scholarly journals Identification and Analysis of Informative Single Nucleotide Polymorphisms in 16S rRNA Gene Sequences of the Bacillus cereus Group

2016 ◽  
Vol 54 (11) ◽  
pp. 2749-2756 ◽  
Author(s):  
Janetta R. Hakovirta ◽  
Samantha Prezioso ◽  
David Hodge ◽  
Segaran P. Pillai ◽  
Linda M. Weigel

Analysis of 16S rRNA genes is important for phylogenetic classification of known and novel bacterial genera and species and for detection of uncultivable bacteria. PCR amplification of 16S rRNA genes with universal primers produces a mixture of amplicons from all rRNA operons in the genome, and the sequence data generally yield a consensus sequence. Here we describe valuable data that are missing from consensus sequences, variable effects on sequence data generated from nonidentical 16S rRNA amplicons, and the appearance of data displayed by different software programs. These effects are illustrated by analysis of 16S rRNA genes from 50 strains of theBacillus cereusgroup, i.e.,Bacillus anthracis,Bacillus cereus,Bacillus mycoides, andBacillus thuringiensis. These species have 11 to 14 rRNA operons, and sequence variability occurs among the multiple 16S rRNA genes. A single nucleotide polymorphism (SNP) previously reported to be specific toB. anthraciswas detected in someB. cereusstrains. However, a different SNP, at position 1139, was identified as being specific toB. anthracis, which is a biothreat agent with high mortality rates. Compared with visual analysis of the electropherograms, basecaller software frequently missed gene sequence variations or could not identify variant bases due to overlapping basecalls. Accurate detection of 16S rRNA gene sequences that include intragenomic variations can improve discrimination among closely related species, improve the utility of 16S rRNA databases, and facilitate rapid bacterial identification by targeted DNA sequence analysis or by whole-genome sequencing performed by clinical or reference laboratories.

2005 ◽  
Vol 71 (10) ◽  
pp. 6308-6318 ◽  
Author(s):  
Helen A. Vrionis ◽  
Robert T. Anderson ◽  
Irene Ortiz-Bernad ◽  
Kathleen R. O'Neill ◽  
Charles T. Resch ◽  
...  

ABSTRACT The geochemistry and microbiology of a uranium-contaminated subsurface environment that had undergone two seasons of acetate addition to stimulate microbial U(VI) reduction was examined. There were distinct horizontal and vertical geochemical gradients that could be attributed in large part to the manner in which acetate was distributed in the aquifer, with more reduction of Fe(III) and sulfate occurring at greater depths and closer to the point of acetate injection. Clone libraries of 16S rRNA genes derived from sediments and groundwater indicated an enrichment of sulfate-reducing bacteria in the order Desulfobacterales in sediment and groundwater samples. These samples were collected nearest the injection gallery where microbially reducible Fe(III) oxides were highly depleted, groundwater sulfate concentrations were low, and increases in acid volatile sulfide were observed in the sediment. Further down-gradient, metal-reducing conditions were present as indicated by intermediate Fe(II)/Fe(total) ratios, lower acid volatile sulfide values, and increased abundance of 16S rRNA gene sequences belonging to the dissimilatory Fe(III)- and U(VI)-reducing family Geobacteraceae. Maximal Fe(III) and U(VI) reduction correlated with maximal recovery of Geobacteraceae 16S rRNA gene sequences in both groundwater and sediment; however, the sites at which these maxima occurred were spatially separated within the aquifer. The substantial microbial and geochemical heterogeneity at this site demonstrates that attempts should be made to deliver acetate in a more uniform manner and that closely spaced sampling intervals, horizontally and vertically, in both sediment and groundwater are necessary in order to obtain a more in-depth understanding of microbial processes and the relative contribution of attached and planktonic populations to in situ uranium bioremediation.


