Identification of Escherichia coli through analysis of 16S rRNA and 16S-23S rRNA internal transcribed spacer region sequences

A bacterial strain, designated BzDS03 was isolated from water sample, collected from Dal Lake Srinagar. The strain was characterized by using 16S ribosomal RNA gene and 16S-23S rRNA internal transcribed spacer region sequences. Phylogenetic analysis showed that 16S rRNA sequence of the isolate formed a monophyletic clade with genera Escherichia. The closest phylogenetic relative was Escherichia coli with 99% 16S rRNA gene sequence similarity. The result of Ribosomal database project's classifier tool revealed that the strain BzDS03 belongs to genera Escherichia.16S rRNA sequence of isolate was deposited in GenBank with accession number FJ961336. Further analysis of 16S-23S rRNA sequence of isolate confirms that the identified strain BzDS03 be assigned as the type strain of Escherichia coli with 98% 16S-23S rRNA sequence similarity. The GenBank accession number allotted for 16S-23S rRNA intergenic spacer sequence of isolate is FJ961337.     

Characterization of a Newly Isolated Highly Effective 3,5,6-Trichloro-2-pyridinol Degrading Strain Cupriavidus pauculus P2

A bacterial strain P2 capable of degrading 3,5,6-trichloro-2-pyridinol (TCP) was isolated and characterized. Phylogenetic analysis based on 16S rRNA gene sequence indicated that it belonged to the genus of Cupriavidus, because it showed the highest sequence similarity to Cupriavidus pauculus LMG 3413(T) (99.7 %) and DNA-DNA relatedness value between strain P2 and C. pauculus LMG 3413(T) was 76.8 %. In combination with morphological, physiological and biochemical characters, strain P2 was identified as C. pauculus. It could use TCP as the sole carbon source and energy source for its growth. It showed a high average degradation rate of 10 mg/L h in mineral salt medium amended with TCP (50-800 mg/L). During TCP degradation, chloridion was released into the medium in two obvious discontinuous stages. Along with this, two colorful metabolites were produced. Finally, the molarity of the total released chloridion was three times that of the initial TCP in the medium. This is the first report of TCP-degrading strain from the genus of Cupriavidus and the detection of two colorful metabolites during TCP degradation. Strain P2 might be a promising candidate for its application in the bioremediation of TCP-polluted environments.     

一株番茄青枯菌拮抗菌的鉴定及抗病效果初探

从形态、生理生化、16S rDNA3个方面确定了番茄青枯菌拮抗菌株3-1-16的分类地位。光学显微镜下观察到菌体为杆状细胞,革兰氏染色均匀,并可见菌体染成蓝紫色。透射电镜进一步观察到细胞内有许多颗粒状物质,无伴胞晶体。Biolog鉴定,3-1-16与巨大芽孢杆菌(Bacillus megaterium)具有最高相似率为98%。16S rRNA分析,3-1-16与巨大芽孢杆菌MO31同源性最高为99.4%。聚类分析显示3-1-16与3株巨大芽孢杆菌聚成一支,支持度为100%。生理生化特征及培养特征测定结果表明,菌株3-1-16鉴定为巨大芽孢杆菌(Bacillus megaterium)。盆栽试验表明该菌株对番茄青枯病防病效果达到81.3%。     

Development of PCR primers specific for the amplification and direct sequencing of gyrB genes from microbacteria, order Actinomycetales

PCR primer sets were developed for the specific amplification and sequence analyses encoding the gyrase subunit B (gyrB) of members of the family Microbacteriaceae, class Actinobacteria. The family contains species highly related by 16S rRNA gene sequence analyses. In order to test if the gene sequence analysis of gyrB is appropriate to discriminate between closely related species, we evaluate the 16S rRNA gene phylogeny of its members. As the published universal primer set for gyrB failed to amplify the responding gene of the majority of the 80 type strains of the family, three new primer sets were identified that generated fragments with a composite sequence length of about 900 nt. However, the amplification of all three fragments was successful only in 25% of the 80 type strains. In this study, the substitution frequencies in genes encoding gyrase and 16S rDNA were compared for 10 strains of nine genera. The frequency of gyrB nucleotide substitution is significantly higher than that of the 16S rDNA, and no linear correlation exists between the similarities of both molecules among members of the Microbacteriaceae. The phylogenetic analyses using the gyrB sequences provide higher resolution than using 16S rDNA sequences and seem able to discriminate between closely related species.     

