Culture-independent analysis of the soil bacterial assemblage at the Great Salt Plains of Oklahoma

The Great Salt Plains (GSP) of Oklahoma is a natural inland terrestrial hypersaline environment that forms evaporite crusts of mainly NaCl. Previous work described GSP bacterial assemblages through the phylogenetic and phenetic characterization of 105 isolates from 46 phylotypes. The current report describes the same bacterial assemblages through culture-independent 16S rRNA gene clone libraries. Although from similar hypersaline mud flats, the bacterial libraries from two sites, WP3 and WP6, were quite different. The WP3 library was dominated by cyanobacteria, mainly Cyanothece and Euhalothece. The WP6 library was rich in anaerobic sulfur-cycle organisms, including abundant Desulfuromonas. This pattern likely reflects differences in abiotic factors, such as frequency of flooding and hydrologic push. While more than 100 OTUs were identified, the assemblages were not as diverse, based on Shannon indexes, as bacterial communities from oligohaline soils. Since natural inland hypersaline soils are relatively unstudied, it was not clear what kind of bacteria would be present. The bacterial assemblage is predominantly genera typically found in hypersaline systems, although some were relatives of microbes common in oligohaline and marine environments. The bacterial clones did not reflect wide functional diversity, beyond phototrophs, sulfur metabolizers, and numerous heterotrophs.     

Phylogenetic-affiliation, antimicrobial potential and PKS gene sequence analysis of moderately halophilic Streptomyces sp. inhabiting an Indian saltpan

A Gram-positive, moderately halophilic Streptomyces strain, designated JAJ06, was isolated from saltpan soil collected at Tuticorin, India, and subjected to a polyphasic characterization with an insight into their biotechnological importance. Growth characteristics and antimicrobial com-pound producing capabilities of Streptomyces sp. JAJ06 were observed on various International Streptomyces Project (ISP) media and production media. Optimum growth was observed on modified ISP 4 medium supplemented with 4% NaCl (w/v) at 29 °C incubated for 7 days. Maximum antibacterial compound production with good mycelial growth was observed on starch-yeast extract-peptone medium prepared with seawater (90%, v/v). The 16S rRNA gene based phylogenetic affiliation was determined by using various bioinformatics tools and the strain was identified as Streptomyces sp. JAJ06 with 99% sequence similarity to Streptomyces radiopugnans(T) . An antimicrobial assay of antimicrobial compound derived from Streptomyces sp. JAJ06 against a set of bacteria and a yeast strain revealed antimicrobial activity with significant minimal inhibitory concentrations. The potential antimicrobial compound produced by Streptomyces sp. JAJ06 was found to be polyketide in nature. Cloning and sequence analysis of 613-bp fragment of ketosynthase gene from the type-II polyketide operon revealed that Streptomyces sp. JAJ06 has the KSα gene with 91% sequence similarity to the type II polyketide synthase gene of Streptomyces peucetius.     

Molecular diversity of peanut‐nodulating rhizobia in soils of Argentina

RSα sequencing is a valuable tool for identification of bacterial strains, and for evaluating the genetic structure of indigenous rhizobial populations. The purpose of this study was to evaluate, qualitatively, the presence or absence of RSα fragment in peanut‐nodulating strains isolated from plants grown at four sites in central Argentina. RSα fragment was found in only three of 26 indigenous strains, and in one of three inoculant strains analyzed. In contrast to results from studies of other symbiotic nitrogen‐fixing bacteria, such as soybean‐nodulating strains, no correlation was found between generation time and presence of RSα sequence. Phylogenetic analysis of the 16S rRNA gene sequence grouped peanut‐nodulating strains into two clusters, Bradyrhizobium japonicum vs. B. elkanii, and showed divergence among strains positive for RSα sequence. Our results confirm the genetic diversity previously reported for various peanut‐nodulating rhizobial strains, and indicate that the RSα fragment is not applicable as a marker or tool for competition assays at the field or ecological level. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Microbial diversity analysis of former salterns in southern Taiwan by 16S rRNA-based methods