1999 ◽  
Vol 65 (11) ◽  
pp. 5042-5049 ◽  
Author(s):  
Kuk-Jeong Chin ◽  
Dittmar Hahn ◽  
Ulf Hengstmann ◽  
Werner Liesack ◽  
Peter H. Janssen

ABSTRACT Most-probable-number (liquid serial dilution culture) counts were obtained for polysaccharolytic and saccharolytic fermenting bacteria in the anoxic bulk soil of flooded microcosms containing rice plants. The highest viable counts (up to 2.5 × 108 cells per g [dry weight] of soil) were obtained by using xylan, pectin, or a mixture of seven mono- and disaccharides as the growth substrate. The total cell count for the soil, as determined by using 4′,6-diamidino-2-phenylindole staining, was 4.8 × 108cells per g (dry weight) of soil. The nine strains isolated from the terminal positive tubes in counting experiments which yielded culturable populations that were equivalent to about 5% or more of the total microscopic count population belonged to the divisionVerrucomicrobia, theCytophaga-Flavobacterium-Bacteroides division, clostridial cluster XIVa, clostridial cluster IX, Bacillus spp., and the class Actinobacteria. Isolates originating from the terminal positive tubes of liquid dilution series can be expected to be representatives of species whose populations in the soil are large. None of the isolates had 16S rRNA gene sequences identical to 16S rRNA gene sequences of previously described species for which data are available. Eight of the nine strains isolated fermented sugars to acetate and propionate (and some also fermented sugars to succinate). The closest relatives of these strains (except for the two strains of actinobacteria) were as-yet-uncultivated bacteria detected in the same soil sample by cloning PCR-amplified 16S rRNA genes (U. Hengstmann, K.-J. Chin, P. H. Janssen, and W. Liesack, Appl. Environ. Microbiol. 65:5050–5058, 1999). Twelve other isolates, which originated from most-probable-number counting series indicating that the culturable populations were smaller, were less closely related to cloned 16S rRNA genes.


2021 ◽  
Author(s):  
Antti Juhani Rissanen ◽  
Moritz Buck ◽  
Sari Peura

A putative novel methanotrophic genus, Candidatus Methylumidiphilus (Methylococcales), was recently shown to be ubiquitous and one of the most abundant methanotrophic genera in water columns of oxygen-stratified lakes and ponds of boreal and subarctic area. However, it has probably escaped detection in many previous studies using 16S rRNA gene amplicon sequencing due to insufficient database coverage, which is because Ca. Methylumidiphilus lacks cultured representatives and previously analysed metagenome assembled genomes (MAGs) affiliated with it do not contain 16S rRNA genes. Therefore, we screened MAGs affiliated with the genus for their 16S rRNA gene sequences in a recently published lake and pond MAG dataset. Among 66 MAGs classified as Ca. Methylumidiphilus (with completeness over 40% and contamination less than 5%) originating from lakes in Finland, Sweden and Switzerland as well as from ponds in Canada, we could find 5 MAGs each containing one 1532 bp long sequence spanning the V1-V9 regions of the 16S rRNA gene. After removal of sequence redundancy, this resulted in two unique 16S rRNA gene sequences. These sequences represented two different putative species, i.e. Ca. Methylumidiphilus alinenensis (Genbank accession: OK236221) as well as another so far unnamed species of Ca. Methylumidiphilus (Genbank accession: OK236220). We suggest that including these two sequences in reference databases will enhance 16S rRNA gene - based detection of members of this genus from environmental samples.


2012 ◽  
Vol 78 (9) ◽  
pp. 3416-3423 ◽  
Author(s):  
Koji Mori ◽  
Takao Iino ◽  
Ken-Ichiro Suzuki ◽  
Kaoru Yamaguchi ◽  
Yoichi Kamagata

ABSTRACTAmong methanogens, only 2 genera,MethanosaetaandMethanosarcina, are known to contribute to methanogenesis from acetate, andMethanosaetais a specialist that uses acetate specifically. However,Methanosaetastrains so far have mainly been isolated from anaerobic digesters, despite the fact that it is widespread, not only in anaerobic methanogenic reactors and freshwater environments, but also in marine environments, based upon extensive 16S rRNA gene-cloning analyses. In this study, we isolated an aceticlastic methanogen, designated strain 03d30qT, from a tidal flat sediment. Phylogenetic analyses based on 16S rRNA andmcrAgenes revealed that the isolate belongs to the genusMethanosaeta. Unlike the other knownMethanosaetaspecies, this isolate grows at Na+concentrations of 0.20 to 0.80 M, with an optimum concentration of 0.28 M. Quantitative estimation using real-time PCR detected the 16S rRNA gene of the genusMethanosaetain the marine sediment, and relative abundance ranged from 3.9% to 11.8% of the total archaeal 16S rRNA genes. In addition, the number ofMethanosaetaorganisms increased with increasing depth and was much higher than that ofMethanosarcinaorganisms, suggesting that aceticlastic methanogens contribute to acetate metabolism to a greater extent than previously thought in marine environments, where sulfate-reducing acetate oxidation prevails. This is the first report on marineMethanosaetaspecies, and based on phylogenetic and characteristic studies, the name “Methanosaeta pelagica” sp. nov. is proposed for this novel species, with type strain 03d30q.