Biodegradation of N-Methylpyrrolidone by Paracoccus sp. NMD-4 and its degradation pathway

N-Methylpyrrolidone (NMP), a kind of nitrogen-containing heterocyclic pollutant, is widely used in chemical industry. Microbial degradation is an important environmental fate process in soil and water, however, the microbial metabolic mechanism is still unknown. Strain NMD-4, capable of utilizing NMP as the sole source of carbon and nitrogen, was isolated from the activated sludge of a pesticide plant in Jiangsu, China, and identified as Paracoccus sp. based on its physiological–biochemical properties, as well as 16S rRNA gene sequence analysis. The degradation characteristic of NMP by strain NMD-4 was studied in a liquid culture, and the metabolic pathway of NMP by the strain was investigated. Two metabolites, 1-methyl-2,5-pyrrolidinedione and succinic acid, were detected and identified by liquid chromatography-mass spectrometry analysis, and a plausible microbial degradation pathway of NMP was proposed by the first time.     

Characterization of the First Oral <Emphasis Type="Italic">Vagococcus</Emphasis> Isolate from a Root-Filled Tooth with Periradicular Lesions

Strain Endo-EH was isolated from a root-filled tooth associated with periradicular lesions. After subculturing on Columbia blood agar, phenotypic and genomic characterizations using different biochemical test systems, automated ribotyping, MALDI-TOF mass spectronomy, antibiotic susceptibility testing, and 16S rRNA gene sequence analysis were applied for further analysis. Phenotypic characterization identified this strain as Vagococcus fluvialis. Riboprint pattern analysis and 16S rRNA sequencing clearly separated it from relevant genera such as Enterococcus and Tetragenococcus and also from other Vagococcus species. This taxon is a new entry to the list of more than 200 microbial species detected in infected root canal systems.     

Combined use of cultivation-dependent and cultivation-independent methods indicates that members of most haloarchaeal groups in an Australian crystallizer pond are cultivable

Haloarchaea are the dominant microbial flora in hypersaline waters with near-saturating salt levels. The haloarchaeal diversity of an Australian saltern crystallizer pond was examined by use of a library of PCR-amplified 16S rRNA genes and by cultivation. High viable counts (10(6) CFU/ml) were obtained on solid media. Long incubation times (> or =8 weeks) appeared to be more important than the medium composition for maximizing viable counts and diversity. Of 66 isolates examined, all belonged to the family Halobacteriaceae, including members related to species of the genera Haloferax, Halorubrum, and Natronomonas. In addition, isolates belonging to a novel group (the ADL group), previously detected only as 16S rRNA genes in an Antarctic hypersaline lake (Deep Lake), were cultivated for the first time. The 16S rRNA gene library identified the following five main groups: Halorubrum groups 1 and 2 (49%), the SHOW (square haloarchaea of Walsby) group (33%), the ADL group (16%), and the Natronomonas group (2%). There were two significant differences between the organisms detected in cultivation and 16S rRNA sequence results. Firstly, Haloferax spp. were frequently isolated on plates (15% of all isolates) but were not detected in the 16S rRNA sequences. Control experiments indicated that a bias against Haloferax sequences in the generation of the 16S rRNA gene library was unlikely, suggesting that Haloferax spp. readily form colonies, even though they were not a dominant group. Secondly, while the 16S rRNA gene library identified the SHOW group as a major component of the microbial community, no isolates of this group were obtained. This inability to culture members of the SHOW group remains an outstanding problem in studying the ecology of hypersaline environments.     

