Identification of Comamonas species using 16S rRNA gene sequence

A bacterial strain Bz02 was isolated from a water sample collected from river Gomti at the Indian city of Lucknow. We characterized the strain using 16S rRNA sequence. Phylogenetic analysis showed that the strain formed a monophyletic clade with members of the genus Comamonas. The closest phylogenetic relative was Comamonas testosteroni with 95% 16S rRNA gene sequence similarity. It is proposed that the identified strain Bz02 be assigned as the type strain of a species of the genus Comamonas (Comamonas sp Bz02) based on 16S rRNA gene sequence search in Ribosomal Database Project, small subunit rRNA and large subunit rRNA databases together with the phylogenetic tree analysis. The sequence is deposted in GenBank with the accession number {"type":"entrez-nucleotide","attrs":{"text":"FJ211417","term_id":"209164434"}}FJ211417.     

Genetic Characterization of Clinical Acanthamoeba Isolates from Japan using Nuclear and Mitochondrial Small Subunit Ribosomal RNA

Because of an increased number of Acanthamoeba keratitis (AK) along with associated disease burdens, medical professionals have become more aware of this pathogen in recent years. In this study, by analyzing both the nuclear 18S small subunit ribosomal RNA (18S rRNA) and mitochondrial 16S rRNA gene loci, 27 clinical Acanthamoeba strains that caused AK in Japan were classified into 3 genotypes, T3 (3 strains), T4 (23 strains), and T5 (one strain). Most haplotypes were identical to the reference haplotypes reported from all over the world, and thus no specificity of the haplotype distribution in Japan was found. The T4 sub-genotype analysis using the 16S rRNA gene locus also revealed a clear sub-conformation within the T4 cluster, and lead to the recognition of a new sub-genotype T4i, in addition to the previously reported sub-genotypes T4a-T4h. Furthermore, 9 out of 23 strains in the T4 genotype were identified to a specific haplotype ({"type":"entrez-nucleotide","attrs":{"text":"AF479533","term_id":"28194589","term_text":"AF479533"}}AF479533), which seems to be a causal haplotype of AK. While heterozygous nuclear haplotypes were observed from 2 strains, the mitochondrial haplotypes were homozygous as T4 genotype in the both strains, and suggested a possibility of nuclear hybridization (mating reproduction) between different strains in Acanthamoeba. The nuclear 18S rRNA gene and mitochondrial 16S rRNA gene loci of Acanthamoeba spp. possess different unique characteristics usable for the genotyping analyses, and those specific features could contribute to the establishment of molecular taxonomy for the species complex of Acanthamoeba.     

Streptomyces antibioticalis, a Novel Species from a Sanitary Landfill Soil

A novel isolate belonging to the genus Streptomyces, strain SL-4^T, was isolated from soil sample collected from a sanitary landfill, New Delhi, India. The taxonomic status of this isolate was studied by polyphasic approach including morphological, physiological and chemo-taxonomic characterization. Spore chains of SL-4^T were open loops, hooks or extended spirals of wide diameter (retinaculiperti). The cell wall peptidoglycan of the isolate SL-4^T contained L,L-diaminopimelic acid, suggesting that the strain has a cell wall of chemotype-I. The polar lipid profile of the isolate was of Type II, with phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannosides. The 16SrRNA gene sequence similarity between SL-4^T and its phylogenetic relatives Streptomyces atrovirens NRRLB 16357^T ({"type":"entrez-nucleotide","attrs":{"text":"DQ026672","term_id":"68137786","term_text":"DQ026672"}}DQ026672), S. albogriseolus NRRLB 1305^T ({"type":"entrez-nucleotide","attrs":{"text":"AJ494865","term_id":"21742835","term_text":"AJ494865"}}AJ494865), S viridodiastaticus NBRC 13106^T ({"type":"entrez-nucleotide","attrs":{"text":"AB184317","term_id":"90960133","term_text":"AB184317"}}AB184317), S. caelestis NRRL 2418^T ({"type":"entrez-nucleotide","attrs":{"text":"X80824","term_id":"587528","term_text":"X80824"}}X80824), S. flavoviridis NBRC 12772^T ({"type":"entrez-nucleotide","attrs":{"text":"AB184842","term_id":"90960663","term_text":"AB184842"}}AB184842), S. pilosus NBRC 12807^T ({"type":"entrez-nucleotide","attrs":{"text":"AB184161","term_id":"90959977","term_text":"AB184161"}}AB184161) and S. longispororuber NBRC 13488^T ({"type":"entrez-nucleotide","attrs":{"text":"AB184440","term_id":"90960256","term_text":"AB184440"}}AB184440) was 99.65, 99.65, 99.64, 99.23, 99.15, 99.14 and 99.13 % respectively. Subsequent DNA–DNA hybridization experiments with the test strain and its clade members showed 55.27, 44.27, 36.86, and 15.65 % relatedness between SL-4^T and its relatives S. atrovirens,S. albogriseolus, S. viridodiastaticus and S. longispororuber respectively. The genotypic and phenotypic data was analyzed to verify possibility of the isolate SL-4^T representing novel member of the genus Streptomyces, for which the name S. antibioticalis is being proposed. The type strain is SL-4^T (=CCM 7434^T=MTCC 8588^T).     

