16S rRNA Similarity Research Articles

Abundant fungi dominate the complexity of microbial networks in soil of contaminated site: High-precision community analysis by full-length sequencing

During the past decade, the characterization of microbial community in soil of contaminated sites was primarily done by high-throughput short-read amplicon sequencing. However, due to the similarity of 16S rRNA and ITS genes amplicon sequences, the short-read approach often limits the microbial composition analysis at the species level. Here, we simultaneously performed full-length and short-read amplicon sequencing to clarify the community composition and ecological status of different microbial taxa in contaminated soil from a high-resolution perspective. We found that (1) full-length 16S rRNA gene sequencing gave better resolution for bacterial identification at all levels, while there were no significant differences between the two sequencing platforms for fungal identification in some samples. (2) Abundant taxa were vital for microbial co-occurrences network constructed by both full-length and short-read sequencing data, and abundant fungal species such as Mortierella alpine, Fusarium solani, Mrakia frigida, and Chaetomium homopilatum served as the keystone species. (3) Heavy metal correlated with the microbial community significantly, and bacterial community and its abundant taxa were assembled by deterministic process, while the other taxa were dominated by stochastic process. These findings contribute to the understanding of the ecological mechanisms and microbial interactions in site soil ecosystems and demonstrate that full-length sequencing has the potential to provide more details of microbial community.

Phylogenetic Tree 16S rRNA Gene of Acinetobacter soli Isolated from the Prepuce of Aceh Cattle

BACKGROUND: In the pre-seed area of healthy Aceh cattle, it is possible to be contaminated with pathogenic bacteria that can interfere with the reproductive system. This study is needed to identify these pathogenic bacteria using a molecular approach, in an effort to prevent infection. AIM: The aim of the present study was to construct phylogenetic tree relationships of Acinetobacter soli identified in the preputial area of Aceh cattle by molecular analysis using 16S rRNA gene sequencing. MATERIALS AND METHODS: A total of 75 preputial specimens were obtained from Indrapuri’s Breeding and Forages Center of Aceh Cattles, Indrapuri district, Banda Aceh, Indonesia. The samples were processed for culture using standard conventional methods. The extraction of genomic DNA and the amplification of the 16S rRNA gene were assayed using polymerase chain reaction. A phylogenetic tree was constructed using distance matrices using the neighbor-joining model of the molecular evolutionary genetic analysis software 6.1 software. RESULTS: The results showed that of 75 preputial swab samples, 18 (24%) were positive for A. soli isolates. There was a 100% sequence similarity to A. soli prototype strain B1 and a 99% similarity to Acinetobacter parvus prototype strain LUH4616, Acinetobacter baylyi strain B2, A. venetianus strain ATCC 31012, as well as a 99% similarity to Acinetobacter baumannii strain DSM 30007, the strain ATCC 19606, and the strain JCM 6841, respectively. We concluded that A. soli-positive presentation in the preparation of Aceh cattle has 100% sequence similarity of 16S rRNA with A. soli strain B1. CONCLUSIONS: The conclusion of this study is that, based on the construction of a phylogenetic tree, it shows that 24% of the bacterial isolate is related to A. soli. It is essential to conduct a regular survey for bacterial contamination and to increase worker awareness and education about hygiene standards.

Development of Real-Time PCR Assay to Specifically Detect 22 Bifidobacterium Species and Subspecies Using Comparative Genomics.

Bifidobacterium species are used as probiotics to provide beneficial effects to humans. These effects are specific to some species or subspecies of Bifidobacterium. However, some Bifidobacterium species or subspecies are not distinguished because similarity of 16S rRNA and housekeeping gene sequences within Bifidobacterium species is very high. In this study, we developed a real-time polymerase chain reaction (PCR) assay to rapidly and accurately detect 22 Bifidobacterium species by selecting genetic markers using comparative genomic analysis. A total of 210 Bifidobacterium genome sequences were compared to select species- or subspecies-specific genetic markers. A phylogenetic tree based on pan-genomes generated clusters according to Bifidobacterium species or subspecies except that two strains were not grouped with their subspecies. Based on pan-genomes constructed, species- or subspecies-specific genetic markers were selected. The specificity of these markers was confirmed by aligning these genes against 210 genome sequences. Real-time PCR could detect 22 Bifidobacterium specifically. We constructed the criterion for quantification by standard curves. To further test the developed assay for commercial food products, we monitored 26 probiotic products and 7 dairy products. Real-time PCR results and labeling data were then compared. Most of these products (21/33, 63.6%) were consistent with their label claims. Some products labeled at species level only can be detected up to subspecies level through our developed assay.