多项分类方法鉴定西藏地区藏灵菇中的乳酸球菌

【目的】采用多项分类法对16株分离自藏灵菇中的乳酸球菌进行准确鉴定。【方法】首先应用传统的生理生化试验,之后采用16S-23S rRNA间区序列多态性分析和变性梯度凝胶电泳(DGGE)进行了鉴定,最后,通过16S rRNA基因序列分析进行验证。【结果】将16株菌株初步鉴定为3个菌群:片球菌群、乳球菌群和肠球菌群,进一步鉴定为14株耐久肠球菌,1株乳酸片球菌,1株乳酸乳球菌乳酸亚种,16S rRNA基因序列分析验证的结果与前3种试验方法的结果相一致。【结论】试验结果表明传统的生理生化鉴定和16S-23S rRNA间区序列多态性分析和变性梯度凝胶电泳(DGGE)相结合的多项分类方法有利于乳酸球菌种间的准确鉴定。     

Diversity and antibiotic resistance patterns of Sphingomonadaceae isolates from drinking water

Sphingomonadaceae (n = 86) were isolated from a drinking water treatment plant (n = 6), tap water (n = 55), cup fillers for dental chairs (n = 21), and a water demineralization filter (n = 4). The bacterial isolates were identified based on analysis of the 16S rRNA gene sequence, and intraspecies variation was assessed on the basis of atpD gene sequence analysis. The isolates were identified as members of the genera Sphingomonas (n = 27), Sphingobium (n = 28), Novosphingobium (n = 12), Sphingopyxis (n = 7), and Blastomonas (n = 12). The patterns of susceptibility to five classes of antibiotics were analyzed and compared for the different sites of isolation and taxonomic groups. Colistin resistance was observed to be intrinsic (92%). The highest antibiotic resistance prevalence values were observed in members of the genera Sphingomonas and Sphingobium and for beta-lactams, ciprofloxacin, and cotrimoxazole. In tap water and in water from dental chairs, antibiotic resistance was more prevalent than in the other samples, mainly due to the predominance of isolates of the genera Sphingomonas and Sphingobium. These two genera presented distinct patterns of association with antibiotic resistance, suggesting different paths of resistance development. Antibiotic resistance patterns were often related to the species rather than to the site or strain, suggesting the importance of vertical resistance transmission in these bacteria. This is the first study demonstrating that members of the family Sphingomonadaceae are potential reservoirs of antibiotic resistance in drinking water.     

Culturable aerobic bacteria from the upstream region of a karst water rivulet

The composition of 681 aerobic and heterotrophic strains that were isolated on two different media was assessed at four sampling points along a ~300 m stretch of a karst water rivulet. Based on partial sequence analysis of 16S rRNA genes, members of 35 genera were identified; however, only a few species dominated as their representatives were repeatedly isolated at different sampling sites. Determination of the phylum affiliation showed that the isolates included members of Bacteriodetes (especially the genus Flavobacterium) and Proteobacteria (mainly Pseudomonas and Stenotrophomonas). MALDI-TOF analysis and/or similarities of partial sequences of flavobacterial strains resulted in the generation of almost complete 16S rRNA gene sequences for 100 isolates, about 60 of which may represent novel phylospecies. The number as well as the intra-phylum distribution of the isolates changed with distance from the discharge site. While phylogenetically restricted at the spring, diversity increased at downstream sampling sites.     

Genome-wide phylogenetic analysis of Gluconobacter, Acetobacter, and Gluconacetobacter

Phylogenetic relationships among three genera, Gluconobacter, Acetobacter, and Gluconacetobacter, of acetic acid bacteria (AAB) are still unclear, although phylogenetic analysis using 16S rRNA gene sequence has shown that Gluconacetobacter diverged first from the ancestor of these three genera. Therefore, the relationships among these three genera were investigated by genome-wide phylogenetic analysis of AAB. Contrary to the results of 16S rRNA gene analysis, phylogenetic analysis of 293 enzymes involved in metabolism clearly showed that Gluconobacter separated first from its common ancestor with Acetobacter and Gluconacetobacter. In addition, we defined 753 unique orthologous proteins among five known complete genomes of AAB, and phylogenetic analysis was carried out using concatenated gene sequences of these 753 proteins. The result also showed that Gluconobacter separated first from its common ancestor with Acetobacter and Gluconacetobacter. Our results strongly suggest that Gluconobacter was the first to diverge from the common ancestor of Gluconobacter, Acetobacter, and Gluconacetobacter, a relationship that is in good agreement with the physiologies and habitats of these genera.     

