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PubMed2006

Myroides pelagicus sp. nov., isolated from seawater in Thailand.

An aerobic, Gram-negative, non-motile, yellow-to-orange pigmented and rod-shaped bacterium, designated strain SM1(T), was isolated from seawater in Thailand and subjected to a polyphasic taxonomic study. Phylogenetic analyses based on 16S rRNA gene sequences revealed that the novel isolate shared 93-95 % sequence similarity with species of the genus Myroides. The DNA-DNA relatedness values of strain SM1(T) with Myroides odoratimimus JCM 7460(T) and Myroides odoratus JCM 7458(T) were below 70 %. The DNA G+C content of strain SM1(T) was 33.6 mol%, the major menaquinone was MK-6 and the major fatty acids were C(15 : 0) iso, C(17 : 1)omega9c iso and C(17 : 0) iso 3-OH. Based on its phenotypic and chemotaxonomic characteristics, strain SM1(T) was classified as representing a novel species of the genus Myroides, for which the name Myroides pelagicus sp. nov. is proposed. The type strain is SM1(T) (=IAM 15337(T)=KCTC 12661(T)).

Bacteroidetes↗

PubMed2006

Pedobacter ginsengisoli sp. nov., a DNase-producing bacterium isolated from soil of a ginseng field in South Korea.

A Gram-negative, strictly aerobic, rod-shaped, non-motile, non-spore-forming bacterial strain, designated Gsoil 104T, was isolated from a soil sample from a ginseng field in Pocheon Province (South Korea) and was characterized taxonomically by using a polyphasic approach. On the basis of 16S rRNA gene sequence similarities, strain Gsoil 104T was shown to belong to the family Sphingobacteriaceae, being related to Pedobacter africanus DSM 12126T (97.0%), Pedobacter caeni LMG 22862T (96.9%), Pedobacter cryoconitis DSM 14825T (96.8%) and Pedobacter heparinus DSM 2366T (96.6%). The phylogenetic distance from any other Pedobacter species with a validly published name was greater than 3.4% (i.e.<96.6% 16S rRNA gene sequence similarity). DNA-DNA hybridization experiments showed that values for DNA-DNA relatedness between strain Gsoil 104T and its phylogenetically closest neighbours were below 37%. The G+C content of the genomic DNA was 43.6 mol%. The predominant respiratory quinone was MK-7. The major fatty acids were C16:1omega7c, iso-C15:0, C16:0, iso-C17:0 3-OH and iso-C15:0 2-OH. These chemotaxonomic data support the affiliation of strain Gsoil 104T to the genus Pedobacter. On the basis of its phenotypic properties and phylogenetic distinctiveness, strain Gsoil 104T represents a novel species in the genus Pedobacter, for which the name Pedobacter ginsengisoli sp. nov. is proposed. The type strain is Gsoil 104T (=KCTC 12576T=LMG 23399T).

Bacteroidetes↗

PubMed2004

Changes in oral microbial profiles after periodontal treatment as determined by molecular analysis of 16S rRNA genes.

Terminal RFLP (T-RFLP) analysis was used to investigate changes in the oral microbiota in saliva and subgingival plaque samples from one patient with aggressive periodontitis (subject A) and two patients with chronic periodontitis (subjects B and C) before and 3 months after periodontal treatment. Substantial changes in the T-RFLP patterns of subgingival plaque samples of subjects B and C were noted after 3 months of improved oral hygiene and full-mouth supra- and subgingival scaling and root planing. However, there was little change in the subgingival microbiota of subject A. Although the proportions of terminal restriction fragments (T-RFs) larger than 1000 bp were notable in the T-RFLP patterns generated after digestion with HhaI of the samples from two subjects before treatment (subject B, 35.5 %; subject C, 29.6 %), the proportions of these T-RFs were significantly reduced or not detected after treatment (subject B, none; subject C, 4.1 %). Real-time PCR showed a significant change in the proportions of target bacteria in subgingival plaque samples of subject B. After 3 months, the Porphyromonas gingivalis population was markedly reduced (3.1 x 10(-3) %), whereas the proportion of Porphyromonas gingivalis before treatment was 7.6 %. The proportions of Tannerella forsythensis, Treponema denticola and Treponema socranskii were also markedly diminished after treatment. Similarly, the proportion of the T-RF presumed to represent Porphyromonas gingivalis was 5.9 % and became undetectable after 3 months. Analysis of 16S rRNA gene clone libraries from subgingival plaque samples of subject B before and after treatment showed a notable change in the subgingival microbiota. These results were in agreement with the T-RFLP analysis data and showed that the T-RFs larger than 1000 bp represent Peptostreptococcus species. Our results indicate that T-RFLP analysis is useful for evaluation of the effects of medical treatment of periodontitis.