Partial rpoB Gene Sequencing Identification and Probiotic Potential of Floricoccus penangensis ML061-4 Isolated from Assam Tea (Camellia sinensis var. assamica)

Assam tea or Miang is a local name of Camellia sinensis var. assamica in northern Thailand. By the local wisdom, Assam tea leaves are used as the raw material in tea fermentation to produce “Fermented Miang” consumed by people in northern Thailand and the countries nearby. In this study, twenty-eight bacterial isolates were obtained from Assam tea leaf samples collected from Nan province, Thailand. Bacterial isolates were identified within 6 genera including Bacillus, Floricoccus, Kocuria, Lysinibacillus, Micrococcus and Staphylococcus. Among these, the strain ML061-4 shared 100.0 and 99.4% similarity of 16S rRNA and rpoB gene sequence with F. penangensis JCM 31735T, respectively. This is the first discovery of F. penangensis in Thailand. F. penangensis ML061-4 exhibited probiotic characteristics including lactic acid production (9.19 ± 0.10 mg/ml), antibacterial activities (Escherichia coli ATCC 25922 and E. coli O157:H7 DMST 12743), acid and bile salt tolerance (71.1 and 54.9%, respectively), autoaggregation (97.0%), coaggregation (66.0% with E. coli O157:H7), cell surface hydrophobicity (90.0%), bacterial adhesion (82.9% with Lactobacillus plantarum FM03-1), competitive inhibition (17.8% with E. coli O157:H7) and competitive exclusion (34.9% with E. coli O157:H7). Overall, the data suggested that F. penangensis ML061-4 had a great potential to be a probiotic.

Inoculation of Sinorhizobium saheli YH1 Leads to Reduced Metal Uptake for Leucaena leucocephala Grown in Mine Tailings and Metal-Polluted Soils

Metalliferous mine tailings have a negative impact on the soil environment near mining areas and render cultivable lands infertile. Phytoremediation involving the synergism of legume and rhizobia provides a useful technique in tackling this issue with cost-effective, environmentally friendly, and easy-to-use features under adverse soil conditions. Leucaena leucocephala has been found to build symbiotic relationships with native rhizobia in the iron-vanadium-titanium oxide (V-Ti magnetite) mine tailing soil. Rhizobia YH1, isolated from the root nodules of L. leucocephala, was classified as Sinorhizobium saheli according to similarity and phylogenetic analyses of 16S rRNA, housekeeping and nitrogen fixation genes. Besides nitrogen fixation, S. saheli YH1 also showed capabilities to produce indole-acetic acid (IAA) (166.77 ± 2.03 mg l−1) and solubilize phosphate (104.41 ± 7.48 mg l−1). Pot culture experiments showed that strain YH1 increased the biomass, plant height and root length of L. leucocephala by 67.2, 39.5 and 27.2% respectively. There was also an average increase in plant N (10.0%), P (112.2%) and K (25.0%) contents compared to inoculation-free control. The inoculation of YH1 not only reduced the uptake of all metals by L. leucocephala in the mine tailings, but also resulted in decreased uptake of Cd by up to 79.9% and Mn by up to 67.6% for plants grown in soils contaminated with Cd/Mn. It was concluded that S. saheli YH1 possessed multiple beneficial effects on L. leucocephala grown in metalliferous soils. Our findings highlight the role of S. saheli YH1 in improving plant health of L. leucocephala by reducing metal uptake by plants grown in heavy metal-polluted soils. We also suggest the idea of using L. leucocephala-S. saheli association for phytoremediation and revegetation of V-Ti mine tailings and soils polluted with Cd or Mn.

