Full-length 16S rRNA Sequences Research Articles

Phyllosphere bacterial communities of trichome-bearing and trichomeless Arabidopsis thaliana leaves

This study aimed to investigate whether the presence of trichomes as conspicuous physical attributes of the leaf surface affects the microbial community composition on Arabidopsis thaliana leaves. The A. thaliana ecotype Col-0 and its trichomeless gl1 mutant were grown in growth cabinets under climate-controlled conditions. The gl1 mutant showed a similar wax composition as the Col-0 wild type with slightly reduced amounts of C(29), C(31) and C(33) alkanes by GC/MS and GC/FID analyses. 120 bacterial isolates representing 39 bacterial genera were obtained from A. thaliana Col-0 leaf surfaces. Phylogenetic analysis of nearly full-length 16S rRNA sequences from 29 selected isolates confirmed their affiliation to the Proteobacteria (Alpha-, Beta-, Gamma-), Actinobacteria, Bacteroidetes and Firmicutes. The bacterial diversity on A. thaliana ecotype Col-0 and its gl1 mutant, devoid of trichomes, were further compared by denaturing gradient gel electrophoresis (DGGE). Banding patterns and sequencing of representative DGGE bands revealed the presence of phylotypes related to Sphingomonas (Alphaproteobacteria), Methylophilus (Betaproteobacteria) and Dyadobacter (Bacteroidetes) which are common phyllosphere inhabitants. Furthermore, wildtype and trichomeless mutant plants were exposed to outdoor conditions for 4-5weeks. The DGGE gels showed only minor differences between the two plant lines, thus suggesting that trichomes per se do not affect bacterial diversity on Arabidopsis leaves under the experimental conditions tested.

Brevibacterium frigoritolerans a novel entomopathogen of Anomala dimidiata and Holotrichia longipennis (Scarabaeidae: Coleoptera)

We describe the isolation, biochemical characterization, phylogenetic analysis, and pathogenecity of a novel entomopathogenic bacterium Brevibacterium frigoritolerans to first instar larvae of Anomala dimidiata and Holotrichia longipennis. The almost full length 16S rRNA sequence of the bacterium has 99% identity with the type strain of B. frigoritolerans, while phylogenetic analysis revealed that the isolate formed a tightly linked branch with the type strain of B. frigoritolerans. Under in vitro bioassay conditions, the isolate infected and caused 89±5.4 and 74±7.7% mortality, in first instar larvae of A. dimidiata and H. longipennis, respectively. The infected larvae exhibited bacteremia like symptoms and mortality occurred between the second and fifth weeks after inoculation. This is an early report on the entomopathogenic potential of the hitherto lesser-known bacterium Brevibacterium frigoritolerans.

Molecular characterisation of the microbial community of a full-scale bioreactor treating Bayer liquor organic waste

Sodium oxalate is an organic impurity produced during the Bayer refining of bauxite as a result of the degradation of humic materials associated with the ore. Physico-chemical oxalate destruction techniques, such as combustion, are often expensive and often pose greater environmental risk than the storage of solid oxalate waste. Biodegradation is a more economical and environmentally friendly way to degrade oxalate, but the microbial communities responsible for oxalate degradation have remained largely uncharacterised. In the present work, the microbial community of a full-scale bioreactor achieving complete degradation of oxalate was characterised using 16S rRNA gene clone libraries followed by phylogenetic analysis of the cloned near full-length 16S rRNA sequences. The community was dominated by species belonging to the α-, β- and γ- Proteobacteria groups. Novel oxalate-degrading bacteria belonging to the genus Halomonas and the β- Proteobacteria group were isolated from the microbial community and are currently being characterised.