Phylogenetic relationships among genera of Aphidiinae (Hymenoptera: Braconidae) based on DNA sequence of the mitochondrial 16S rRNA gene

Phylogenetic relationships among forty‐nine taxa representing twenty‐four genera of Aphidiinae (Hymenoptera: Braconidae) were investigated using DNA sequence of a portion of the mitochondrial 16S rRNA gene and parsimony analysis. Seven species in six other subfamilies of Braconidae were used as outgroup. The results suggested that members of Aphidiinae are monophyletic. The basal lineage of Aphidiinae was Aclitus in weighted and unweighted parsimony analyses and Praini was basal relative to Ephedrini. With the exception of Pauesia and Aphidius, all genera were monophyletic. The results support generic status for Euaphidius, but not for Lysaphidus. Diaeretus leucopterus was internal to a clade composed of three Pauesia species, suggesting that the latter genus may be paraphyletic. A combined analysis that included DNA sequence of 16S rRNA, NADH1 dehydrogenase and 28S rRNA resulted in more robust cladograms with topologies similar to those inferred from the 16S rRNA gene sequence alone. The results are compared to previously proposed phylogenies of Aphidiinae based on morphological and molecular characters.     

Identification of Comamonas species using 16S rRNA gene sequence

A bacterial strain Bz02 was isolated from a water sample collected from river Gomti at the Indian city of Lucknow. We characterized the strain using 16S rRNA sequence. Phylogenetic analysis showed that the strain formed a monophyletic clade with members of the genus Comamonas. The closest phylogenetic relative was Comamonas testosteroni with 95% 16S rRNA gene sequence similarity. It is proposed that the identified strain Bz02 be assigned as the type strain of a species of the genus Comamonas (Comamonas sp Bz02) based on 16S rRNA gene sequence search in Ribosomal Database Project, small subunit rRNA and large subunit rRNA databases together with the phylogenetic tree analysis. The sequence is deposted in GenBank with the accession number {"type":"entrez-nucleotide","attrs":{"text":"FJ211417","term_id":"209164434"}}FJ211417.     

Isolation and Identification of a Pathogen of Silkworm Bombyx mori

A pathogenic bacterial strain, ST-1, was isolated from a naturally infected silkworm. The strain was identified on the basis of its physiological and biochemical properties and the results of sequence analysis of its 16S rRNA gene. The results of the 16S rRNA gene sequence analysis revealed that ST-1 shared the highest sequence identity (more than 99%) with Pseudomonas chlororaphis subsp. aurantiaca. ST-1 bacteria were gram-negative and 0.7-0.9 × 1.3-1.5 μm long, short rods with rounded ends. The strain could utilize sodium citrate, malonate, D-glucose, sucrose, D-fructose, D-mannose, and L-arabinose. Pathogenicity of ST-1 for silkworm could be depicted as a linear regression of the logarithm (y) of ST-1 concentration against probability (x) (y = 0.4040 + 0.0600x). The median lethal concentration (LC(50)) was 2.12 × 10(4) cfu/ml. In conclusion, ST-1 was identified as Ps. chlororaphis subsp. aurantiaca. This is the first report that Ps. aurantiaca is a pathogen for silkworm Bombyx mori.     

1株抗菌植物内生菌EJH-2菌株的分离和鉴定

目的从中药植物金银花的组织中分离到1株具有抗菌活性的植物内生菌EJH-2菌株,并对其进行了分子生物学鉴定。方法通过总DNA提取,PCR扩增,得到1300bp的16S rRNA序列。PCR产物序列通过BLAST软件在NCBI网站中进行同源性比较。通过Bioedit7．0和Treedrawing软件绘制系统发育树。结果EJH-2的16SrRNA序列和数据库中的类多粘芽胞杆菌KCTC 1663菌株的序列的同源性为99．76％。在系统发育树中,EJH-2菌株和多粘类芽胞杆菌在同一分支。结论EJH-2菌株应归属于多粘类芽胞杆菌（Paenibacillus polymyxa）。     