Molecular cloning and characterization of pigeon (Columba liva) ubiquitin and ubiquitin-conjugating enzyme genes from pituitary gland library

In the study of the regulation of incubation, broodiness and laying performance in pigeons (Columba liva), a cDNA library, which was enriched with full-length brooding-related genes, was constructed by SMART LD-PCR techniques using the pituitary glands of incubating White King pigeons. The titers of optimal primary libraries were 1.54×10⁶ pfu/mL and 1.80×10⁶ pfu/mL and the titers of amplified libraries were 1.89×10⁸ pfu/mL and 2.32×10⁹ pfu/mL. The percentages of recombinant clones of primary libraries and amplified libraries were all over 90%. A positive clone was sequenced and named ubiquitin based on the highly similar from other species. The fragment has the four initial codons of ATG, a termination codon of TAA and a signal sequence of AATAAA for adding the poly-A tail. The open reading frame of 918bp encodes 305 amino acids (NCBI accession number is {"type":"entrez-nucleotide","attrs":{"text":"EU981283","term_id":"197693971","term_text":"EU981283"}}EU981283). Recombinant pigeon ubiquitin protein was efficiently expressed with the form of insoluble inclusion bodies in E. coli BL21 transformed with a pET28a⁺ expression vector containing the DNA sequence encoding mature pigeon ubiquitin. The molecular weight of expressed protein is the same as predicted size of approximately 35kD. To improve the efficiency of cloning full-length cDNA, strategies of RACE combined with cDNA library were used. The length of pigeons ubiquitin-conjugating enzyme gene obtained was 1263 bp containing a complete open reading frame of 435 bp that encodes 144 aa (NCBI accession number is {"type":"entrez-nucleotide","attrs":{"text":"EU914824","term_id":"197130954","term_text":"EU914824"}}EU914824). The results of this study not only provide a starting point for further study of ubiquitin function in pigeon species, but also provide a starting point for investigating the brooding mechanisms of pigeons.     

Design of tandem genes cluster and expression vector for biosynthesis of soybean isoflavones

A tandem gene cluster CHS-CHI-IFS (rIFS) for secondary metabolites of plant isoflavones was constructed by using the chalcone synthase (CHS), chalcone isomerase (CHI), and isoflavone synthase (IFS) (GenBank accession numbers {"type":"entrez-nucleotide","attrs":{"text":"EU526827","term_id":"170783761","term_text":"EU526827"}}EU526827, {"type":"entrez-nucleotide","attrs":{"text":"EU526829","term_id":"170783765","term_text":"EU526829"}}EU526829, {"type":"entrez-nucleotide","attrs":{"text":"EU526830","term_id":"170783767","term_text":"EU526830"}}EU526830) in a single recombination event with the pET22b vector. The resulting expression vector pET-rIFS was heterogeneously expressed. The highlights of the vector include ease of handling, high efficiency and universal application among diverse plant species. To the best of our knowledge, this is the first attempt at developing a novel method of constructing tandem gene cluster for future research involving secondary metabolism of isoflavones and isoflavones engineering.Key words: Isoflavones biosynthesis, Novel method, Secondary metabolism, Tandem gene cluster     

Genome Sequence of the Oligotrophic Marine Gammaproteobacterium HTCC2143, Isolated from the Oregon Coast

Strain HTCC2143 was isolated from Oregon Coast surface waters using dilution-to-extinction culturing. Here we present the genome of strain HTCC2143 from the BD1-7 clade of the oligotrophic marine Gammaproteobacteria group. The genome of HTCC2143 contains genes for carotenoid biosynthesis and proteorhodopsin and for proteins that have potential biotechnological significance: epoxide hydrolases, Baeyer-Villiger monooxygenases, and polyketide synthases.Strain HTCC2143 was sampled and isolated from surface waters (depth, 10 m) off the Coastal Pacific Ocean, Newport, OR (44°36′0"N, 124°6′0"W). In the course of dilution-to-extinction culture studies on coastal microbial communities, strain HTCC2143 was isolated in a pristine seawater-based medium (2). Phylogenetic analysis of 16S rRNA gene sequences placed strain HTCC2143 in the BD1-7 clade of the oligotrophic marine Gammaproteobacteria (OMG) group (2) and indicated that it is related to Dasania marina, isolated from Arctic marine sediment (3, 8). The HTCC2143 16S rRNA gene sequence is 95.3% similar to that of D. marina ({"type":"entrez-nucleotide","attrs":{"text":"AY771747","term_id":"157384056","term_text":"AY771747"}}AY771747) and is 96.6% similar to that of environmental gene clone 20m-45 ({"type":"entrez-nucleotide","attrs":{"text":"GU061297","term_id":"262072220","term_text":"GU061297"}}GU061297), taken from intertidal beach seawater of the Yellow Sea, South Korea. Other closer relatives of HTCC2143 included uncultured gammaproteobacterial clones from seafloor lava (clone P0X3b5B06 from Hawaii South Point X3, {"type":"entrez-nucleotide","attrs":{"text":"EU491383","term_id":"169640639","term_text":"EU491383"}}EU491383; 96.3%) (9), deep-sea sediment (Ucp1554 from the South Atlantic Ocean, Cape Basin, {"type":"entrez-nucleotide","attrs":{"text":"AM997645","term_id":"283475537","term_text":"AM997645"}}AM997645; 95.9%) (10), Yellow Sea sediment (95.8%; D8S-33, {"type":"entrez-nucleotide","attrs":{"text":"EU652559","term_id":"186462116","term_text":"EU652559"}}EU652559), and Arctic sediment (from Kings Bay, Svalbard, Norway; clone SS1_B_07_55, {"type":"entrez-nucleotide","attrs":{"text":"EU050825","term_id":"156453922","term_text":"EU050825"}}EU050825; 95.7%).Genomic DNA was prepared at Oregon State University and sequenced by the J. Craig Venter Institute. The finished contigs were automatically annotated with a system based on the program GenDB (5) and manually annotated as described in previous reports (7, 12). The annotation is available at http://bioinfo.cgrb.oregonstate.edu/microbes/. The draft genome of strain HTCC2143 comprises 3,925,629 bases and 3,662 predicted coding sequences with a G+C content of 47.0%. The genome of HTCC2143 was predicted to contain 40 tRNAs, 1 16S rRNA, 2 5S rRNAs, and 2 23S rRNA genes. Four genes for selenocysteine metabolism were found, including a selenophosphate-dependent tRNA 2-selenouridine synthase and an l-seryl-tRNA(Sec) selenium transferase (EC 2.9.1.1).Strain HTCC2143 had genes for a complete tricarboxylic acid cycle, glycolysis, a pentose phosphate pathway, and an Entner-Doudoroff pathway. Genes were present for a high-affinity phosphate transporter and a pho regulon for sensing of environmental inorganic phosphate availability, as well as genes from the NUDIX (nucleoside diphosphate linked to some other moiety X) hydrolase domain family (1) that reflects the metabolic complexity of prokaryotes (4). Genes for ammonium transporters, nitrate reductase, and sulfate reductase were also present in the HTCC2143 genome.Carotenoid and proteorhodopsin genes were also found in the genome, as well as genes for polyketide synthase modules and related proteins. Carotenoid and proteorhodopsin genes were reported previously from another member of the OMG group, strain HTCC2207, a SAR92 clade isolate (11). HTCC2143 also encoded two epoxide hydrolases, two cyclohexanone monooxygenases (CHMOs) and a cyclododecanone monooxygenase (CDMO). CDMOs and CHMOs are members of the Baeyer-Villiger monooxygenase (BVMO) family. BVMOs are “green” alternatives to the chemically mediated Baeyer-Villiger reactions that allow the conversion of ketones into esters or of cyclic ketones into lactones (6).This genome provides further evidence that dilution-to-extinction culturing methods that make use of low-nutrient media that are similar to the conditions of the natural environment can result in the isolation of novel, environmentally significant organisms with potential biotechnological value (13).     