Unveiling an abundant core microbiota in the human adult colon by a phylogroup-independent searching approach

The potential presence of widespread and stable bacterial core phylogroups in the human colon has promoted considerable attention. Despite major efforts, no such phylogroups have yet been identified. Therefore, using a novel phylogroup- and tree-independent approach, we present a reanalysis of 1,114,722 V2 region and 71,550 near full-length 16S rRNA sequences from a total of 210 human beings, with widespread geographic origin, ethnic background and diet, in addition to a wide range of other mammals. We found two highly prevalent core phylogroups (cores 1 and 2), belonging to the clostridial family Lachnospiraceae. These core phylogroups showed a log-normal distribution among human individuals, while non-core phylogroups showed more skewed distributions towards individuals with low levels compared with the log-normal distribution. Molecular clock analyses suggest that core 2 co-evolved with the radiation of vertebrates, while core 1 co-evolved with the mammals. Taken together, the stability, prevalence and potential functionality support the fact that the identified core phylogroups are pivotal in maintaining gut homeostasis and health.

Gene cloning and characterization of a xylanase from a newly isolated Bacillus subtilis strain R5

A novel mesophilic strain, R5, was isolated from Osaka, Japan. The growth temperature of the strain ranged from 10 to 40 degrees C with optimal growth at 30 degrees C. Cells of strain R5 were highly motile small rods. The full-length 16S rRNA sequence was 99% homologous to that of Bacillus subtilis strain 168. The optimum pH and NaCl concentration for growth of the strain were 7.0 and 3%, respectively. Based on the biochemical characteristics and 16S rRNA sequences R5 was identified as a strain of B. subtilis. The strain R5 produced protease, cellulase, amylase, lipase/esterase, xylanase and a biosurfactant extracellularly. The gene encoding xylanase was cloned and expressed in Escherichia coli. The gene encoded a protein consisting of 213 amino acids with a relative molecular mass of 23 kDa. The gene product was purified and examined for enzymatic characteristics. The recombinant enzyme exhibited highest activity at temperatures ranging from 40 to 50 degrees C and at pH 6.0. The enzyme activity was enhanced in the presence of metal cations. The V(max) and K(m) values of the recombinant enzyme towards xylan from beech-wood were 5550 nkat/mg and 4.5 mg/mL, respectively.

Lynamicins A−E, Chlorinated Bisindole Pyrrole Antibiotics from a Novel Marine Actinomycete

A series of chlorinated bisindole pyrroles, lynamicins A-E (1-5), was discovered from a novel marine actinomycete, NPS12745, which was isolated from a marine sediment collected off the coast of San Diego, California. Close to full length 16S rRNA sequence analysis indicated that NPS12745 is a novel strain of a recently described marine actinomycete with the proposed genus name Marinispora. The antimicrobial spectrum of these compounds was evaluated against a panel of 11 pathogens, which demonstrated that these substances possess broad-spectrum activity against both Gram-positive and Gram-negative organisms. Significantly, compounds 1-5 were active against drug-resistant pathogens such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium.

Accurate taxonomy assignments from 16S rRNA sequences produced by highly parallel pyrosequencers

The recent introduction of massively parallel pyrosequencers allows rapid, inexpensive analysis of microbial community composition using 16S ribosomal RNA (rRNA) sequences. However, a major challenge is to design a workflow so that taxonomic information can be accurately and rapidly assigned to each read, so that the composition of each community can be linked back to likely ecological roles played by members of each species, genus, family or phylum. Here, we use three large 16S rRNA datasets to test whether taxonomic information based on the full-length sequences can be recaptured by short reads that simulate the pyrosequencer outputs. We find that different taxonomic assignment methods vary radically in their ability to recapture the taxonomic information in full-length 16S rRNA sequences: most methods are sensitive to the region of the 16S rRNA gene that is targeted for sequencing, but many combinations of methods and rRNA regions produce consistent and accurate results. To process large datasets of partial 16S rRNA sequences obtained from surveys of various microbial communities, including those from human body habitats, we recommend the use of Greengenes or RDP classifier with fragments of at least 250 bases, starting from one of the primers R357, R534, R798, F343 or F517.