2013 ◽  
Vol 63 (Pt_5) ◽  
pp. 1589-1596 ◽  
Author(s):  
Anahit Penesyan ◽  
Sven Breider ◽  
Peter Schumann ◽  
Brian J. Tindall ◽  
Suhelen Egan ◽  
...  

Two Gram-reaction-negative, rod-shaped, motile bacteria, designated strains U82 and U95T, were isolated from the marine alga Ulva australis collected at Sharks Point, Clovelly, a rocky intertidal zone near Sydney, Australia. Both strains were oxidase- and catalase-positive, formed brown- to black-pigmented colonies and required NaCl for growth. Phylogenetic analysis based on nearly complete 16S rRNA gene sequences revealed that these strains belong to the Roseobacter clade within the Alphaproteobacteria . The 16S rRNA genes of both strains were identical across the sequenced 1326 nt, but showed differences in the intergenic spacer region (ITS) between the 16S and the 23S rRNA genes. At the genomic level the DNA G+C contents of strains U82 and U95T were identical (52.6 mol%) and they had a DNA–DNA hybridization value of 83.7 %, suggesting that these strains belong to the same species. The closest described phylogenetic neighbour to strains U82 and U95T was Thalassobius aestuarii DSM 15283T with 95.8 % 16S rRNA gene sequence similarity. Other close relatives include further species of the genera Thalassobius and Shimia . Strains U82 and U95T were negative for bacteriochlorophyll a production, showed antibacterial activity towards other marine bacteria, were resistant to the antibiotics gentamicin and spectinomycin and were unable to hydrolyse starch or gelatin. The major fatty acids (>1 %) were 18 : 1ω7c, 16 : 0, 18 : 2, 10 : 0 3-OH, 12 : 0, 20 : 1 2-OH and 18 : 0. The polar lipid pattern indicated the presence of phosphatidylglycerol, phosphatidylcholine, two unidentified aminolipids and four unidentified phospholipids. Both strains produced ubiquinone 10 (Q-10) as the sole respiratory lipoquinone. Based on their phenotypic and phylogenetic characteristics, it is suggested that strains U82 and U95T are members of a novel species within a new genus for which the name Epibacterium ulvae gen. nov., sp. nov. is proposed. The type strain of the type species is U95T ( = DSM 24752T = LMG 26464T).


2014 ◽  
Vol 81 (2) ◽  
pp. 604-613 ◽  
Author(s):  
David Wilkins ◽  
Xiao-Ying Lu ◽  
Zhiyong Shen ◽  
Jiapeng Chen ◽  
Patrick K. H. Lee

ABSTRACTMethanogenic archaea play a key role in biogas-producing anaerobic digestion and yet remain poorly taxonomically characterized. This is in part due to the limitations of low-throughput Sanger sequencing of a single (16S rRNA) gene, which in the past may have undersampled methanogen diversity. In this study, archaeal communities from three sludge digesters in Hong Kong and one wastewater digester in China were examined using high-throughput pyrosequencing of the methyl coenzyme M reductase (mcrA) and 16S rRNA genes.Methanobacteriales,Methanomicrobiales, andMethanosarcinaleswere detected in each digester, indicating that both hydrogenotrophic and acetoclastic methanogenesis was occurring. Two sludge digesters had similar community structures, likely due to their similar design and feedstock. Taxonomic classification of themcrAgenes suggested that these digesters were dominated by acetoclastic methanogens, particularlyMethanosarcinales, while the other digesters were dominated by hydrogenotrophicMethanomicrobiales. The proposed euryarchaeotal orderMethanomassiliicoccalesand the uncultured WSA2 group were detected with the 16S rRNA gene, and potentialmcrAgenes for these groups were identified. 16S rRNA gene sequencing also recovered several crenarchaeotal groups potentially involved in the initial anaerobic digestion processes. Overall, the two genes produced different taxonomic profiles for the digesters, while greater methanogen richness was detected using themcrAgene, supporting the use of this functional gene as a complement to the 16S rRNA gene to better assess methanogen diversity. A significant positive correlation was detected between methane production and the abundance ofmcrAtranscripts in digesters treating sludge and wastewater samples, supporting themcrAgene as a biomarker for methane yield.