Human Infections with Spirometra decipiens Plerocercoids Identified by Morphologic and Genetic Analyses in Korea

Tapeworms of the genus Spirometra are pseudophyllidean cestodes endemic in Korea. At present, it is unclear which Spirometra species are responsible for causing human infections, and little information is available on the epidemiological profiles of Spirometra species infecting humans in Korea. Between 1979 and 2009, a total of 50 spargana from human patients and 2 adult specimens obtained from experimentally infected carnivorous animals were analyzed according to genetic and taxonomic criteria and classified as Spirometra erinaceieuropaei or Spirometra decipiens depending on the morphology. Morphologically, S. erinaceieuropaei and S. decipiens are different in that the spirally coiled uterus in S. erinaceieuropaei has 5-7 complete coils, while in S. decipiens it has only 4.5 coils. In addition, there is a 9.3% (146/1,566) sequence different between S. erinaceieuropaei and S. decipiens in the cox1 gene. Partial cox1 sequences (390 bp) from 35 Korean isolates showed 99.4% (388/390) similarity with the reference sequence of S. erinaceieuropaei from Korea (G1724; GenBank {"type":"entrez-nucleotide","attrs":{"text":"KJ599680","term_id":"646228164","term_text":"KJ599680"}}KJ599680) and an additional 15 Korean isolates revealed 99.2% (387/390) similarity with the reference sequences of S. decipiens from Korea (G1657; GenBank {"type":"entrez-nucleotide","attrs":{"text":"KJ599679","term_id":"646228151","term_text":"KJ599679"}}KJ599679). Based on morphologic and molecular databases, the estimated population ratio of S. erinaceieuropaei to S. decipiens was 35: 15. Our results indicate that both S. erinaceieuropaei and S. decipiens found in Korea infect humans, with S. erinaceieuropaei being 2 times more prevalent than S. decipiens. This study is the first to report human sparganosis caused by S. decipiens in humans in Korea.     

High Prevalence and Significant Association of ESBL and QNR Genes in Pathogenic Klebsiella pneumoniae Isolates of Patients from Kolkata, India

Pathogenic Klebsiella pneumoniae, resistant to beta-lactam and quinolone drugs, is widely recognized as important bacteria causing array of diseases. The resistance property is obtained by acquisition of plasmid encoded bla_TEM, bla_SHV, bla_CTX-M, QNRA, QNRB and QNRS genes. The aim of this study was to document the prevalence and association of these resistant genes in K. pneumoniae infecting patients in India. Approximately 97 and 76.7 % of the 73 K. pneumoniae isolates showed resistance towards beta-lactam and quinolone drugs respectively. Bla genes were detected in 74 % of K. pneumoniae isolates; with prevalence in the following order: bla_TEM > bla_SHV > bla_CTXM. QNR genes were detected in 67 % samples. Chi-square analysis revealed significant association between presence of bla and qnr genes in our study (P value = 0.000125). Sequence analysis of some bla_TEM, bla_SHV, bla_CTX-M and QNRB PCR products revealed presence of bla_TEM1 (GenBank accession: {"type":"entrez-nucleotide","attrs":{"text":"JN193522","term_id":"343796760","term_text":"JN193522"}}JN193522), bla_TEM116 ({"type":"entrez-nucleotide","attrs":{"text":"JN193523","term_id":"343796762","term_text":"JN193523"}}JN193523 and {"type":"entrez-nucleotide","attrs":{"text":"JN193524","term_id":"343796764","term_text":"JN193524"}}JN193524), bla_SHV11, bla_CTXM72 variants ({"type":"entrez-nucleotide","attrs":{"text":"JF523199","term_id":"329024835","term_text":"JF523199"}}JF523199) and QNRB1 ({"type":"entrez-nucleotide","attrs":{"text":"JN193526","term_id":"343796802","term_text":"JN193526"}}JN193526 and {"type":"entrez-nucleotide","attrs":{"text":"JN193527","term_id":"343796804","term_text":"JN193527"}}JN193527) in our samples.     