First isolation of Nocardia crassostreae from Pacific oyster Crassostrea gigas in Europe

In summer 2006 an extensive mortality of Pacific oysters Crassostrea gigas occurred in Lake Grevelingen, the Netherlands. A sample of Pacific oysters was investigated for the presence of shellfish pathogens as potential causes of the mortality. Yellow-green lesions were observed in several oysters upon clinical inspection. Histopathology showed that 6 out of 36 oysters had a suspected bacterial infection, including 4 Nocardia-like infections. Two bacterial species, Vibrio aestuarianus and Nocardia crassostreae, were isolated from haemolymph samples and identified using PCR and sequencing of the 16S rRNA gene. This is the first isolation of N. crassostreae from shellfish in European waters. The near full-length 16S rRNA sequence of this Dutch Nocardia sp. isolate was identical to other known N. crassostreae isolates from the west coast of North America. The primary cause of oyster mortality was thought to be the physiological stress from environmental conditions, including prolonged high water temperatures and low oxygen levels. The multiple bacterial species isolated from the diseased Pacific oysters may have been a secondary cause.

Identification of environmental plasmid-bearing Vibrio species isolated from polluted and pristine marine reserves of Hong Kong, and resistance to antibiotics and mercury

Fifty environmental isolates of Vibrio species were isolated from water samples of Mai Po Nature Reserve and the Cape d'Aguilar Marine Reserve in Hong Kong and screened for the presence of plasmid. Mai Po is a wastewater-impacted area while the Cape d'Aguilar Marine Reserve is pristine natural marine water. Plasmid was found in Vibrio isolates from both sites at similar frequencies and each site showed distinctive plasmid profiles. These plasmid-bearing Vibrio isolates were identified as different species of the Vibrio genus by both biochemical test and subsequently full-length 16S rRNA sequences. Antibiotic resistance test showed that all these plasmid-bearing Vibrio isolates showed multiple resistance to 21 antibiotics tested. In addition, selective isolates also showed tolerance to 10 microM Hg 2+ in culture medium and they generally harbored large plasmid(s) (>30 kb). Our results show that the high frequency of plasmid in Vibrio species of both polluted and pristine environments may be ecologically important to the survival of these bacteria in the environment. The specific functioning of the cryptic plasmids remains the focus of current investigations.

RRNA and dnaK relationships of Bradyrhizobium sp. Nodule Bacteria from four Papilionoid legume trees in Costa Rica

Enzyme electrophoresis and sequencing of rRNA and dnaK genes revealed high genetic diversity among root nodule bacteria from the Costa Rican trees Andira inermis, Dalbergia retusa, Platymiscium pinnatum (Papilionoideae tribe Dalbergieae) and Lonchocarpus atropurpureus (Papilionoideae tribe Millettieae). A total of 21 distinct multilocus genotypes [ETs (electrophoretic types)] was found among the 36 isolates analyzed, and no ETs were shared in common by isolates from different legume hosts. However, three of the ETs from D. retusa were identical to Bradyrhizobium sp. isolates detected in prior studies of several other legume genera in both Costa Rica and Panama. Nearly full-length 16S rRNA sequences and partial 23S rRNA sequences confirmed that two isolates from D. retusa were highly similar or identical to Bradyrhizobium strains isolated from the legumes Erythrina and Clitoria (Papilionoideae tribe Phaseoleae) in Panama. rRNA sequences for five isolates from L. atropurpureus, P. pinnatum and A. inermis were not closely related to any currently known strains from Central America or elsewhere, but had affinities to the reference strains Bradyrhizobium japonicum USDA 110 (three isolates) or to B. elkanii USDA 76 (two isolates). A phylogenetic tree for 21 Bradyrhizobium strains based on 603 bp of the dnaK gene showed several significant conflicts with the rRNA tree, suggesting that genealogical relationships may have been altered by lateral gene transfer events.

