RESUMEN
Multilocus sequence analysis (MLSA) is an important method for identification of taxa that are not well differentiated by 16S rRNA gene sequences alone. In this procedure, concatenated sequences of selected genes are constructed and then analyzed. The effects that the number and the order of genes used in MLSA have on reconstruction of phylogenetic relationships were examined. The recA, rpoA, gapA, 16S rRNA gene, gyrB, and ftsZ sequences from 56 species of the genus Vibrio were used to construct molecular phylogenies, and these were evaluated individually and using various gene combinations. Phylogenies from two-gene sequences employing recA and rpoA in both possible gene orders were different. The addition of the gapA gene sequence, producing all six possible concatenated sequences, reduced the differences in phylogenies to degrees of statistical (bootstrap) support for some nodes. The overall statistical support for the phylogenetic tree, assayed on the basis of a reliability score (calculated from the number of nodes having bootstrap values of ≥ 80 divided by the total number of nodes) increased with increasing numbers of genes used, up to a maximum of four. No further improvement was observed from addition of the fifth gene sequence (ftsZ), and addition of the sixth gene (gyrB) resulted in lower proportions of strongly supported nodes. Reductions in the numbers of strongly supported nodes were also observed when maximum parsimony was employed for tree construction. Use of a small number of gene sequences in MLSA resulted in accurate identification of Vibrio species.
Asunto(s)
Tipificación de Secuencias Multilocus/métodos , Vibrio/clasificación , Vibrio/genética , Proteínas Bacterianas/genética , Análisis por Conglomerados , Filogenia , ARN Ribosómico 16S/genéticaRESUMEN
Five strains representing a novel family within the Gammaproteobacteria were isolated from the estuarine grasses Spartina alterniflora and Juncus roemerianus. All strains were facultatively anaerobic, Gram-negative, short, motile, polar monotrichous rods that were mesophilic, oxidase-negative, catalase-positive, had DNA G+C contents of 41.5-44.4 mol% and required seawater salts or NaCl. Growth was observed at pH 3.5-8.0. Polar lipids diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine, aminophospholipid, phospholipids and unidentified aminolipids were found in the representative strain S-G2-2(T). The major menaquinone and ubiquinone were MK-8 (100 %) and Q-8 (93 %), respectively. Predominant fatty acids present were C(12 : 0) aldehyde and/or unknown fatty acid 10.9525 (MIDI designation) and/or iso-C(16 : 1) I/C(14 : 0) 3-OH, C(16 : 1)ω7c/C(16 : 1)ω6c, C(16 : 0), C(17 : 0) cyclo and C(18 : 1)ω7c and/or C(18 : 1)ω6c. The nearly full-length 16S rRNA gene sequences of the strains were very similar (99-100 % similarity), and the strains were identified as members of the same species by DNA-DNA relatedness measurements. 16S rRNA gene sequence analysis revealed that the strains formed a monophyletic lineage within the order Alteromonadales. All five strains fixed N(2). Analysis of partial nifH gene sequences also revealed a monophyletic lineage within the Gammaproteobacteria, and the sequences were dissimilar to those of any previously described diazotroph. Differences between the novel strains and other members of the Alteromonadales include the inability to produce cytochrome oxidase. The novel strains were metabolically versatile. On the basis of the information described above, the new genus and species Celerinatantimonas diazotrophica gen. nov., sp. nov. are proposed to accommodate the five strains within a new family, Celerinatantimonadaceae fam. nov. The type strain of Celerinatantimonas diazotrophica is S-G2-2(T) (â=âATCC BAA-1368(T) â=âDSM 18577(T)).
Asunto(s)
Gammaproteobacteria/clasificación , Gammaproteobacteria/aislamiento & purificación , Fijación del Nitrógeno , ADN Bacteriano/genética , Ácidos Grasos/metabolismo , Gammaproteobacteria/genética , Gammaproteobacteria/metabolismo , Datos de Secuencia Molecular , Filogenia , ARN Ribosómico 16S/genéticaRESUMEN
Strains of Vibrio spp. isolated from roots of the estuarine grasses Spartina alterniflora and Juncus roemerianus produce the phytohormone indole-3-acetic acid (IAA). The colorimetric Salkowski assay was used for initial screening of IAA production. Gas chromatography-mass spectroscopy (GC-MS) was then employed to confirm and quantify IAA production. The accuracy of IAA quantification by the Salkowski assay was examined by comparison to GC-MS assay values. Indole-3-acetamide, an intermediate in IAA biosynthesis by the indole-3-acetamide pathway, was also identified by GC-MS. Multilocus sequence typing of concatenated 16S rRNA, recA, and rpoA genes was used for phylogenetic analysis of environmental isolates within the genus Vibrio. Eight Vibrio type strains and five additional species-level clades containing a total of 16 environmental isolates and representing five presumptive new species were identified as IAA-producing Vibrio species. Six additional environmental isolates similar to four of the Vibrio type strains were also IAA producers. To our knowledge, this is the first report of IAA production by species of the genus Vibrio or by bacteria isolated from an estuarine environment.