The microbiota diversity of the former salterns in southern Taiwan was investigated by denaturing gradient gel electrophoresis (DGGE) and fluorescence in situ hybridization (FISH). Soil samples from three salterns were analyzed using DGGE and 16S rRNA from 502 colonies representing 5 archaea and 18 bacteria taxonomic groups. Each representative taxonomic group was further identified, whereas 8.7% of clones were unclassified microorganisms. Chromohalobacter, Halomonas and Virgibacillus are dominant in the Biemen saltern, Chiguensis saltern and Szutsau saltern, respectively. During FISH analysis, several taxonomic-specific probes were used. The DAPI-stained-cell count in the Szutsao saltern had a higher number of microorganisms (4.58 x 10(7) cell/cm(3)) than the other salterns. Archaea occupied 2.7-6.6% whereas bacteria accounted for 37.2-52.9% of total microbial population at the three sites. Among these three sampling sites, the Szutsao saltern had the highest diversity in halophilic microbial composition, as indicated by DGGE and FISH.     

Phylogenetic analysis of Pasteurella multocida subspecies and molecular identification of feline P. multocida subsp. septica by 16S rRNA gene sequencing

Pasteurella multocida is commonly found in the oral cavity of cats and dogs. In humans it is known as an opportunistic pathogen after bites from these animals. Phenotypic identification of P. multocida based on biochemical reactions is often limited and usually only done on a species level, even though 3 subspecies are described. For molecular taxonomy and diagnostic purposes a phylogenetic analysis of the three subspecies of P. multocida based on their 16S rRNA (rrs) gene sequence was therefore carried out. We found P. multocida subsp. septica on a distinguished branch on the phylogenetic tree of Pasteurellaceae, due to a 1.5% divergence of its rrs gene compared to the two other, more closely related subspecies multocida and gallicida. This phylogenetic divergence can be used for the identification of P. multocida subsp. septica by rrs gene determination since they form a phylogenetically well isolated and defined group as shown with a set of feline isolates. Comparison to routine phenotypic identification shows the advantage of the sequence-based identification over conventional methods. It is therefore helpful for future unambiguous identification and molecular taxonomy of P. multocida as well as for epidemiological investigations.     

Development of molecular driven screening for desulfurizing microorganisms targeting the dszB desulfinase gene

COnsensus DEgenerate Hybrid Oligonucleotide Primers (CODEHOP) were developed for the detection of the dszB desulfinase gene (2′-hydroxybiphenyl-2-sulfinate desulfinase; EC 3.13.1.3) by polymerase chain reaction (PCR), which allow to reveal larger diversity than traditional primers. The new developed primers were used as molecular monitoring tool to drive a procedure for the isolation of desulfurizing microorganisms. The primers revealed a large dszB gene diversity in environmental samples, particularly in diesel-contaminated soil that served as inoculum for enrichment cultures. The isolation procedure using the dibenzothiophene sulfone (DBTO₂) as sole sulfur source reduced drastically the dszB gene diversity. A dszB gene closely related to that carried by Gordonia species was selected. The desulfurization activity was confirmed by the production of desulfurized 2-hydroxybiphenyl (2-HBP). Metagenomic 16S rRNA gene sequencing showed that the Gordonia genus was represented at low abundance in the initial bacterial community. Such observation highlighted that the culture medium and conditions represent the bottleneck for isolating novel desulfurizing microorganisms. The new developed primers constitute useful tool for the development of appropriate cultural-dependent procedures, including medium and culture conditions, to access novel desulfurizing microorganisms useful for the petroleum industry.     