Effect of Freezing on PCR Amplification of 16S rRNA Genes from Microbes Associated with Black Band Disease of Corals

Molecular analysis of black band disease of corals revealed that samples frozen immediately after collection yielded more proteobacterial 16S rRNA sequences, while unfrozen samples produced more cyanobacterial and sulfur-oxidizing bacterial sequences. These results suggest the need to use multiple approaches for preparation of samples to characterize this complex polymicrobial disease.Black band disease (BBD) is a polymicrobial disease that affects corals on reefs worldwide. It consists of a migrating microbial mat dominated by cyanobacteria that lyses coral tissue, leading to coral colony death, and is one of the most destructive of coral diseases. Microscopic examination of BBD samples consistently reveals an abundance of nonheterocystous, filamentous cyanobacteria and colorless gliding bacteria with internal elemental sulfur granules characteristic of the genus Beggiatoa (6, 17, 18). It is thought that these are key players in the etiology of BBD. However, with one exception (2), previous molecular studies of BBD consistently detected very low proportions of cyanobacteria (4, 8, 9, 19, 20) and only one study has detected Beggiatoa (19). Instead, all molecular BBD studies indicate a highly variable and diverse composition of heterotrophic bacteria, mostly members of the Alphaproteobacteria.It is unknown why the dominant cyanobacteria and filamentous sulfur-oxidizing bacteria observable microscopically in BBD samples are poorly or not at all detected by molecular methods. It is possible that freezing of the samples in these studies is the cause for low detection of BBD cyanobacteria and sulfur oxidizers. Freezing is the common method of sample processing to extract DNA for microbial community analysis of BBD and has been used in all previous molecular studies. However, this approach may impart a bias to detection of specific BBD bacteria. Suomalainen et al. (22) reported that freezing of samples targeting the fish pathogen Flavobacterium columnare destroyed DNA, suggested to be due to the release of DNase and other enzymes from the cell, leaving most of the F. columnare DNA undetectable by PCR. They noted that DNA from bacteria such as Escherichia coli was not affected (22). Bissett et al. (3) speculated that freezing sediments prior to DNA extraction lysed Beggiatoa filaments and caused their DNA to be lost (3). A recent report showed that algae and cyanobacteria with large cell sizes, including filamentous strains, could not be sufficiently cryopreserved (5). While the above-described studies showed or speculated that freezing of samples affects the detection of some microorganisms in environmental samples, none of these studies included detailed investigation of the mechanism responsible for the effect of freezing or of the effect of freezing on the assessment of microbial community composition.In the present study, we investigated the effect of freezing on molecular analysis of the BBD microbial community by using DNA extracts of frozen and unfrozen BBD samples from two coral hosts (Siderastrea siderea and Diploria clivosa), using both universal and cyanobacterium-specific primers targeting the 16S rRNA gene. BBD samples (i.e., the BBD microbial mat) were collected by suctioning the mat off the coral surface using individual sterile syringes while scuba diving. Samples were transferred to 2-ml cryovials (after decanting seawater) upon return to shore and either immediately frozen and stored at −20°C until DNA extraction or maintained at ambient temperature with DNA extracted within 1 h of collection. Eleven samples were collected from reefs of the Florida Keys (United States), Lee Stocking Island (Bahamas), and St. Croix (United States Virgin Islands).Genomic DNA was extracted by the bead-beating method as previously described (12, 19, 20). Frozen samples were first thawed at room temperature, and 500-μl aliquots were directly transferred into multimix lysing matrix tubes by using trimmed pipette tips, excluding any water. Unfrozen samples were transferred to multimix lysing matrix tubes in the same way. The extracted DNA was verified by gel electrophoresis, and DNA extracts from frozen samples were stored at −20°C, whereas DNA extracts from unfrozen samples were kept at 4°C until used for PCR amplification.DNA extracted from both frozen and unfrozen samples was amplified by PCR using universal bacterial primers 27F and 1492R (14) and cyanobacterium-specific primers CYA359F and CYA781R(B) (15) targeting 16S rRNA genes. The purification of PCR products, cloning, and sequencing of plasmid inserts were done as described previously (20). Primer M13F (11) or CYA359F (15) was used to obtain partial sequences, and an additional primer, 518F (13), M13R (11), or CYA781R(B) (15), was used to obtain full-length sequences. Sequence editing, BLAST (1), and phylogenetic analysis using ARB (10) were done as described previously (19, 20). Sequences that matched at similarity identity values of 97% and above were considered to be of the same operational taxonomic unit. Representative gene sequences that were closely related to cyanobacterial sequences were subjected to maximum-parsimony, neighbor-joining, and maximum-likelihood phylogenetic analyses, and a consensus tree was produced based on maximum-parsimony analysis.The results for universal bacterial primers indicated that all of the frozen BBD samples except one (Fig. ​(Fig.1,1, clone