Marker genes that are less conserved in their sequences are useful for predicting genome-wide similarity levels between closely related prokaryotic strains.

BackgroundThe 16s rRNA gene is so far the most widely used marker for taxonomical classification and separation of prokaryotes. Since it is universally conserved among prokaryotes, it is possible to use this gene to classify a broad range of prokaryotic organisms. At the same time, it has often been noted that the 16s rRNA gene is too conserved to separate between prokaryotes at finer taxonomic levels.ResultsIn this paper, we examine how well levels of similarity of 16s rRNA and 73 additional universal or nearly universal marker genes correlate with genome-wide levels of gene sequence similarity. We demonstrate that the percent identity of 16s rRNA predicts genome-wide levels of similarity very well for distantly related prokaryotes, but not for closely related ones. In closely related prokaryotes, we find that there are many other marker genes for which levels of similarity are much more predictive of genome-wide levels of gene sequence similarity. Finally, we show that the identities of the markers that are most useful for predicting genome-wide levels of similarity within closely related prokaryotic lineages vary greatly between lineages. However, the most useful markers are always those that are least conserved in their sequences within each lineage.ConclusionsOur results show that by choosing markers that are less conserved in their sequences within a lineage of interest, it is possible to better predict genome-wide gene sequence similarity between closely related prokaryotes than is possible using the 16s rRNA gene. We point readers towards a database we have created (POGO-DB) that can be used to easily establish which markers show lowest levels of sequence conservation within different prokaryotic lineages.Electronic supplementary materialThe online version of this article (doi:10.1186/s40168-016-0162-5) contains supplementary material, which is available to authorized users.

Whole-genome prokaryotic clustering based on gene lengths

The fast-growing number of complete genome sequences prompts the development of new phylogenetic approaches. Until recently, understanding the phylogeny of prokaryotes was based on the comparison of highly conserved genes. Several novel whole-genome methods have been proposed during the last few years. Here, we present a novel method of taxonomic analysis, constructed on the basis of gene content and lengths of orthologous genes of 66 completely sequenced genomes of unicellular organisms using Clusters of Orthologous Groups ( COGs). Our input data consist of average protein lengths related to ∼5000 COGs from 66 genomes. We clustered these data, using an application of the information bottleneck method for unsupervised clustering. This approach is not a regular distance-based method, distinguishing it from other recently published whole-genome based clustering techniques. Although our comprehensive genome clustering is independent of phylogenies based on the level of homology of individual genes, it correlates well with the standard “tree of life” based on sequence similarity of 16s rRNA.

Genetic and Functional Diversity among Fluorescent Pseudomonads Isolated from the Rhizosphere of Banana

Fluorescent pseudomonads from banana rhizospheric soil were isolated and screened for the production of enzymes and hormones such as phosphatase, indole-3-acetic acid (IAA), 1-aminocyclopropane-1-carboxylate (ACC) deaminase, protease, and antifungal metabolites. Of 95 isolates, 50 (52%) isolates solubilized tri-calcium phosphate (TCP), 63 (66%) isolates produced plant growth hormone IAA, 10 (11%) isolates exhibited ACC deaminase, and 23 (24%) isolates produced protease. Isolates were screened for antifungal activity toward phytopathogenic fungi. Gene-specific primers have identified the putative antibiotic producing isolates. These putative isolates were grown in the production media and production of antibiotics was confirmed by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). Genotypic analysis by BOX (bacterial repetitive BOX element)-polymerase chain reaction (PCR) resulted into three distinct genomic clusters at a 50% similarity level and 62 distinct BOX profiles. Based on the sequence similarity of 16S rRNA and construction of subsequent phylogenetic tree analysis, isolates were designated as Pseudomonas monteilii, P. plecoglossicida, P. fluorescens, P. fulva, P. mosselii, P. aeruginosa, P. alcaligenes, and P. pseudoalcaligenes. Present study revealed the genetic and functional diversity among isolates of fluorescent pseudomonads associated with rhizospheric soil of banana and also identified P. monteilii as dominant species. The knowledge on genetic and functional diversity of fluorescent pseudomonads associated with banana rhizosphere is useful to understand their ecological role and for their utilization in sustainable agriculture.