Adult↗

PubMed2006

Novel subgingival bacterial phylotypes detected using multiple universal polymerase chain reaction primer sets.

INTRODUCTION: Molecular ecological analysis based on 16S rRNA gene sequence analysis is well established for the characterisation of complex bacterial communities. However, 'universal' PCR primers can introduce biases into the analysis of the species composition of clone libraries because of mismatches between the primer and target organism sequences. In this study, three universal primer sets were compared for the analysis of the microflora in subgingival plaque. METHODS: Three subgingival plaque samples were collected from two subjects with localised severe chronic periodontitis. Half of each sample was cultured while DNA was extracted from the remaining half and 16S rDNA amplified with universal primer pairs 27F, 1525R (A); 27F, 1492R (B) and 530F, 1525R (C). Amplified genes were cloned, sequenced and identified by comparison with 16S rRNA databases. RESULTS: 137 taxa were identified among 177 isolates and 417 clones sequenced. Of these, 86 were detected only by the molecular technique whereas 26 were found only by culture. Sequences from 81 taxa did not correspond to those of named species and of these, 38 were not represented in the nucleotide databases. 16S RNA genes for these 38 taxa were sequenced and deposited with GenBank. CONCLUSION: The use of three sets of universal primers allowed the identification of 38 novel bacterial phylotypes. There were marked differences in the composition of the libraries generated by the different primer sets. A combination of molecular and cultural techniques is recommended to maximise the coverage of detection of bacterial taxa in oral samples.

Bacteria↗

PubMed2006

Molecular analysis of bacteria in asymptomatic and symptomatic endodontic infections.

The purpose of the present study was to use terminal restriction fragment length polymorphism analysis and the 16S rRNA gene clone library to investigate the diversity of the microbiota associated with asymptomatic and symptomatic endodontic infections and to compare the bacterial community structure in these two clinical conditions. Samples were taken from asymptomatic endodontic infections associated with chronic periradicular lesions and from symptomatic infections clinically diagnosed as acute abscesses. 16S rRNA genes from DNA isolated from clinical samples were used to construct clone libraries or were subjected to terminal restriction fragment length polymorphism analysis. Sequence analysis of 186 clones revealed 42 taxa; 23 (55%) were uncultivated phylotypes, of which seven were unique to endodontic infections. Clone sequencing and terminal restriction fragment length polymorphism analysis revealed that the most commonly detected taxa were Fusobacterium nucleatum (including terminal restriction fragment types 1 and 2), Peptostreptococcus micros/Peptostreptococcus sp. oral clone AJ062/BS044/FG014, Prevotella species, Dialister species, Mogibacterium species, Lachnospiraceae oral clone 55A-34, Filifactor alocis, Megasphaera sp. oral clone CS025/BS073, and Veillonella sp. oral clone BP1-85/Veillonella dispar/V. parvula. Bacteroides-like sp. oral clone X083/Bacteroidales oral clone MCE7_20 and Dialister sp. oral clone BS016/MCE7_134 were detected only in asymptomatic teeth. On the other hand, F. nucleatum terminal restriction fragment type 2, Prevotella intermedia, Dialister pneumosintes, and some phylotypes were exclusively detected in symptomatic samples. Bacterial profiles of symptomatic endodontic infections generated by terminal restriction fragment length polymorphism analysis were clearly different from those of asymptomatic infections. Overall, the average number of terminal restriction fragments in symptomatic samples was significantly larger than in asymptomatic samples. Molecular analysis of the microbiota associated with symptomatic or asymptomatic endodontic infections indicates that the endodontic bacterial diversity is greater than previously described by culture methods and that the structure of the microbiota differ significantly between asymptomatic and symptomatic infections.