library E) were dominated (44 to 87%) by Alphaproteobacteria (Fig. ​(Fig.1;1; see Tables S1 and S2 in the supplemental material). We previously (19) compared the 16S rRNA gene sequences retrieved from seven of these libraries (Fig. ​(Fig.1,1, libraries A to G), all of which were obtained from frozen BBD samples from the host S. siderea, to investigate the diversity of BBD microorganisms between BBD infections. In the present study, we focus on the differences in results obtained using frozen versus unfrozen BBD samples from S. siderea (Fig. ​(Fig.1,1, libraries G and H) and a second coral host, D. clivosa (Fig. ​(Fig.1,1, libraries I and J). The S. siderea samples (libraries G and H) were taken from different host colonies on the same reef (Butler Bay Reef site), whereas the D. clivosa clone libraries were constructed from subsamples of one BBD sample.Open in a separate windowFIG. 1.Dominant bacterial phylogenetic groups, based on 16S rRNA gene sequence types and universal primers, present in clone libraries produced from frozen and unfrozen BBD samples from the coral hosts Siderastrea siderea and Diploria clivosa. The numbers above the bars represent the numbers of sequences in the respective clone libraries. Libraries A to H, frozen (A to G) and unfrozen (H) BBD from S. siderea. Libraries I and J, frozen (I) and unfrozen (J) BBD from D. clivosa. Sampling locations and sampling dates: libraries A and B, Horseshoe Reef, Lee Stocking Island, Bahamas, 19 July 2004; C, Rainbow Garden Reef, Lee Stocking Island, Bahamas, 16 July 2004; D, Watson''s Reef, Florida Keys, 3 May 2005; E, G, and H, Butler Bay Reef site, St. Croix, U.S. Virgin Islands (USVI), 22 October 2005, 1 June 2005, and 5 June 2006, respectively; F, Frederiksted Reef site, St. Croix, USVI, 1 June 2005; I and J, Frederiksted Reef site, St. Croix, USVI, 7 August 2006. All of the sequences from clone libraries A to G have been previously published by Sekar et al. (19, 20).This approach yielded strikingly different results for the two methods. For example, the clone library produced from one frozen sample (Fig. ​(Fig.1,1, library G) from S. siderea contained only one (of 60) cyanobacterium-related sequence (see {"type":"entrez-nucleotide","attrs":{"text":"EF123584","term_id":"132537907","term_text":"EF123584"}}EF123584 [GenBank sequence accession no.] in Table S1 in the supplemental material), which was phylogenetically related to a sequence from an uncultured planktonic Synechococcus sp. (GenBank sequence accession no. {"type":"entrez-nucleotide","attrs":{"text":"AY172810","term_id":"30525542","term_text":"AY172810"}}AY172810; Fig. ​Fig.2).2). In contrast, the clone library from the corresponding unfrozen sample (Fig. ​(Fig.1,1, library H) was dominated by a cyanobacterial ribotype which represented 37% of the clones. This ribotype was closely related to an Oscillatoria ribotype (GenBank sequence accession no. {"type":"entrez-nucleotide","attrs":{"text":"AY038527","term_id":"21322142","term_text":"AY038527"}}AY038527/{"type":"entrez-nucleotide","attrs":{"text":"AF473936","term_id":"18996878","term_text":"AF473936"}}AF473936) detected in almost all reported BBD molecular studies (2, 4, 7, 23). The sequence was confirmed as the BBD Oscillatoria sequence by phylogenetic analysis using two representative clone sequences (GenBank sequence accession no. {"type":"entrez-nucleotide","attrs":{"text":"EF123639","term_id":"132537962","term_text":"EF123639"}}EF123639 and {"type":"entrez-nucleotide","attrs":{"text":"EF123644","term_id":"132537967","term_text":"EF123644"}}EF123644) (Fig. ​(Fig.2).2). The unfrozen S. siderea clone library additionally produced a dominant epsilonproteobacterial ribotype (14 of 15 clones) (see Table S1 in the supplemental material) that was not detected in the corresponding frozen sample. Phylogenetic analysis of two representative sequences (GenBank sequence accession no. {"type":"entrez-nucleotide","attrs":{"text":"EF123607","term_id":"132537930","term_text":"EF123607"}}EF123607 and {"type":"entrez-nucleotide","attrs":{"text":"EF123613","term_id":"132537936","term_text":"EF123613"}}EF123613, not shown in Fig. ​Fig.2)2) determined that the sequence was related to a sequence from the sulfur-oxidizing bacterium “Candidatus Arcobacter sulfidicus” (GenBank sequence accession no. {"type":"entrez-nucleotide","attrs":{"text":"AY035822","term_id":"18057056","term_text":"AY035822"}}AY035822) (24), a species known to deposit filamentous sulfur (21) and reported previously in BBD (9).Open in a separate windowFIG. 2.Phylogenetic tree derived from the 16S rRNA gene sequences closely related to Synechococcus spp., Xenococcus spp., and Oscillatoria spp. sequences detected in BBD and their neighbors. The tree topology is based on the maximum-parsimony analysis. The bar represents 10% estimated sequence divergence. Boldface type indicates sequences from this study, designated as follows. FRSSBA, UFSSBA, FRSSCY, and UFSSCY indicate sequences retrieved from frozen (FR) and unfrozen (UF) samples of S. siderea (SS) using universal bacterial primers (BA) and cyanobacterium-specific (CY) primers for 16S rRNA gene amplification. FRDCBA, UFDCBA, and UFDCCY indicate sequences retrieved from frozen and unfrozen samples of Diploria clivosa (DC), and the same primer designations are used as for S. siderea sequences. GenBank sequence accession numbers are listed for all sequences. Asterisks designate sequences corresponding to the sequence from the BBD Oscillatoria discussed in the text.Again in