Publications

4833

Three strictly anaerobic, methanogenic strains JH1T, JH4 and JH8 were isolated from rumen of the Korean native cattle (HanWoo; Bos taurus coreanae) in South Korea. The colonies were circular, opaque, and slightly yellowish. Phylogenetic analyses of 16S rRNA gene and mcrA (encoding α subunit of methyl-coenzyme M reductase) sequences confirmed the affiliation of the novel strains with the Methanobacteriales , and Methanobrevibacter wolinii SHT was the most closely related species. The 16S rRNA gene and mcrA sequence similarities between strains JH1T, JH4 and JH8 and M. wolinii SHT were 96.2 and 89.0 % respectively, and DNA–DNA hybridization of the isolates and M. wolinii DSM 11976T showed a 20 % reassociation. Strain JH1T exhibited 92 % DNA–DNA relatedness with strains JH4 and JH8, and their 16S rRNA gene and mcrA sequences were identical. Cells stained Gram-positive and were non-motile rods, 1.5–1.8 µm long and 0.6 µm wide. The strains were able to use H2/CO2 and formate. The optimum temperature and pH ranges for growth were 37–40 °C and pH 6.5–7.0. The DNA G+C content of strain JH1T was 28 mol%. Based on data from this study using a polyphasic approach, the three strains represent a novel species of genus Methanobrevibacter , for which the name Methanobrevibacter boviskoreani sp. nov. is proposed. The type strain is JH1T ( = KCTC 4102T = JCM 18376T).

Two Gram-negative, facultatively anaerobic, rod-shaped bacteria, strains EF212Tand PS125T, were isolated from the octocoralsEunicea fuscaandPlexaurasp., respectively. EF212Twas isolated from a specimen ofE. fuscacollected off the coast of Florida, USA, and PS125Twas isolated from a specimen ofPlexaurasp. collected off the coast of Bimini, Bahamas. Analysis of the nearly full-length 16S rRNA gene sequences showed that these novel strains were most closely related toEndozoicomonas montiporaeCL-33T,E. elysicolaMKT110TandE. numazuensisHC50T(EF212T, 95.6–97.2 % identity; PS125T, 95.1–96.4 % identity). DNA–DNA hybridization values among EF212T, PS125T,E. montiporaeLMG 24815TandE. elysicolaKCTC 12372Twere far below the 70 % cut-off, with all values for duplicate measurements being less than 35 %. Both EF212Tand PS125Trequired NaCl for growth and showed optimal growth at 2–3 % NaCl, 22-30 °C and pH 8.0. The predominant cellular fatty acids were summed feature 3 (C16 : 1ω6cand/or C16 : 1ω7c), summed feature 8 (C18 : 1ω6cand/or C18 : 1ω7c), C16 : 0and C14 : 0. The DNA G+C content of EF212Twas 48.6 mol% and that of PS125Twas 47.5 mol%. In addition to the genotypic differences observed between the two novel strains and related type strains, phenotypic and chemotaxonomic experiments also revealed differences between strains. Thus, strains EF212Tand PS125Trepresent novel species of the genusEndozoicomonas, for which the namesEndozoicomonas euniceicolasp. nov. andEndozoicomonas gorgoniicolasp. nov., respectively, are proposed. The type strains are EF212T( = NCCB 100458T = DSM 26535T) forEndozoicomonas euniceicolasp. nov. and PS125T( = NCCB 100438T = CECT 8353T) forEndozoicomonas gorgoniicolasp. nov. An emended description of the genusEndozoicomonasis also provided to encompass differences observed in the results of genotypic, chemotaxonomic and phenotypic tests compared from the original and amended genus descriptions.