Intraspecific 16S rRNA gene diversity among clinical isolates of Neisseria species

In the present work, nearly the entire 16S rRNA gene sequences of 46 clinical samples of Neisseria spp. were determined, and the aligned sequences were analyzed to investigate the diversity of 16S rRNA genes in each commensal Neisseria species. Two 16S rRNA types were identified in two Neisseria sicca strains, three 16S rRNA types in five Neisseria macacae strains, fourteen 16S rRNA types in twenty Neisseria flavescens isolates, and fourteen 16S rRNA types in nineteen Neisseria mucosa isolates. The number of nucleotides that were different between 16S rRNA sequences within specie ranged from 1 to 15. We found high intraspecific sequence variation in 16S rRNA genes of Neisseria spp. strains.     

Culture-dependent and -independent molecular analysis of the bacterial community within uranium ore

The bacterial community structure within a uranium ore was investigated using culture-dependent and -independent clone library analysis and denaturing gradient gel electrophoresis of 16S rRNA genes. The major aerobic heterotrophic bacteria were isolated and identified, and their resistance to uranium and other heavy metals was characterized. Together with near neutral pH, moderate organic carbon content, elevated U and other heavy metals (V, Ni, Mn, Cu, etc.), the ore showed high microbial counts and phylotype richness. The bacterial community mainly consisted of uncultured Proteobacteria, with the predominance of γ - over β - and α -subdivisions, along with Actinobacteria and Firmicutes. A phylogenetic study revealed that nearly one-third of the community was affiliated to as yet uncultured and unidentified bacteria having a closer relationship to Pseudomonas. Lineages of Burkholderiaceae and Moraxellaceae were relatively more abundant in the total community, while genera affiliated to Xanthomonadaceae and Microbacteriaceae and Exiguobacterium were detected in the culturable fraction. More than 50% of the bacterial isolates affiliated to Stenotrophomonas, Microbacterium, Acinetobacter, Pseudomonas and Enterobacter showed resistance to uranium and other heavy metals. The study showed for the first time that uranium ore harbors major bacterial groups related to organisms having a wide range of environmentally significant functional attributes, and the most abundant members are possibly new groups/taxa. These findings provide new insights into U-ore geomicrobiology that could be useful in biohydrometallurgy and bioremediation applications.     

Uncultured bacterial diversity in tropical maize (Zea mays L.) rhizosphere

Structure of maize (Zea mays L.) rhizosphere bacteria was evaluated to explore the feasibility of identifying novel rhizosphere bacteria using culture-independent method based on direct amplification and analysis of 16S rRNA gene (rRNA) sequences and especially to obtain a better understanding of bacterial community structure and diversity from maize. A total of 274 sequences were analyzed and assigned 48.00% Proteobacteria, 10.30% Actinobacteria, 9.90% Bacteroidetes, 6.60% Verrucomicrobia, 4.80% Acidobacteria, 1.80% Firmicutes, 1.50% Chloroflexi, 1.50% TM7, 1.10% Deinococcus-Thermus, 0.70% Planctomycetes, 0.70% Gemmatimonadetes and 0.40% Cyanobacteria. Economically important phyla Actinobacteria was second most dominant group after Proteobacteria, in our clone library. It would be interesting to hypothesize that root exudates from maize rhizosphere favors growth of Actinobacteria like microbes to eliminate pathogenic bacteria and decompose plant matter, for enhanced plant and soil health. An additional 12.8% of clone library (35 operational taxonomical units (OTUs) from 43 clones) with less than 94% similarity to any GenBank sequence could not be assigned to any known phylum and may represent unidentified bacterial lineages and suggests that a large amount of the rhizobacterial diversity remains to be characterized by culturing.     