clone library I, from the frozen subsample of D. clivosa (see Table S2 in the supplemental material), the Alphaproteobacteria were dominant (44%) and cyanobacteria represented in low percentages (4%). These cyanobacterial sequences were phylogenetically related to sequences of Leptolyngbya spp. (not shown in Fig. ​Fig.2)2) and a planktonic cyanobacterium Xenococcus sp. (GenBank sequence accession no. {"type":"entrez-nucleotide","attrs":{"text":"AF132783","term_id":"5814229","term_text":"AF132783"}}AF132783) (Fig. ​(Fig.2;2; see Table S2 in the supplemental material). The library from the unfrozen BBD subsample of D. clivosa (see Table S2 in the supplemental material) was dominated by Gammaproteobacteria (35%), followed by cyanobacteria (24%) which had the same cyanobacterial sequence type (BBD Oscillatoria) observed in the unfrozen S. siderea sample (see Table S2 in the supplemental material). For corroboration of these results, we constructed an additional clone library, using universal primers, from an unfrozen BBD sample from S. siderea collected during June 2007; in this sample, 47% of the sequences were also related to the sequence from BBD Oscillatoria.The use of cyanobacterium-specific primers produced results similar to the overall pattern we detected using universal primers. Frozen BBD from S. siderea produced 27 sequences, of which 24 were closely related to sequences from planktonic Synechococcus spp. and Xenococcus sp. (see Table S3 in the supplemental material). This was confirmed by phylogenetic analysis (Fig. ​(Fig.2)2) using representative sequences (GenBank sequence accession no. {"type":"entrez-nucleotide","attrs":{"text":"EU019432","term_id":"157704222","term_text":"EU019432"}}EU019432, {"type":"entrez-nucleotide","attrs":{"text":"EU019435","term_id":"157704225","term_text":"EU019435"}}EU019435, {"type":"entrez-nucleotide","attrs":{"text":"EU019439","term_id":"157704229","term_text":"EU019439"}}EU019439, {"type":"entrez-nucleotide","attrs":{"text":"EU019442","term_id":"157704232","term_text":"EU019442"}}EU019442, {"type":"entrez-nucleotide","attrs":{"text":"EU019449","term_id":"157704239","term_text":"EU019449"}}EU019449, and {"type":"entrez-nucleotide","attrs":{"text":"EU019455","term_id":"157704245","term_text":"EU019455"}}EU019455). In contrast, all of the sequences (n = 37) obtained from unfrozen S. siderea samples were closely related to the sequence from the BBD Oscillatoria (see Table S3 in the supplemental material). Representative sequences (GenBank sequence accession no. {"type":"entrez-nucleotide","attrs":{"text":"EU019460","term_id":"157704250","term_text":"EU019460"}}EU019460 and {"type":"entrez-nucleotide","attrs":{"text":"EU019467","term_id":"157704257","term_text":"EU019467"}}EU019467) confirmed this phylogenetic affiliation (Fig. ​(Fig.2).2). Similarly, each of 38 sequences obtained from the unfrozen subsample of D. clivosa with cyanobacterium-specific primers was closely related to the sequence from the BBD Oscillatoria (see Table S3 in the supplemental material), again confirmed by phylogenetic analysis using two representative sequences (GenBank sequence accession no. {"type":"entrez-nucleotide","attrs":{"text":"EU019508","term_id":"157704298","term_text":"EU019508"}}EU019508 and {"type":"entrez-nucleotide","attrs":{"text":"EU019515","term_id":"157704305","term_text":"EU019515"}}EU019515) (Fig. ​(Fig.22).There was very little overlap (6 to 10%) between sequences obtained from frozen versus unfrozen BBD samples collected from both coral hosts when considering all of the BBD bacterial sequences detected (see Tables S1 and S2 in the supplemental material). Only four sequences were common to both frozen and unfrozen clone libraries (6% of 62 sequences detected within 136 clones) for S. siderea and seven sequences (10% of 69 sequences detected within 108 clones) for D. clivosa. Statistical analysis (ANOSIM) showed that the sequence types differed significantly between frozen and unfrozen clone libraries (R = 0.987; P = 0.022). Overall, all frozen libraries (libraries A to G and I) were 69% similar to each other, while the two unfrozen libraries (libraries H and J) were 58% similar.The results of our study are significant for the ongoing investigations into the etiology of BBD. While it is well known that the BBD microbial community consists of photoautotrophs (cyanobacteria), sulfate-reducing bacteria, sulfur-oxidizing bacteria, and heterotrophs (16), we are just beginning to understand the roles of these functional groups in the disease process. A first step in this understanding is the valid and repeatable detection of specific members of the BBD consortium. In summary, we show here that unfrozen samples produce better results for detection of BBD cyanobacteria and sulfur-oxidizing bacteria, while frozen samples are best for detection of heterotrophic proteobacterial sequences. The latter is particularly important because of the consistent finding of Proteobacteria associated with toxic dinoflagellates (19, 20), as well as other marine invertebrate pathogens (4), in BBD. We have not studied the mechanism behind the freezing effect (e.g., release of DNase), which is outside the scope of this study. Though the current study was done with BBD samples, the effect of freezing on other microbial mats or biofilms cannot be ignored. Based on the results of this study, we suggest using multiple sample-processing approaches to characterize the microbial communities associated with BBD and other microbial mats.     