Effects of temperature and biostimulation on oil‐degrading microbial communities in temperate estuarine waters

Improved strategies for oil-spill remediation will follow a better understanding of the nature, activities and regulating parameters of petroleum hydrocarbon-degrading microbial communities in temperate marine environments. The addition of crude oil to estuarine water resulted in an immediate change in bacterial community structure, increased abundance of hydrocarbon-degrading microorganisms and a rapid rate of oil degradation, suggesting the presence of a pre-adapted oil-degrading microbial community and sufficient supply of nutrients. Relatively rapid degradation was found at 4 degrees C, the lowest temperature tested; and it was temperature rather than nutrient addition that most influenced the community structure. A detailed phylogenetic analysis of oil-degrading microcosms showed that known hydrocarbonoclastic organisms like Thalassolituus and Cycloclasticus, as well as proposed oil degraders like Roseobacter, were present at both 4 degrees C and 20 degrees C, demonstrating the thermo-versatility of such organisms. Clones related to Oleispira antarctica (98% 16S rRNA similarity), a psychrophilic alkane degrader, were dominant in the 4 degrees C oil-degrading community, whereas other clones constituting a different clade and showing 94% similarity 16S rRNA with O. antarctica were found in situ. These findings demonstrate the potential for intrinsic bioremediation throughout the course of the year in temperate estuarine waters, and highlight the importance of both versatile psychrotolerant and specialized psychrophilic hydrocarbon-degrading microbes in effecting this process at low temperatures.

Sphingomonas sp. strain SB5 degrades carbofuran to a new metabolite by hydrolysis at the furanyl ring.

Microorganisms capable of degrading carbofuran were isolated from soils and examined for the degradation of this pesticide at ring structure. An isolate that could degrade carbofuran and carbofuran-7-phenol was selected for further studies. The 16S rRNA analysis results showed that the isolate belongs to the genus of Sphingomonas, close to dioxin and dicamba degraders, and is named Sphingomonas sp. SB5. SB5 did not show any similarity of 16S rRNA to known carbofuran degraders. When time-course degradation of carbofuran by SB5 was examined by solvent extraction combined with liquid chromatographic analysis, almost complete disappearance of carbofuran was observed within 12 h, giving several accumulative metabolites. Bacterial cultures incubated with carbofuran-7-phenol suggested that the accumulated metabolites were derived from carbofuran-7-phenol. The control without SB5 and kanamycin-treated SB5 did not show any metabolite, suggesting a biological involvement in the degradation of carbofuran. GC/MS and LC/MS analyses identified 2-hydroxy-3-(3-methylpropan-2-ol) phenol as one of the accumulated metabolites, suggesting that the strain SB5 could degrade carbofuran-7-phenol by hydrolysis at the furanyl ring. This is the first report to identify 2-hydroxy-3-(3-methylpropan-2-ol) phenol as a new product derived biologically from carbofuran-7-phenol.

Characteristics of Newly Isolated Nitrifying Bacteria from Rhizoplane of Paddy Rice.

An ammonia-oxidizing bacterium, strain NRS527 and nitrite-oxidizing bacterium, strain NRB 5220 were newly isolated from rhizoplane of paddy rice. The cells of strain NRS527 are spiral (0.2-0.3 μm wide, with 3-6 turns), gram negative, obligately aerobic, and chemolithotrophic. Intracytoplasmic membranes, a characteristic of ammmonia-oxidizing bacteria are absent. The optimum concentration of ammonium sulfate (substrate) in the medium is 38 mM. The G+C content of the total DNA is 59.18 mol%. The similarity of 16S rRNA (%) to Nitrosospira briensis C-128 is 94.71. The supplemental effect of organic compounds could not be recognized. The cells of strain NRB5220 are rod shaped (0.5-0.8 ×1.0-2.0 μm), gram negative, and obligately aerobic. The G+C content is 60.37 mol%. The similarity of 16S rRNA (%) to Nitrobacter agilis ATCC14123 is 97.38. The optimum concentration of sodium nitrite (substrate) in the medium is 22 mM. Growth of strain NRB5220 is hastened when peptone and yeast extract are added to the medium as nutrients. Both strains were resistant to tetracycline at 33 μg/ml. The new nitrifying bacteria isolated from rhizoplane of paddy rice were identified as Nitrosospira sp. NRS527 and Nitrobacter sp. NRB5220.