Adolescent↗

PubMed1975

Cellulases from Sporocytophaga myxococcoides. Purification and Properties.

Two extracellular cellulases active on carboxymethylcellulose have been isolated from the culture supernatant of Sporocytophaga myxococcoides by a series of gel-filtration and ion-exchange chromatography steps. Cellulase II, being present in highest amount, had a molecular weight determined by gel electrophoresis of 52000, pI 4.75 and a relatively broad pH optimum (5.5--7.5). Cellulase I had a molecular weight of 46000. pI was 7.5 and the pH optimum 6.5--7.5. Both cellulases had a very low carbohydrate content, possibly present as impurities. They had fairly similar amino acid compositions. The specific acitivity of cellulase I was about 6 times higher than that of cellulase II. Both cellulases acted as endoglucanases. A cell-associated cellulase, present in amounts corresponding to about 10% of total activity, was partly purified. It showed similarities with cellulase II.

Amino Acids↗

PubMed1994

Intracellular expression of Vitreoscilla hemoglobin alters Escherichia coli energy metabolism under oxygen-limited conditions.

An earlier stoichiometric analysis of oxygen-limited metabolism of Escherichia coli expressing cloned Vitreoscilla hemoglobin (VHb) suggested improved efficiency of ATP production relative to wild-type controls [Khosla, C., Curtis, J. E., DeModena, J., Rinas, J. & Bailey, J. E. (1990) BioTechnol. 8, 849-853]. This hypothesis has been further examined by determining several energetic parameters of different VHb-expressing E. coli (VHb+) strains relative to controls not expressing VHb (VHb-). The H+/O ratio, the transmembrane delta pH, and the ATP content of VHb+ constructs are 1.5, 1.6 and 2 times, respectively, corresponding values in VHb- controls. VHb was expressed using a low-copy-number vector in E. coli mutant strains lacking cytochrome o, cytochrome d, or both terminal oxidases; significant growth enhancement due to VHb expression was observed only in the strain having functional cytochrome o and lacking cytochrome d. All of these data obtained using different E. coli strains are consistent with a model of VHb action that hypothesizes enhancement by VHb of activity of the lower oxygen-affinity, higher proton-pumping-efficiency cytochrome o terminal oxidase under oxygen-limited growth conditions.

Aerobiosis↗

PubMed2004

Quantifying 3H-thymidine incorporation rates by a phylogenetically defined group of marine planktonic bacteria (Bacteriodetes phylum).