The interpersonal and intrapersonal diversity of human-associated microbiota in key body sites

The human body harbors 10 to 100 trillion microbes, mainly bacteria in our gut, which greatly outnumber our own human cells. This bacterial assemblage, referred to as the human microbiota, plays a fundamental role in our well-being. Deviations from healthy microbial compositions (dysbiosis) have been linked with important human diseases, including inflammation-linked disorders, such as allergies, obesity, and inflammatory bowel disease. Characterizing the temporal variations and community membership of the healthy human microbiome is critical to accurately identify the significant deviations from normality that could be associated with disease states. However, the diversity of the human microbiome varies between body sites, between patients, and over time. Environmental differences have also been shown to play a role in shaping the human microbiome in different cultures, requiring that the healthy human microbiome be characterized across life spans, ethnicities, nationalities, cultures, and geographic locales. In this article we summarize our knowledge on the microbial composition of the 5 best-characterized body sites (gut, skin, oral, airways, and vagina), focusing on interpersonal and intrapersonal variations and our current understanding of the sources of this variation.     

Temporal bacterial diversity and detection of putative methanotrophs in surface mats of Lonar crater lake

The phylogenetic diversity of bacterial communities in microbial mats of two different seasons from saline and hyperalkaline Lonar Lake was investigated using 16S rRNA gene library analysis. Arthrospira (Cyanobacteria) related clones (>80% of total clones) dominated libraries of both the seasons. Clear differences were found in both the seasons as the operational taxonomic units (OTUs) related to Fusibacter (LAI‐1 and LAI‐59) and Tindallia magadiensis (LAI‐27) found in post‐monsoon were not found in the pre‐monsoon library. Likewise, OTUs related to Planococcus rifietensis (LAII‐67), Bordetella hinzii (LAII‐2) and Methylobacterium variabile (LAII‐25) found in the pre‐monsoon were not found in post‐monsoon. The study was extended to identify methanotrophs in the surface mats. Libraries constructed with type I and type II methanotroph specific 16S rRNA gene primers showed the presence of clones (LAMI‐99 and LAMII‐2) closely related to Methylomicrobium buryaticum and Beijerinckiaceae family members. Denaturing gradient gel electrophoresis (DGGE) fingerprinting based on protein‐coding genes (pmoA and mxaF) further confirmed the detection of Methylomicrobium sp. Hence, we report here for the first time the detection of putative methanotrophs in surface mats of Lonar Lake. The finding of clones related to organisms with interesting functional attributes such as assimilation of C₁ compounds (LAII‐25, LAMI‐39, LAMI‐99 and LAMII‐2), non‐sulfur photosynthetic bacteria (LAMII‐43) and clones distantly affiliated to organisms of heavily polluted environments (LAI‐59 and LAMII‐52), is of significant note. These preliminary results would direct future studies on the functional dynamics of microbial mat associated food web chain in the extreme environment. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

HAIN基因分型试剂盒鉴定临床非结核杆菌的应用研究

目的 采用HAIN基因分型试剂盒鉴定临床非结核分枝杆菌,评价其优缺点.方法 收集浙江、安徽等地74株临床非结核分枝杆菌,采用HAIN基因分型试剂盒鉴定临床非结核分枝杆菌,并用16S rRNA基因测序方法对其进行比较和评价.结果 74株非结核分枝杆菌HAIN基因分型试剂盒鉴定结果为:31株胞内分枝杆菌,12株脓肿分枝杆菌,8株偶发分枝杆菌,6株堪萨斯分枝杆菌,5株鸟分枝杆菌,3株耻垢分枝杆菌,2株草分枝杆菌,2株瘰疬分枝杆菌,1株戈登分枝杆菌,另外有4株菌株只能鉴定为分枝杆菌属,不能鉴定到种.8株结核分枝杆菌鉴定准确.与16SrRNA基因测序相比较,HAIN基因分型试剂盒除了4株菌株只能鉴定为分枝杆菌属以外,其余70株非结核分枝杆菌均能鉴定到种,鉴定符合率为94.59％;并且它能进一步区分脓肿分枝杆菌和龟分枝杆菌,以及堪萨斯分枝杆菌和胃分枝杆菌.如果单纯鉴定HAIN基因分型试剂盒菌株范围之内的临床最常见非结核分枝杆菌菌株,鉴定符合率为100％.结论 HAIN基因分型试剂盒鉴定临床非结核分枝杆菌时间短、操作简单、结果准确,可在临床推广使用.     