ABSTRACT “ Candidatus Cardinium hertigii” ( Bacteroidetes ) is a maternally inherited endosymbiont known from several arthropods. Its mechanisms for persistence in host populations are mostly reproductive manipulation, though it has been occasionally reported to improve fitness parameters in several hosts. In Culicoides (Diptera: Ceratopogonidae) biting midges, the prevalence of “ Candidatus Cardinium” infection was documented as moderate, with no detectable sex bias. We therefore investigated whether “ Candidatus Cardinium” affects important fitness parameters, such as survival and body size, in Culicoides imicola , a dominant vector species. Field-collected midges were trapped and analyzed for survival under different environmental conditions and antibiotic treatment, taking into account “ Candidatus Cardinium” infection status and parity status (i.e., parous or nulliparous). Additionally, wing lengths were measured as a proxy parameter for body size and analyzed together with “ Candidatus Cardinium” infection data. The findings revealed no difference in survival of Culicoides infected with “ Candidatus Cardinium” and that of uninfected midges in both parity states and under all tested conditions: optimal, starvation, heat, and antibiotic treatment. Beyond survival, no wing length difference was found for “ Candidatus Cardinium”-infected versus uninfected midges. In aggregate, these findings support our conclusion that “ Candidatus Cardinium” does not have an overt effect on the survival and size of adult C. imicola midges. “ Candidatus Cardinium” may affect immature stages or may alter adult reproductive performance.

A new species of Hassallia (Cyanobacteria, Nostocales, Microchaetaceae) from a supralittoral tropical marine biotope is described. Hassallia littoralis is a false-branched nostocalean cyanobacterium with caespitose free filaments or with fasciculated individual filaments not in a common sheath. Filaments are mainly heteropolar, bearing mono- and bi-pored heterocysts and isopolar or heteropolar hormogonia. The sheath is often widening, with pronounced rounded terminals. This new species is defined according to molecular, morphological and ecological criteria, considering data from different stages of its life cycle as well as the 16S rRNA partial gene sequence.

Cyanobacteria were responsible for the oxygenation of the ancient atmosphere; however, the evolution of this phylum is enigmatic, as relatives have not been characterized. Here we use whole genome reconstruction of human fecal and subsurface aquifer metagenomic samples to obtain complete genomes for members of a new candidate phylum sibling to Cyanobacteria, for which we propose the designation ‘Melainabacteria’. Metabolic analysis suggests that the ancestors to both lineages were non-photosynthetic, anaerobic, motile, and obligately fermentative. Cyanobacterial light sensing may have been facilitated by regulators present in the ancestor of these lineages. The subsurface organism has the capacity for nitrogen fixation using a nitrogenase distinct from that in Cyanobacteria, suggesting nitrogen fixation evolved separately in the two lineages. We hypothesize that Cyanobacteria split from Melainabacteria prior or due to the acquisition of oxygenic photosynthesis. Melainabacteria remained in anoxic zones and differentiated by niche adaptation, including for symbiosis in the mammalian gut.