Combined Use of Cultivation-Dependent and Cultivation-Independent Methods Indicates that Members of Most Haloarchaeal Groups in an Australian Crystallizer Pond Are Cultivable

Haloarchaea are the dominant microbial flora in hypersaline waters with near-saturating salt levels. The haloarchaeal diversity of an Australian saltern crystallizer pond was examined by use of a library of PCR-amplified 16S rRNA genes and by cultivation. High viable counts (10⁶ CFU/ml) were obtained on solid media. Long incubation times (≥8 weeks) appeared to be more important than the medium composition for maximizing viable counts and diversity. Of 66 isolates examined, all belonged to the family Halobacteriaceae, including members related to species of the genera Haloferax, Halorubrum, and Natronomonas. In addition, isolates belonging to a novel group (the ADL group), previously detected only as 16S rRNA genes in an Antarctic hypersaline lake (Deep Lake), were cultivated for the first time. The 16S rRNA gene library identified the following five main groups: Halorubrum groups 1 and 2 (49%), the SHOW (square haloarchaea of Walsby) group (33%), the ADL group (16%), and the Natronomonas group (2%). There were two significant differences between the organisms detected in cultivation and 16S rRNA sequence results. Firstly, Haloferax spp. were frequently isolated on plates (15% of all isolates) but were not detected in the 16S rRNA sequences. Control experiments indicated that a bias against Haloferax sequences in the generation of the 16S rRNA gene library was unlikely, suggesting that Haloferax spp. readily form colonies, even though they were not a dominant group. Secondly, while the 16S rRNA gene library identified the SHOW group as a major component of the microbial community, no isolates of this group were obtained. This inability to culture members of the SHOW group remains an outstanding problem in studying the ecology of hypersaline environments.     

A broad-host-range, generalized transducing phage (SN-T) acquires 16S rRNA genes from different genera of bacteria

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 x 10(-9) transductants/PFU. Our findings further implicate transduction in the horizontal transfer of 16S rRNA genes between different species or genera of bacteria.     

产氢产乙酸菌ZR-1 的分离鉴定及产酸特性

采用改良的亨盖特厌氧操作技术, 从有机废水污泥中分离到一株耐低温高效产氢产乙酸菌ZR-1。经过对其形态学观察、生理生化特征研究及16S rRNA 序列比对, 初步鉴定为梭状芽胞杆菌属的乙二醇梭菌(Clostridium glycolicum)。通过单因子实验, 在厌氧条件下对该菌株的培养温度、pH、最适底物、金属离子的影响等产酸条件进行了优化。结果表明该菌株最适生长温度37 °C,最佳培养基初始pH 值8.5, 最适发酵底物丁酸盐, Mn²⁺对其产酸有一定的激活作用。最适培养条件下丁酸盐降解率达到12.7%, H₂ 含量达到了28.73%。     

Estimation of the abundance of an uncultured soil bacterial strain by a competitive quantitative PCR method.

Strain EA25 was identified in a clone library of bacterial 16S rRNA gene sequences that had been amplified from DNA extracted from soil collected in eastern Washington State. EA25 was subsequently shown to be related to members of the genera Planctomyces and Chlamydia and most closely related (93% similarity) to strain MC18, a strain identified in an Australian soil sample (W. Liesack and E. Stackebrandt, J. Bacteriol. 174:5072-5078, 1992). A competitive quantitative PCR method developed by Zachar et al. (V. Zachar, R.A. Thomas, and A.S. Goustin, Nucleic Acids Res. 21:2017-2018, 1993) was used to estimate the abundance of this uncultured strain in soil. An estimation of the abundance of EA25 was based on the number of copies of the sequence in the DNA extracted and the efficiency of the DNA extraction. In addition, amplification rates of Escherichia coli DNAs added to soil were shown to be similar to those of DNAs from laboratory cultures of E. coli. The number of EA25 16S rRNA genes was estimated to be 2.17 x 10(8) copies per g of soil, suggesting that strains similar to EA25 and the similar Australian strain could be widely distributed and present in significant numbers in soils from temperate regions. This represents the first enumeration of 16S rDNA copies from an uncultured strain in soil.