, morphological characteristics and phylogenetic relationships of seven Anabaena strains isolated from two plateau lakes, Lakes Dianchi and Erhai, were investigated. Morphological characteristics such as morphology of filament, cellular shapes and sizes, relative position of heterocytes and akinetes, and presence or absence of aerotopes, were described for these seven strains. Phylogenetic relationships were determined by constructing 16S rRNA gene tree using the neighbor-joining algorithm. The seven strains were morphologically identified as three groups, and phylogenetic analysis based on 16S rRNA gene sequences also showed that these seven strains were in three groups. Strains EH-2, EH-3, and EH-4 were in group A belonging to the Anabaena circinalis and A. crassa group, and strains DC-1, DC-2, and EH-1 were in group B and identified as A. flos-aquae. Strain DC-3 without aerotopes was significantly different from the other isolated strains and was determined as A. cylindrica.

Cloning and sequence analysis of Sox genes in a tetraploid cyprinid fish, Tor douronensis

A PCR survey for Sox genes in a young tetraploid fish Tor douronensis (Teleostei: Cyprinidae) was performed to access the evolutionary fates of important functional genes after genome duplication caused by polyploidization event. Totally 13 Sox genes were obtained in Tor douronensis, which represent SoxB, SoxC and SoxE groups. Phylogenetic analysis of Sox genes in Tor douronensis provided evidence for fish-specific genome duplication, and suggested that Sox19 might be a teleost specific Sox gene member. Sequence analysis revealed most of the nucleotide substitutions between duplicated copies of Sox genes caused by tetraploidization event or their orthologues in other species are silent substitutions. It would appear that the sequences are under purifying selective pressure, strongly suggesting that they represent functional genes and supporting selection against all null allele at either of two duplicated loci of Sox4a, Sox9a and Sox9b. Surprising variations of the intron length and similarities of two duplicated copies of Sox9a and Sox9b, suggest that Tor douronensis might be an allotetraploidy.