Asunto(s)
Ácidos Indolacéticos/metabolismo , Magnoliopsida/microbiología , Reguladores del Crecimiento de las Plantas/metabolismo , Poaceae/microbiología , Vibrio/aislamiento & purificación , Vibrio/metabolismo , Análisis por Conglomerados , ADN Bacteriano/química , ADN Bacteriano/genética , Cromatografía de Gases y Espectrometría de Masas , Datos de Secuencia Molecular , Filogenia , ARN Ribosómico 16S/genética , Rec A Recombinasas/genética , Análisis de Secuencia de ADN , Espectrofotometría , Vibrio/clasificación , Vibrio/genéticaRESUMEN
Methods to assess the diversity of the diazotroph assemblage in the rhizosphere of the salt marsh cordgrass, Spartina alterniflora were examined. The effectiveness of nifH PCR-denaturing gradient gel electrophoresis (DGGE) was compared to that of nifH clone library analysis. Seventeen DGGE gel bands were sequenced and yielded 58 nonidentical nifH sequences from a total of 67 sequences determined. A clone library constructed using the GC-clamp nifH primers that were employed in the PCR-DGGE (designated the GC-Library) yielded 83 nonidentical sequences from a total of 257 nifH sequences. A second library constructed using an alternate set of nifH primers (N-Library) yielded 83 nonidentical sequences from a total of 138 nifH sequences. Rarefaction curves for the libraries did not reach saturation, although the GC-Library curve was substantially dampened and appeared to be closer to saturation than the N-Library curve. Phylogenetic analyses showed that DGGE gel band sequencing recovered nifH sequences that were frequently sampled in the GC-Library, as well as sequences that were infrequently sampled, and provided a species composition assessment that was robust, efficient, and relatively inexpensive to obtain. Further, the DGGE method permits a large number of samples to be examined for differences in banding patterns, after which bands of interest can be sampled for sequence determination.
Asunto(s)
Bacterias/clasificación , Bacterias/genética , Biodiversidad , ADN Bacteriano/genética , Poaceae/microbiología , Bacterias/aislamiento & purificación , Proteínas Bacterianas/genética , Análisis por Conglomerados , Cartilla de ADN/genética , ADN Bacteriano/química , Electroforesis en Gel de Campo Pulsado , Datos de Secuencia Molecular , Oxidorreductasas/genética , Filogenia , Raíces de Plantas/microbiología , Análisis de Secuencia de ADN , Homología de Secuencia , Microbiología del SueloRESUMEN
Marine infaunal burrows and tubes greatly enhance solute transport between sediments and the overlying water column and are sites of elevated microbial activity. Biotic and abiotic controls of the compositions and activities of burrow and tube microbial communities are poorly understood. The microbial communities in tubes of the marine infaunal polychaete Diopatria cuprea collected from two different sediment habitats were examined. The bacterial communities in the tubes from a sandy sediment differed from those in the tubes from a muddy sediment. The difference in community structure also extended to the sulfate-reducing bacterial (SRB) assemblage, although it was not as pronounced for this functional group of species. PCR-amplified 16S rRNA gene sequences recovered from Diopatra tube SRB by clonal library construction and screening were all related to the family Desulfobacteriaceae. This finding was supported by phospholipid fatty acid analysis and by hybridization of 16S rRNA probes specific for members of the genera Desulfosarcina, Desulfobacter, Desulfobacterium, Desulfobotulus, Desulfococcus, and Desulfovibrio and some members of the genera Desulfomonas, Desulfuromonas, and Desulfomicrobium with 16S rRNA gene sequences resolved by denaturing gradient gel electrophoresis. Two of six SRB clones from the clone library were not detected in tubes from the sandy sediment. The habitat in which the D. cuprea tubes were constructed had a strong influence on the tube bacterial community as a whole, as well as on the SRB assemblage.