2015 ◽  
Vol 65 (Pt_1) ◽  
pp. 90-94 ◽  
Author(s):  
P. García-Fraile ◽  
M. Chudíčková ◽  
O. Benada ◽  
J. Pikula ◽  
M. Kolařík

During the study of bacteria associated with bats affected by white-nose syndrome hibernating in caves in the Czech Republic, we isolated two facultatively anaerobic, Gram-stain-negative bacteria, designated strains 12T and 52T. Strains 12T and 52T were motile, rod-like bacteria (0.5–0.6 µm in diameter; 1–1.3 µm long), with optimal growth at 20–35 °C and pH 6–8. On the basis of the almost complete sequence of their 16S rRNA genes they should be classified within the genus Serratia ; the closest relatives to strains 12T and 52T were Serratia quinivorans DSM 4597T (99.5 % similarity in 16S rRNA gene sequences) and Serratia ficaria DSM 4569T (99.5 % similarity in 16S rRNA gene sequences), respectively. DNA–DNA relatedness between strain 12T and S. quinivorans DSM 4597T was only 37.1 % and between strain 52T and S. ficaria DSM 4569T was only 56.2 %. Both values are far below the 70 % threshold value for species delineation. In view of these data, we propose the inclusion of the two isolates in the genus Serratia as representatives of Serratia myotis sp. nov. (type strain 12T = CECT 8594T = DSM 28726T) and Serratia vespertilionis sp. nov. (type strain 52T = CECT 8595T = DSM 28727T).


2011 ◽  
Vol 77 (21) ◽  
pp. 7749-7756 ◽  
Author(s):  
Jacoline Gerritsen ◽  
Harro M. Timmerman ◽  
Susana Fuentes ◽  
L. Paul van Minnen ◽  
Henk Panneman ◽  
...  

ABSTRACTProphylactic probiotic therapy has shown beneficial effects in an experimental rat model for acute pancreatitis on the health status of the animals. Mechanisms by which probiotic therapy interferes with severity of acute pancreatitis and associated sepsis, however, are poorly understood. The aims of this study were to identify the probiotic-induced changes in the gut microbiota and to correlate these changes to disease outcome. Duodenum and ileum samples were obtained from healthy and diseased rats subjected to pancreatitis for 7 days and prophylactically treated with either a multispecies probiotic mixture or a placebo. Intestinal microbiota was characterized by terminal-restriction fragment length polymorphism (T-RFLP) analyses of PCR-amplified 16S rRNA gene fragments. These analyses showed that during acute pancreatitis the host-specific ileal microbiota was replaced by an “acute pancreatitis-associated microbiota.” This replacement was not reversed by administration of the probiotic mixture. An increase, however, was observed in the relative abundance of a novel bacterial phylotype most closely related toClostridium lituseburenseand referred to as commensal rat ileum bacterium (CRIB). Specific primers targeting the CRIB 16S rRNA gene sequence were developed to detect this phylotype by quantitative PCR. An ileal abundance of CRIB 16S rRNA genes of more than 7.5% of the total bacterial 16S rRNA gene pool was correlated with reduced duodenal bacterial overgrowth, reduced bacterial translocation to remote organs, improved pancreas pathology, and reduced proinflammatory cytokine levels in plasma. Our current findings and future studies involving this uncharacterized bacterial phylotype will contribute to unraveling one of the potential mechanisms of probiotic therapy.