In April of 2012, tobacco (Nicotiana tabacum L.) plants with symptoms resembling those associated with viral infection were observed in commercial fields in the Department of El-Paraíso, Honduras. Symptoms on affected plants included apical leaf curling and stunting, overall chlorosis and plant stunting, young plant deformation with cabbage-like leaves, wilting, and internal vascular necrosis of stems and leaf petioles. All cultivars grown were affected, with disease incidence ranging from 5 to 80% of symptomatic plants per field. The fields were also heavily infested with the psyllid Bactericera cockerelli. This psyllid is a serious pest of solanaceous crops in the United States, Mexico, Central America, and New Zealand and has been shown to transmit the bacterium “Candidatus Liberibacter solanacearum” to potato, tomato, and other solanaceous species (2,3). Tobacco (cv. Habano criollo) plant samples were collected from one field in the municipality of Trojes. Initial testing of the plant samples for viruses, including Tobacco mosaic virus, Impatiens necrotic spot virus, Cucumber mosaic virus, and Potato virus Y, using Immunostrips (Agdia, Elkhart, IN) were negative. Total DNA was then extracted from leaf tissues of a total of 13 plants, including eight symptomatic plants and five asymptomatic plants with the cetyltrimethylammonium bromide (CTAB) buffer extraction method (2,4). The DNA samples were tested by PCR using specific PCR primer pairs OA2/OI2c and OMB 1482f/2086r, to amplify a portion of 16S rDNA and the outer membrane protein (OMB) gene of “Ca. L. solanacearum,” respectively (2). All eight (100%) symptomatic plant samples were positive for “Ca. L. solanacearum” with both sets of primer pairs. “Ca. L. solanacearum” was not detected in the asymptomatic plants. The 16S rDNA and OMB gene amplicons of two plant samples each were cloned and four clones of each of the four amplicons were sequenced. BLASTn analysis of the consensus sequences confirmed “Ca. L. solanaeacrum” in the tobacco samples. The 16S rDNA consensus sequences (1,168 bp) of all amplicons were identical and showed 100% identity with several 16S rDNA sequences of “Ca. L. solanacearum” in GenBank (e.g., Accession Nos. HM245242, JF811596, and JX559779). The consensus sequence of the OMB amplicon (605 bp) showed 97 to 100% homology with a number of “Ca. L. solanacearum” OMB sequences in GenBank, including Accession Nos. CP002371, FJ914617, JN848754 and JN848752. The tobacco-associated consensus 16S rDNA and OMB sequences from this study were deposited in GenBank as Accession Nos. KC768320 and KC768328, respectively. To our knowledge, this is the first report of “Ca. L. solanacearum” associated with tobacco in Honduras, where this cash crop is economically important. This bacterium has also caused millions of dollars in losses to potato, tomato, and several other solanaceous crops in North and Central America and New Zealand, particularly in regions where B. cockerelli is present (3). Furthermore, “Ca. L. solanacearum” has caused significant economic damage to carrot crops in Europe, where it is transmitted by the psyllids Trioza apicalis in northern Europe (4) and B. trigonica in the Mediterranean region (1). References: (1) A. Alfaro-Fernandez et al. Plant Dis. 96:581, 2012. (2) J. M. Crosslin. Southwest. Entomol. 36:125, 2011. (3) J. E. Munyaneza. Am. J. Pot. Res. 89:329, 2012. (4) J. E. Munyaneza et al. J. Econ. Entomol. 103:1060, 2010.

A novel anaerobic bacterium that could ferment amino acids and organic acids was isolated from an anaerobic, propionate-oxidizing enrichment culture originating from soil of a rice field in Japan. Cells of the isolate, designated strain 4F6ET, were Gram-staining-negative, oxidase- and catalase-negative, non-spore-forming, vibrio-shaped, motile rods (0.8×2.0–2.5 µm) with two or three lateral flagella. Growth occurred at 20–42 °C (optimum at 37–40 °C), at pH 6.4–8.4 (optimum at pH 7.3) and at 0–1.5 % (w/v) NaCl (optimum at 0–0.5 %). Good growth occurred on glycine, serine, cysteine, pyruvate and citrate, whereas poor growth was observed on threonine, glutamine, l-malate, α-ketoglutarate, peptone and Casamino acids. In co-culture with the hydrogen-utilizing methanogen Methanobacterium formicicum JCM 10132T, strain 4F6ET oxidized alanine, valine, leucine, isoleucine, methionine, aspartate, glutamate, histidine, asparagine and fumarate. Yeast extract was required for growth. The G+C content of genomic DNA was 61.9 mol%. A phylogenetic analysis based on comparison of the 16S rRNA gene sequence showed that the type strains of Fretibacterium fastidiosum , Aminobacterium colombiense and Aminobacterium mobile , members of the family Synergistaceae , were the closest relatives of strain 4F6ET, with low sequence similarities (89.3, 89.5 and 86.2 %, respectively). Strain 4F6ET contained iso-C13 : 0 (24.43 %), iso-C15 : 0 (16.47 %) and C19 : 1ω11c/C19 : 1ω9c (16.32 %) as the major fatty acids, which differed from those of F. fastidiosum , Aminobacterium colombiense and Aminobacterium mobile . On the basis of phenotypic, chemotaxonomic and phylogenetic differences between strain 4F6ET and the type strains of F. fastidiosum and Aminobacterium species, we propose that strain 4F6ET represents a novel genus and species, Aminivibrio pyruvatiphilus gen. nov., sp. nov. The type strain of Aminivibrio pyruvatiphilus is strain 4F6ET ( = JCM 18417T = DSM 25964T).