Divergent Bradyrhizobium symbionts on Tachigali versicolor from Barro Colorado Island, Panama

Relationships of root-nodule bacteria from the tree Tachigali versicolor (legume subfamily Caesalpinioideae) were analyzed for 20 isolates sampled from juvenile plants growing on Barro Colorado Island (BCI), Panama. Bacterial genetic diversity appeared to be low. In the highly polymorphic 5' intervening sequence region of 23S rRNA, all isolates had the same length variant. A 472 bp segment spanning this region was sequenced in four isolates, and all proved to be identical at every nucleotide position. RFLP analysis of a 868 bp fragment of the nitrogenase alpha-subunit gene likewise indicated that all 20 isolates shared an identical set of restriction sites. Phylogenetic analysis of both partial 23S rRNA and nearly full-length 16S rRNA sequences showed that bacterial symbionts of T. versicolor fall into the genus Bradyrhizobium. However, they are divergent from the bradyrhizobia associated with other BCI legumes, as well as from other currently known bacteria in this genus. Inoculation tests with two promiscuously-nodulating legumes showed that bacteria from T. versicolor were unable to form nodules on Vigna unguiculata, but did nodulate Macroptilium atropurpureum, although the nodules lacked nitrogen fixation activity. The association of Tachigali with a divergent lineage of Bradyrhizobium is noteworthy in view of this plant's position within a clade of the mostly non-nodulating "primitive" legume subfamily Caesalpinioideae that gave rise to the predominantly nodulating subfamily Mimosoideae.

Relationships of bradyrhizobia from Platypodium and Machaerium (Papilionoideae: tribe Dalbergieae) on Barro Colorado Island, Panama.

Enzyme electrophoresis and rRNA sequencing indicated that root nodule bacteria from the canopy tree Platypodium elegans and the lianas Machaerium milleflorum and Machaerium arboreum on Barro Colorado Island, Panama, were highly diverse on a local scale. A total of 11 distinct multilocus genotypes [ETs (electrophoretic types)] was found among the 33 isolates analysed. On average, ETs differed from one another at 74% of the 11 enzyme loci assayed, and separate nodules on a single host individual were often occupied by genetically divergent ETs. Certain ETs were sampled multiple times from both Platypodium and Machaerium, suggesting a lack of specificity toward the two legume genera. Within the intervening sequence (IVS) region in the 5' end of 23S rRNA, seven ETs had a length variant similar to that of Bradyrhizobium japonicum USDA 110, and the other four ETs had an IVS region 26-28 bp shorter. Parsimony analysis of both partial 23S rRNA and nearly full-length 16S rRNA sequences indicated that all Platypodium and Machaerium isolates were related to B. japonicum rather than Bradyrhizobium elkanii. The 16S rRNA sequence of one isolate was >99% similar to that of B. japonicum USDA 110, and the closest known relatives for other isolates were Philippine bradyrhizobia from the legumes Stylosanthes and Samanea.

Relationships of bradyrhizobia from the legumes Apios americana and Desmodium glutinosum.

Multilocus enzyme electrophoresis, partial 23S rRNA sequences, and nearly full-length 16S rRNA sequences all indicated high genetic similarity among root-nodule bacteria associated with Apios americana, Desmodium glutinosum, and Amphicarpaea bracteata, three common herbaceous legumes whose native geographic ranges in eastern North America overlap extensively. A total of 19 distinct multilocus genotypes (electrophoretic types [ETs]) were found among the 35 A. americana and 33 D. glutinosum isolates analyzed. Twelve of these ETs (representing 78% of all isolates) were either identical to ETs previously observed in A. bracteata populations, or differed at only one locus. Within both 23S and 16S rRNA genes, several isolates from A. americana and D. glutinosum were either identical to A. bracteata isolates or showed only single nucleotide differences. Growth rates and nitrogenase activities of A. bracteata plants inoculated with isolates from D. glutinosum were equivalent to levels found with native A. bracteata bacterial isolates, but none of the three A. americana isolates tested had high symbiotic effectiveness on A. bracteata. Phylogenetic analysis of both 23S and 16S rRNA sequences indicated that both A. americana and D. glutinosum harbored rare bacterial genotypes similar to Bradyrhizobium japonicum USDA 110. However, the predominant root nodule bacteria on both legumes were closely related to Bradyrhizobium elkanii.