Microbial diversity of mine water at Zhong Tiaoshan copper mine, China

Microbial diversity of mine water at Zhong Tiaoshan copper mine, Shanxi province, China, was analyzed using a culture-independent 16S rRNA gene (rDNA) based on cloning approach. A total of 59 Operational Taxonomic Units (OTUs) were obtained from 226 clones from all three samples (8 OTUs from sample SX1, 25 from SX2 and 26 from SX3). 46 of them were representative OTUs and were sequenced. 93.5% of the total clones had sequences that were less than 5% difference from those in the nucleic acids database. The percentage of overlapping OTUs among samples was from 12.1% to 35.3%. Phylogenetic analysis indicated that 60.62% of the clones were affiliated with members of the Proteobacteria (alpha -3.10%, beta -24.78%, gamma -31.41%, delta -1.33%), whereas 29.20% of the clones were closely related to the Nitrospira (Leptospirillum ferrooxidans 20.80%, Leptospirillum ferriphilum 0.88% and Leptospirillum group III 7.52%, respectively). The rest clones were affiliated with the Firmicutes (2.65%) and the Bacteroidetes (7.52%). The results of Principal Component Analysis (PCA) based on the percentages of OTUs and biogeochemical data revealed that biogeochemical properties affected the diversity of microbial communities in mine water. Especially, the pH value, temperature and different concentrations of elements such as lead, zinc, sulfur, iron and copper seemed to be key factors affecting the composition and structure of microbial communities in this study.     

Comparative evaluation of prokaryotic 16S rDNA clone libraries and SSCP in groundwater samples

A comparison of ribosomal RNA sequence analysis methods based on clone libraries and single-strand conformational polymorphism technique (SSCP) was performed with groundwater samples obtained between 523-555 meters below surface. The coverage of analyzed clones by phylotype-richness estimates was between 88-100%, confirming that the clone libraries were adequately examined. Analysis of individual bands retrieved from SSCP gels identified 1-6 different taxonomic units per band, suggesting that a single SSCP band does often represent more than one single prokaryotic species. The prokaryotic diversity obtained by both methods showed an overall difference of 42-80%. In comparison to SSCP, clone libraries underestimated the phylogenetic diversity and only 36-66% of the phylotypes observed with SSCP were also detected with the clone libraries. An exception was a sample where the SSCP analysis of Archaea identified only half of the phylotypes retrieved by the clone library. Overall, this study suggests that the clone library and the SSCP approach do not provide an identical picture of the prokaryotic diversity in groundwater samples. The results clearly show that the SSCP method, although this approach is prone to generate methodological artifacts, was able to detect significantly more phylotypes than microbial community analysis based on clone libraries.     

Isolation and identification of a Candida digboiensis strain from an extreme acid mine drainage of the Lignite Mine,Gujarat

An extremely acidic mine drainage (AMD) water sample was collected in 1998 and 2008 from Panandhro lignite mine, Gujarat, India. The yeast isolated from this sample was identified using mini API identification system, as a member of genus Candida. The major cellular fatty acids detected by FAME from the isolate are C_16:0 and C_{18:2 cis 9,12}/C_18:0α as 25.23 and 19.5%, respectively. The isolate was identified as Candida digboiensis by 18S rRNA gene sequence analysis and designated as Candida digboiensis SRDyeast1. Phylogenetic analysis using D1/D2 variable domains showed that the closest relative of this strain is Candida blankii with 3% divergence. This organism has been reported for the first time from the lignite mine AMD sample, and for cellular fatty acid analysis. This yeast is able to survive in the AMD sample preserved at 10–42 °C temperature since last 10 years along with iron oxidizing microorganisms. It can grow in the presence of 40% glucose, 10% NaCl and in the pH range of 1 to 10. The isolate is capable of producing enzymes like protease and lipase. This isolate differs from the type strain Candida digboiensis in as many as six physiological and metabolic characteristics. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)