The rate of [(3)H-methyl] thymidine ((3)H-TdR) incorporation into DNA has been applied extensively to measure cell production by bacterial communities in aquatic environments. Here we describe a method to quantify (3)H-TdR incorporation by specific, phylogenetically defined members of the bacterial community. The method involves selectively capturing DNA from targeted groups of bacteria and then quantifying its (3)H radioactivity. The method was applied to measure (3)H-TdR incorporation by the members of the phylum Bacteriodetes whose members, which include the Cytophaga-Flavobacter cluster, are ubiquitous in coastal waters. (3)H-labelled DNA from Bacteriodetes was selectively biotinylated in PCR-like reactions that contained a Bacteriodetes-specific 16S rRNA gene primer, thermostable DNA polymerase and biotinylated dUTP. The biotinylated DNA was then captured on streptavidin-coated beads and its (3)H radioactivity determined by scintillation counting. We have termed this method 'selective nucleic acid polymerase-biotinylation and capture' or 'SNAP-BAC'. Internal (33)P-labelled DNA standards were used to quantify the recovery of (3)H-labelled DNA from the SNAP-BAC reactions. The method was verified by successfully targeting Bacteriodetes in simple laboratory mixtures of (3)H-labelled DNA extracted from pure cultures of Bacteriodetes and gamma-proteobacteria. Field application of this method in Puget Sound and off the Washington coast determined that Bacteriodetes were responsible for 56 +/- 17% and 32 +/- 5% of community (3)H-TdR incorporation (1.3 +/- 0.3 and 9.9 +/- 1.7 pmol l(-1) h(-1)) at these two locations.

Bacteroidetes↗

PubMed2006

Identification and characterization of ectosymbionts of distinct lineages in Bacteroidales attached to flagellated protists in the gut of termites and a wood-feeding cockroach.

Bacterial attachments to nearly the entire surface of flagellated protists in the guts of termites and the wood-feeding cockroach Cryptocercus are often observed. Based on the polymerase chain reaction-amplified 16S rRNA gene sequences, we investigated the phylogenetic relationships of the rod-shaped, attached bacteria (ectosymbionts) of several protist species from five host taxa and confirmed their identity by fluorescence in situ hybridizations. These ectosymbionts are affiliated with the order Bacteroidales but formed three distinct lineages, each of which may represent novel bacterial genera. One lineage consisted of the closely related ectosymbionts of two species of the protist genus Devescovina (Cristamonadida). The second lineage comprised three phylotypes identified from the protist Streblomastix sp. (Oxymonadida). The third lineage included ectosymbionts of the three protist genera Hoplonympha, Barbulanympha and Urinympha in the family Hoplonymphidae (Trichonymphida). The ultrastructural observations indicated that these rod-shaped ectosymbionts share morphological similarities of their cell walls and their point of attachment with the protist but differ in shape. Elongated forms of the ectosymbionts appeared in all the three lineages. The protist cells Streblomastix sp. and Hoplonympha sp. display deep furrows and vane-like structures, but these impressive structures are probably evolutionarily convergent because both the host protists and their ectosymbionts are distantly related.

Animals↗

PubMed2006

Phylogenetic identification and metabolism of marine dimethylsulfide-consuming bacteria.

Microbial consumption is one of the main processes, along with photolysis and ventilation, that remove the biogenic trace gas dimethylsulfide (DMS) from the surface ocean. Although a few isolates of marine bacteria have been studied for their ability to utilize DMS, little is known about the characteristics or phylogenetic affiliation of DMS consumers in seawater. We enriched coastal and open-ocean waters with different carbon sources to stimulate different bacterial communities (glucose-consuming bacteria, methyl group-consuming bacteria and DMS consumers) in order to test how this affected DMS consumption and to examine which organisms might be involved. Dimethylsulfide consumption was greatly stimulated in the DMS addition treatments whereas there was no stimulation in the other treatments. Analysis of microbial DNA by two different techniques (sequenced bands from DGGE gels and clone libraries) showed that bacteria grown specifically with the presence of DMS were closely related to the genus Methylophaga. We also followed the fate of consumed DMS in some of the enrichments. Dimethylsulfide was converted mostly to DMSO in glucose or methanol enrichments, whereas it was converted mostly to sulfate in DMS enrichments, the latter suggesting use of DMS as a carbon and energy source. Our results indicate that unlike the biochemical precursor of DMS, dimethylsulfoniopropionate (DMSP), which is consumed by a broad spectrum of marine microorganisms, DMS seems to be utilized as a carbon and electron source by specialists. This is consistent with the usual observation that DMSP turns over at much higher rates than DMS.

Atlantic Ocean↗

PubMed2006

Spatial distribution of sponge-associated bacteria in the Mediterranean sponge Tethya aurantium.