Characteristics of a high-concentration-ammonium sulfate-requiring ammonia-oxidizing bacterium isolated from deodorization plants of chicken farms

A high-concentration-ammonium sulfate-requiring, ammonia-oxidizing bacterium, strain K1, was newly isolated from packed tower biological deodorization plants of chicken farms. The cells of strain K1 are rods (0.1–1.0 × 1.0–2.0 μm), gram negative, obligately aerobic, and nonmotile. Colonies (1–2 mm in diameter) on a plate culture are reddish, circular, and smooth. Intracytoplasmic membranes characteristic of nitrifying bacteria are present. The G+C content of the total DNA is 48.5 mol%. The similarity of 16S rRNA (%) to N. europaea ATCC 25978T (type strain) is 93.77%. This bacterium has a higher optimal growth temperature (35°C) than is usually the case and tolerance up to 40°C. The optimum concentration of ammonium sulfate in the medium is 303 mM, which should make it applicable for use in deodorization plants for enhancing the efficiency of deodorization. Phosphoglycerate kinase (PGK) and triosephosphate isomerase (TIM) were found to possess high specific activities (5700 and 4 × 10 5 U/mg, respectively) compared to the activities of these enzymes in strain ATCC 25978T (300 and 14 U/mg).

Genome phylogeny based on gene content.

Species phylogenies derived from comparisons of single genes are rarely consistent with each other, due to horizontal gene transfer, unrecognized paralogy and highly variable rates of evolution. The advent of completely sequenced genomes allows the construction of a phylogeny that is less sensitive to such inconsistencies and more representative of whole-genomes than are single-gene trees. Here, we present a distance-based phylogeny constructed on the basis of gene content, rather than on sequence identity, of 13 completely sequenced genomes of unicellular species. The similarity between two species is defined as the number of genes that they have in common divided by their total number of genes. In this type of phylogenetic analysis, evolutionary distance can be interpreted in terms of evolutionary events such as the acquisition and loss of genes, whereas the underlying properties (the gene content) can be interpreted in terms of function. As such, it takes a position intermediate to phylogenies based on single genes and phylogenies based on phenotypic characteristics. Although our comprehensive genome phylogeny is independent of phylogenies based on the level of sequence identity of individual genes, it correlates with the standard reference of prokarytic phylogeny based on sequence similarity of 16s rRNA. Thus, shared gene content between genomes is quantitatively determined by phylogeny, rather than by phenotype, and horizontal gene transfer has only a limited role in determining the gene content of genomes.

Newly isolated marine ammonia-oxidizing bacterium, Nitrosomonas sp. TNO632

A new marine ammonia-oxidizing bacterium (strain TNO632) was isolated in a screening of 80 sea-water samples on gellan gum-solidified plates. The cells of strain TNO632 are rods (0.7 – 0.9 × 1.0 – 2.0 μm), gramnegative, obligately aerobic, and nonmotile. Colonies (1–2 mm in diameter) on a plate culture are reddish, circular, and smooth. Intracytoplasmic membranes characteristic of nitrifying bacteria are present. The G+C content of the total DNA is 53.3 mol%. The similarity of 16S rRNA (%) to Nitrosomonas europaea ATCC 25978% (type strain) is 94.26%. The optimum temperature for maximum growth in marine ASW-medium is 27–29°C. The optimum concentrations of ammonium sulfate, sodium chloride, and magnesium chloride for growth are 26, 100 and 100 mM, respectively. Growth of the strain is imposiible when urea is used as the sole carbon source. Hydroxylamine cytochrome c reductase (HCR) and triosephosphate isomerase (TIM) were detected and found to possess high specific activities (980 and 140 U/mg, respectively) in TNO632 compared to those in ATCC 25978T (450 and 14 U/mg).