2005 ◽  
Vol 71 (12) ◽  
pp. 8301-8304 ◽  
Author(s):  
Amy Beumer ◽  
Jayne B. Robinson

ABSTRACT Genomic analysis has revealed heterogeneity among bacterial 16S rRNA gene sequences within a single species; yet the cause(s) remains uncertain. Generalized transducing bacteriophages have recently gained recognition for their abundance as well as their ability to affect lateral gene transfer and to harbor bacterial 16S rRNA gene sequences. Here, we demonstrate the ability of broad-host-range, generalized transducing phages to acquire 16S rRNA genes and gene sequences. Using PCR and primers specific to conserved regions of the 16S rRNA gene, we have found that generalized transducing phages (D3112, UT1, and SN-T), but not specialized transducing phages (D3), acquired entire bacterial 16S rRNA genes. Furthermore, we show that the broad-host-range, generalized transducing phage SN-T is capable of acquiring the 16S rRNA gene from two different genera: Sphaerotilus natans, the host from which SN-T was originally isolated, and Pseudomonas aeruginosa. In sequential infections, SN-T harbored only 16S rRNA gene sequences of the final host as determined by restriction fragment length polymorphism analysis. The frequency of 16S rRNA gene sequences in SN-T populations was determined to be 1 × 10−9 transductants/PFU. Our findings further implicate transduction in the horizontal transfer of 16S rRNA genes between different species or genera of bacteria.


2020 ◽  
Vol 70 (5) ◽  
pp. 3483-3490 ◽  
Author(s):  
Jianyang Li ◽  
Mingming Qi ◽  
Qiliang Lai ◽  
Chunming Dong ◽  
Xiupian Liu ◽  
...  

Two Gram-stain-negative, short rod-shaped and non-flagellated strains, designated 17-4AT and L52-1-41, were isolated from the surface seawater of the Indian Ocean and South China Sea, respectively. The 16S rRNA genes of the two strains shared sequence similarity of 99.45 %. Strain 17-4AT shared the highest 16S rRNA gene similarity of 98.02 % with Pusillimonas caeni EBR-8-1T, followed by Pusillimonas noertemannii BN9T (97.47 %), Pusillimonas soli MJ07T (96.93 %), Parapusillimonas granuli Ch07T (96.68 %), Pusillimonas ginsengisoli DCY25T (96.65 %), Eoetvoesia caeni PB3-7BT (96.63 %), Paracandidimonas caeni 24T (96.34 %), Castellaniella defragrans 54PinT (96.28 %) and Pusillimonas harenae B201T (96.05 %). L52-1-41 shared the highest 16S rRNA gene similarity of 97.74 % with Pusillimonas caeni EBR-8-1T, followed by Pusillimonas noertemannii BN9T (97.47 %), Pusillimonas soli MJ07T (96.65 %), Parapusillimonas granuli Ch07T (96.41 %), Pusillimonas ginsengisoli DCY25T (96.37 %), Eoetvoesia caeni PB3-7BT (96.35 %), Pusillimonas harenae B201T (96.28 %), and Paracandidimonas caeni 24T (96.06 %). The results of phylogenetic analyses indicated that 17-4AT and L52-1-41 formed a stable, distinct and highly supported lineage affiliated to the genus Pusillimonas . The results of the digital DNA–DNA hybridization (dDDH) and average nucleotide identity (ANI) analyses indicated that they represented a single species. They featured similar genomic DNA G+C contents of 53.2–53.4 mol%. Activities of catalase and oxidase were negative for both strains. The fatty acids patterns of 17-4AT and L52-1-41 were most similar, mostly comprised of C16 : 0, C17 : 0cyclo, C18 : 0, C18 : 1ω9c and summed feature 8 (C18 : 1ω7c and/or C18 : 1  ω6c). The major polar lipids of the two strains were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and unidentified aminolipids. The respiratory quinone of the two strains was Q-8. Hence, on the basis of the phenotypic, chemotaxonomic and genotypic data presented in this study, we proposed the classification of both strains as representatives of a novel species named Pusillimonas maritima sp. nov., with the type strain 17-4AT (=MCCC 1A12670T=KCTC 62121T=NBRC 113794T), and another strain L52-1-41 (=MCCC 1A05046=KCTC 52313).


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