First Report of Northern Root-Knot Nematode,Meloidogyne hapla,Parasitic on Oaks,Quercus brantii and Q. infectoria in Iran

Root-knot nematodes (RKN) are the most serious plant parasitic nematodes having a broad host range exceeding 2,000 plant species. Quercus brantii Lindl. and Q. infectoria Oliv are the most important woody species of Zagros forests in west of Iran where favors sub-Mediterranean climate. National Botanical Garden of Iran (NBGI) is scheduled to be the basic center for research and education of botany in Iran. This garden, located in west of Tehran, was established in 1968 with an area of about 150 ha at altitude of 1,320 m. The Zagros collection has about 3-ha area and it has been designed for showing a small pattern of natural Zagros forests in west of Iran. Brant’s oak (Q. brantii) and oak manna tree (Q. infectoria) are the main woody species in Zagros collection, which have been planted in 1989. A nematological survey on Zagros forest collection in NBGI revealed heavily infection of 24-yr-old Q. brantii and Q. infectoria to RKN, Meloidogyne hapla. The roots contained prominent galls along with egg sac on the surface of each gall. The galls were relatively small and in some parts of root several galls were conjugated, and all galls contained large transparent egg masses. The identification of M. hapla was confirmed by morphological and morphometric characters and amplification of D2-D3 expansion segments of 28S rRNA gene. The obtained sequences of large-subunit rRNA gene from M. hapla was submitted to the GenBank database under the accession number {"type":"entrez-nucleotide","attrs":{"text":"KP319025","term_id":"852381377","term_text":"KP319025"}}KP319025. The sequence was compared with those of M. hapla deposited in GenBank using the BLAST homology search program and showed 99% similarity with those {"type":"entrez-nucleotide","attrs":{"text":"KJ755183","term_id":"667714404","term_text":"KJ755183"}}KJ755183, {"type":"entrez-nucleotide","attrs":{"text":"GQ130139","term_id":"239819330","term_text":"GQ130139"}}GQ130139, {"type":"entrez-nucleotide","attrs":{"text":"DQ328685","term_id":"84794916","term_text":"DQ328685"}}DQ328685, and {"type":"entrez-nucleotide","attrs":{"text":"KJ645428","term_id":"657709843","term_text":"KJ645428"}}KJ645428. The second stage juveniles of M. hapla isolated from Brant’s oak (Q. Brantii) showed the following morphometric characters: (n = 12), L = 394 ± 39.3 (348 to 450) µm; a = 30.9 ± 4 (24.4 to 37.6); b = 4.6 ± 0.44 (4 to 5.1); b΄ = 3.3 ± 0.3 (2.7 to 3.7), c = 8.0 ± 1 (6.2 to 10.3), ć = 5.3 ± 0.8 (3.5 to 6.3); Stylet = 12.1 ± 0.8 (11 to 13) µm; Tail = 50 ± 5.6 (42 to 57) µm; Hyaline 15 ± 1.8 (12 to 18) µm. Oak manna, Q. infectoria population of second stage juveniles clearly possessed short body length and consequently other morphometric features were less than those determined for Q. brantii population, and these features were: (n = 12), L = 359.0 ± 17.3 (319 to 372) µm; a = 28.6 ± 3 (22.8 to 31); b = 5.0 ± 0.3 (4.8 to 5.2); b΄ = 3.3 ± 0.2 (3 to 3.6), c = 8.1 ± 0.5 (7.4 to 8.8), ć = 4.7 ± 0.5 (3.9 to 5.2); Stylet = 11.4 ± 0.7 (10 to 12) µm; Tail = 44 ± 1.8 (42 to 47) µm; Hyaline 12 ± 1.7 (10 to 15) µm. To date two species of Meloidogyne, M. querciana Golden, 1979 and M. christiei Golden and Kaplan, 1986 have been reported to parasitize oaks (Quercus spp.) from the United States of America. M. querciana was found on pin oak Quercus palustris in Virginia. The oak RKN infected pine oak, red oak, and American chestnut heavily in greenhouse tests (Golden, 1979). The other species M. christiei was described from turkey oak and Q. laevis in Florida, which has monospecific host range (Golden and Kaplan, 1986). Both of these RKN species seem to be restricted to the United States of America and have not been reported from other place. According to our knowledge this is the first report of occurrence of M. hapla on Q. brantii and Q. infectoria in the world. This study includes these two oak species to the host range of RKN, M. hapla for the world and expands the information of RKN, M. hapla host ranges on oaks.     

Butlerius butleri Goodey, 1929 (Rhabditida) from Iran with the Phylogenetic Position of the Species

A population of Butlerius butleri Goodey, 1929 was isolated from vermicompost in Kerman in the Kerman Province of Iran during a nematode survey that was conducted during 2014. This population of B. butleri is characterized by the presence of a dorsal thorn-like tooth (4 to 5 μm long), long spicules (44 to 47 μm long), gubernaculum (33 to 37 μm or more than half of the spicule length), three pairs of precloacal papillae, five pairs of postcloacal papillae (papillae V3 and V5 comprising three small papillae), and a long filiform tail (304 to 409 μm in females, 312 to 380 μm in males). Molecular and phylogenetic analysis of B. butleri individuals from this Iranian population based on 18S ribosomal deoxyribonucleic acid (rDNA) sequence placed this species close to Pseudodiplogasteroides compositus ({"type":"entrez-nucleotide","attrs":{"text":"AB597237","term_id":"354681824","term_text":"AB597237"}}AB597237) and an unidentified Pseudodiplogasteroides species ({"type":"entrez-nucleotide","attrs":{"text":"AB597238","term_id":"354681825","term_text":"AB597238"}}AB597238). Measurements, illustrations, and the phylogenetic tree, including the position of B. butleri are provided.     

First report,morphological and molecular characterization of Xiphinema elongatum and X. pachtaicum (Nematoda,Longidoridae) from Ethiopia

A total of six soil samples were collected around rhizosphere of citrus plants during 2010 from Melkassa Agricultural Research Center experimental station, Ethiopia. From these samples two most important ecto-plant parasitic nematodes of the genus Xiphinema were found and analysed. The genus Xiphinema is a large group of the phylum nematoda which constitutes more than 260 species. They are polyphagous root- ectoparasites of many crop plants and some species of this genus cause damage by direct feeding on root tips and transmit nepoviruses. The delimitation and discrimination of two species in the genus is presented, described herein as Xiphinema elongatum and Xiphinema pachtaicum. Morphological and morphometric data were done using light microscopy and results of both species were fit within the previously described nematode species of Xiphinema elongatum and Xiphinema pachtaicum. 18S rDNA were analysed using Bayesian inference (BI) method to reconstruct phylogenetic relationships of the studied Xiphinema sp. ({"type":"entrez-nucleotide","attrs":{"text":"KP407872","term_id":"806981416","term_text":"KP407872"}}KP407872 Xiphinema elongatum and {"type":"entrez-nucleotide","attrs":{"text":"KP407873","term_id":"806981417","term_text":"KP407873"}}KP407873 Xiphinema pachtaicum) with other Xiphinema species. The 18S rDNA sequence of Xiphinema pachtaicum was alike to previously described species from the GenBank but Xiphinema elongatum exhibited very small levels of nucleotides differences (0.4%) which might be possible intra-specific divergence. Though this region of rDNA has less resolution on complex species, its combination with morphological and morphometric analyses, suggests these species as Xiphinema elongatum and Xiphinema pachtaicum with the GenBank accession number of {"type":"entrez-nucleotide","attrs":{"text":"KP407872","term_id":"806981416","term_text":"KP407872"}}KP407872 and {"type":"entrez-nucleotide","attrs":{"text":"KP407873","term_id":"806981417","term_text":"KP407873"}}KP407873, respectively. Short notes, morphological measurements, illustrations, and molecular data are given to these species. These species are reported for the first time from Ethiopia and it provides new geographical information of these organisms.     