Phylogeny and Diversity of Achromatium oxaliferum

Achromatium oxaliferum was first described in 1893 by Schewiakoff as an unusually large bacterium living in freshwater sediments. Up to now no pure culture is available. Physical enrichments of achromatia collected from the acidic Lake Fuchskuhle, which houses a peculiar, smaller variety, and the neutral Lake Stechlin were investigated by the cultivation-independent rRNA approach. PCR in combination with cloning and sequencing was used for the retrieval of 24 partial and 4 nearly full-length 16S rRNA sequences that formed two distinct phylogenetic clusters. Fluorescence-in-situ-hybridization (FISH) with four 16S rRNA-targeted oligonucleotide probes unambiguously assigned the different sequences to either regular, large A. oxaliferum cells or to the smaller Lake Fuchskuhle population, tentatively named "A. minus". The two Achromatium sp. 16S rRNA sequence clusters form a stable deep branch in the gamma subclass of the class Proteobacteria. The closest cultivated relatives are Chromatium vinosum, Rhabdochromatium marinum and Ectothiorhodospira halophila with 16S rRNA similarities of 86.2 to 90.5%. Profound differences in the population structure of achromatia were revealed in the two lakes by FISH. In one sample from Lake Stechlin three genotypes could be visualized, and 49% of the cells were assigned to A. oxaliferum clone AST01, 28% to Achromatium sp. genotype AFK192/AFK433 and 23% to Achromatium sp. genotype AFK192/AST433. In contrast, a morphologically and phylogenetically homogeneous population of "A. minus". was present in Lake Fuchskuhle.

Phylogeny and identification in situ of Nevskia ramosa.

An enrichment of the neuston bacterium Nevskia ramosa was investigated by the cultivation-independent rRNA approach. N. ramosa was first described by Famintzin in 1892 as a rod-shaped, slightly bent bacterium forming typical flat rosettes on the surface of shallow freshwater habitats by unilateral slime formation. PCR in combination with cloning and sequencing was used for retrieving 21 partial and 5 nearly full-length 16S rRNA sequences forming three tight clusters. In situ hybridization with rRNA-targeted oligonucleotide probes allowed us to assign the three sequence clusters to three distinct bacterial populations abundant in the enrichment. The two probes that unambiguously identified the N. ramosa morphotype were derived from a 16S rRNA sequence that had similarities of 87.9 to 88.9% to the rRNA sequences of the most closely related group in the database, Xanthomonas sp. and relatives. N. ramosa currently is the only representative of an independent, deep branch of the gamma subclass of the class Proteobacteria. The two other populations abundant in the enrichment were affiliated with the alpha subclass of the class Proteobacteria. They were most closely related to Blastobacter sp. (97.2% similarity) and Mycoplana bullata (97.6% similarity) and might represent new species in the respective genera.

Phylogeny of Rhizobium galegae with respect to other rhizobia and agrobacteria.

PCR-RFLP with nine restriction enzymes was applied to the 16S and 23S rRNA genes of 42 rhizobial and agrobacterial strains to determine the phylogenetic position of Rhizobium galegae and increase the understanding of the evolution of ribosomal operons. The strains were selected based on previous phylogenetic studies. PCR primers were designed so that they amplified a 2.3 kb fragment of the 23S rRNA gene (excluding the B8 loop). Universal primers rD1 and fD1 were used to amplify the full-length 16S rRNA. The RFLP analysis resulted in 27 and 32 different restriction patterns for 16S and 23S, respectively. The RFLP patterns were transformed to genetic distances and dendrograms were constructed from the data using the unweighted pair group method with averages. The shapes of the dendrograms derived from the analysis of the 16S and 23S rRNA genes correlated well, with only a few strains having different positions. The 23S tree generally had deeper branching than the 16S tree, allowing better discrimination between species and strains. The combined data from the two analyses described 36 genotypes. The eight R. galegae strains formed a homogeneous cluster in all dendrograms. The RFLP analysis was confirmed by partial sequence analysis of the 16S rRNA gene (the first 800 bp), which correlated well with full-length 16S rRNA sequence analysis. The 16S data placed R. galegae near the genus Agrobacterium with Agrobacterium vitis as its nearest neighbour, whereas in the 23S and the combined dendrograms it showed closer affinity to the genus Rhizobium.