The local distribution of the bacterial community associated with the marine sponge Tethya aurantium Pallas 1766 was studied. Distinct bacterial communities were found to inhabit the endosome and cortex. Clear differences in the associated bacterial populations were demonstrated by denaturing gradient gel electrophoresis (DGGE) and analysis of 16S rRNA gene clone libraries. Specifically associated phylotypes were identified for both regions: a new phylotype of Flexibacteria was recovered only from the sponge cortex, while Synechococcus species were present mainly in the sponge endosome. Light conduction via radiate spicule bundles conceivably facilitates the unusual association of Cyanobacteria with the sponge endosome. Furthermore, a new monophyletic cluster of sponge-derived 16S rRNA gene sequences related to the Betaproteobacteria was identified using analysis of 16S rRNA gene clone libraries. Members of this cluster were specifically associated with both cortex and endosome of T. aurantium.

Animals↗

PubMed2007

Characterization of marine bacteria and the activity of their enzyme systems involved in degradation of the algal storage glucan laminarin.

The algal storage glucan laminarin is one of the most abundant carbon sources for marine prokaryotes. Its degradation was investigated in bacteria isolated during and after a spring phytoplankton bloom in the coastal North Sea. On average, 13% of prokaryotes detected by epifluorescence counts were able to grow in Most Probable Number dilution series on laminarin as sole carbon source. Several bacterial strains were isolated from different dilutions, and phylogenetic characterization revealed that they belonged to different phylogenetic groups. The activity of the laminarin-degrading enzyme systems was further characterized in three strains of Vibrio sp. that were able to grow on laminarin as sole carbon source. At least two types of activity were detected upon degradation of laminarin: release of glucose, and release of glucans larger than glucose. The expression of laminarinase activity was dependent on the presence of the substrate, and was repressed by the presence of glucose. In addition, low levels of activity were expressed under starvation conditions. Laminarinase enzymes showed minimal activity on substrates with similar glucosidic bonds to those of laminarin, but different sizes and secondary and/or tertiary structures. The characteristics found in these enzyme systems may help to elucidate factors hampering rapid carbohydrate degradation by prokaryotes.

Bacteria↗

PubMed1994

Phylogeny and lipid composition of Thermonema lapsum, a thermophilic gliding bacterium.

1,490 nucleotides of the 16S rRNA gene of a Gram-negative, thermophilic and gliding bacterium, Thermonema lapsum, have been sequenced. Phylogenetic analysis indicates that T. lapsum is related to cytophaga-flavobacteria-bacteroides (CFB) and is confirmed by the identification signature nucleotides that define this group. Further phylogenetic analysis indicates that T. lapsum forms the deepest branch in the CFB group; this observation was confirmed by the identification of unique nucleotide and nucleotide pairs which separate T. lapsum from all other members of this group. The phospholipid fatty acid (PLFA) profile also confirmed that T. lapsum is related to the cytophaga-flavobacteria-bacteroides group and also to selected members of the genus Flexibacter; the PLFA profile is unique to T. lapsum.

Bacteria, Aerobic↗

PubMed1998

Evidence for substrate binding of a recombinant thermostable xylanase originating from Rhodothermus marinus.

The xynl encoded 5 domain xylanase from the thermophilic bacterium Rhodothermus marinus binds specifically to xylan, beta-glucan and amorphous but not crystalline cellulose. Our results show that the binding is mediated by the full length xylanase, but not by the catalytic domain only. Based on similarities concerning both predicted secondary structure and binding specificity found with one cellulose binding domain of CenC from Cellulomonas fimi, we suggest that the binding is mediated by the two N-terminally repeated domains.

Amino Acid Sequence↗

PubMed2005

Isolation of an algal morphogenesis inducer from a marine bacterium.