Comparative genomics of Riemerella anatipestifer reveals genetic diversity

Background

Riemerella anatipestifer is one of the most important pathogens of ducks. However, the molecular mechanisms of R. anatipestifer infection are poorly understood. In particular, the lack of genomic information from a variety of R. anatipestifer strains has proved severely limiting.

Results

In this study, we present the complete genomes of two R. anatipestifer strains, RA-CH-1 (2,309,519 bp, Genbank accession {"type":"entrez-nucleotide","attrs":{"text":"CP003787","term_id":"403311769","term_text":"CP003787"}}CP003787) and RA-CH-2 (2,166,321 bp, Genbank accession {"type":"entrez-nucleotide","attrs":{"text":"CP004020","term_id":"441482619","term_text":"CP004020"}}CP004020). Both strains are from isolates taken from two different sick ducks in the SiChuang province of China. A comparative genomics approach was used to identify similarities and key differences between RA-CH-1 and RA-CH-2 and the previously sequenced strain RA-GD, a clinical isolate from GuangDong, China, and ATCC11845.

Conclusion

The genomes of RA-CH-2 and RA-GD were extremely similar, while RA-CH-1 was significantly different than ATCC11845. RA-CH-1 is 140,000 bp larger than the three other strains and has 16 unique gene families. Evolutionary analysis shows that RA-CH-1 and RA-CH-2 are closed and in a branch with ATCC11845, while RA-GD is located in another branch. Additionally, the detection of several iron/heme-transport related proteins and motility mechanisms will be useful in elucidating factors important in pathogenicity. This information will allow a better understanding of the phenotype of different R. anatipestifer strains and molecular mechanisms of infection.     

Detection of Wolbachia pipientis, including a new strain containing the wsp gene,in two sister species of Paraphlebotomus sandflies,potential vectors of zoonotic cutaneous leishmaniasis

Individual, naturally occurring Phlebotomus mongolensis and Phlebotomus caucasicus from Iran were screened for infections with the maternally inherited intracellular Rickettsia-like bacterium Wolbachia pipientis via targeting a major surface protein gene (wsp). The main objective of this study was to determine if W. pipientis could be detected in these species. The sandflies were screened using polymerase chain reaction to amplify a fragment of the Wolbachia surface protein gene. The obtained sequences were edited and aligned with database sequences to identify W. pipientis haplotypes. Two strains of Wolbachia were found. Strain Turk 54 (accession {"type":"entrez-nucleotide","attrs":{"text":"EU780683","term_id":"212725653","term_text":"EU780683"}}EU780683) is widespread and has previously been reported in Phlebotomus papatasi and other insects. Strain Turk 07 (accession {"type":"entrez-nucleotide","attrs":{"text":"KC576916","term_id":"499068248","term_text":"KC576916"}}KC576916) is a novel strain, found for first time in the two sister species. A-group strains of W. pipientis occur throughout much of the habitat of these sandflies. It is possible that Wolbachia is transferred via horizontal transmission. Horizontal transfer could shed light on sandfly control because Wolbachia is believed to drive a deleterious gene into sandflies that reduces their natural population density. With regard to our findings in this study, we can conclude that one species of sandfly can be infected with different Wolbachia strains and that different species of sandflies can be infected with a common strain.     

Genetic Identification of Spirometra decipiens Plerocercoids in Terrestrial Snakes from Korea and China

Human sparganosis is a zoonotic disease caused by infection with larval forms (procercoid/plerocercoid) of Spirometra spp. The purpose of this study was to identify Spirometra spp. of infected snakes using a multiplex PCR assay and phylogenetic analysis of mitochondrial DNA sequence data from the spargana of terrestrial snakes obtained from Korea and China. A total of 283 snakes were obtained that included 4 species of Colubridae comprising Rhabdophis tigrinus tigrinus (n=150), Dinodon rufozonatum rufozonatum (n=64), Elaphe davidi (n=2), and Elaphe schrenkii (n=7), and 1 species of Viperidae, Agkistrodon saxatilis (n=60). The snakes were collected from the provinces of Chungbuk, Chungnam, and Gyeongbuk in Korea (n=161), and from China (n=122). The overall infection rate with spargana was 83% (235/283). The highest was recorded for D. rufozonatum rufozonatum (100%), followed by A. saxatilis (85%) and R. tigrinus tigrinus (80%), with a negative result for E. davidi (0%) and E. schrenkii (0%). The sequence identities between the spargana from snakes (n=50) and Spirometra erinaceieuropaei ({"type":"entrez-nucleotide","attrs":{"text":"KJ599680","term_id":"646228164","term_text":"KJ599680"}}KJ599680) or S. decipiens ({"type":"entrez-nucleotide","attrs":{"text":"KJ599679","term_id":"646228151","term_text":"KJ599679"}}KJ599679) control specimens were 90.8% and 99.2%, respectively. Pairwise genetic distances between spargana (n=50) and S. decipiens ranged from 0.0080 to 0.0107, while those between spargana and S. erinaceieuropaei ranged from 0.1070 to 0.1096. In this study, all of the 904 spargana analyzed were identified as S. decipiens either by a multiplex PCR assay (n=854) or mitochondrial cox1 sequence analysis (n=50).