Ulva and Enteromorpha are cosmopolitan and familiar marine algal genera. It is well known that these green macroalgae lose their natural morphology during short-term cultivation under aseptic conditions and during long-term cultivation in nutrient-added seawater and adopt an unusual form instead. These phenomena led to the belief that undefined morphogenetic factors that were indispensable to the foliaceous morphology of macroalgae exist throughout the oceans. We characterize a causative factor, named thallusin, isolated from an epiphytic marine bacterium. Thallusin induces normal germination and morphogenesis of green macroalgae.

Bacteroidetes↗

PubMed2005

Diversity of the human intestinal microbial flora.

The human endogenous intestinal microflora is an essential "organ" in providing nourishment, regulating epithelial development, and instructing innate immunity; yet, surprisingly, basic features remain poorly described. We examined 13,355 prokaryotic ribosomal RNA gene sequences from multiple colonic mucosal sites and feces of healthy subjects to improve our understanding of gut microbial diversity. A majority of the bacterial sequences corresponded to uncultivated species and novel microorganisms. We discovered significant intersubject variability and differences between stool and mucosa community composition. Characterization of this immensely diverse ecosystem is the first step in elucidating its role in health and disease.

Adult↗

PubMed2005

Xanthorhodopsin: a proton pump with a light-harvesting carotenoid antenna.

Energy transfer from light-harvesting carotenoids to chlorophyll is common in photosynthesis, but such antenna pigments have not been observed in retinal-based ion pumps and photoreceptors. Here we describe xanthorhodopsin, a proton-pumping retinal protein/carotenoid complex in the eubacterium Salinibacter ruber. The wavelength dependence of the rate of pumping and difference absorption spectra measured under a variety of conditions indicate that this protein contains two chromophores, retinal and the carotenoid salinixanthin, in a molar ratio of about 1:1. The two chromophores interact strongly, and light energy absorbed by the carotenoid is transferred to the retinal with a quantum efficiency of approximately 40%. The antenna carotenoid extends the wavelength range of the collection of light for uphill transmembrane proton transport.

Amino Acid Sequence↗

PubMed2006

Persistence and growth of fecal Bacteroidales assessed by bromodeoxyuridine immunocapture.

Extraintestinal growth of fecal bacteria can impair accurate assessment of watershed health. Anaerobic fecal bacteria belonging to the order Bacteroidales are attractive candidates for fecal source tracking because they have host-specific distributions and do not grow well in the presence of high oxygen concentrations. Growth of general and human-specific fecal Bacteroidales marker organisms in environmental samples (sewage) and persistence of the corresponding genetic markers were investigated using bromodeoxyuridine (BrdU) DNA labeling and immunocapture, followed by PCR detection. Background amplification of unlabeled controls occasionally occurred when a high number of PCR cycles was used. By using fluorescent detection of PCR products obtained after 15 cycles, which was determined to be quantitative, we enriched for BrdU-labeled DNA and did not detect unlabeled DNA. By using pure cultures of Bacteroides vulgatus, the ability of Bacteroidales bacteria to take up and incorporate BrdU into nascent DNA was confirmed. Fecal Bacteroidales organisms took up and incorporated BrdU into DNA during growth. In sewage incubated aerobically at the in situ temperature, Bacteroidales genetic marker sequences persisted for at least 24 h and Bacteroidales fecal bacteria grew for up to 24 h as well. Detection by PCR using a low, quantitative cycle number decreased the sensitivity of the assay such that we were unable to detect fecal Bacteroidales human-specific marker sequences in unlabeled or BrdU-labeled fractions, even when fluorescent detection was used. Using 30 PCR cycles with unlabeled fractions, human-specific Bacteroidales sequences were detected, and they persisted for up to 24 h in sewage. These data support the utility of BrdU labeling and immunocapture followed by length heterogeneity PCR or fluorescent detection using low numbers of PCR cycles. However, this method may not be sensitive enough to identify cells that are present at low densities in aquatic environments.

Bacteroidetes↗