Unique Restriction Fragment Length Polymorphism Research Articles

Polymorphisms in the hypervariable control region of the mitochondrial DNA differentiate BPH populations.

The brown planthopper (BPH; Nilaparvata lugens) is one of India's most destructive pests of rice. BPH, a monophagous migratory insect, reported from all major rice-growing ecosystems of the country, is capable of traversing large distances and causing massive crop loss. A crucial step for developing viable management strategies is understanding its population dynamics. Very few reliable markers are currently available to screen BPH populations for their diversity. In the current investigation, we developed a combinatorial approach using the polymorphism present within the mitochondrial Control Region of BPH and in the nuclear genome (genomic simple sequence repeats; gSSRs) to unravel the diversity present in BPH populations collected from various rice-growing regions of India. Using two specific primer pairs, the complete Control Region (1112 to 2612 bp) was PCR amplified as two overlapping fragments, cloned and sequenced from BPH individuals representing nine different populations. Results revealed extensive polymorphism within this region due to a variable number of tandem repeats. The three selected gSSR markers also exhibited population-specific amplification patterns. Overall genetic diversity between the nine populations was high (>5%). Further, in silico double-digestion of the consensus sequences of the Control Region, with HpyCH4IV and Tsp45I restriction enzymes, revealed unique restriction fragment length polymorphisms (digital-RFLPs; dRFLPs) that differentiated all the nine BPH populations. To the best of our knowledge, this is the first report of markers developed from the Control Region of the BPH mitogenome that can differentiate populations. Eventually, such reliable and rapid marker-based identification of BPH populations will pave the way for an efficient pest management strategy.

Plant hosts may influence arbuscular mycorrhizal fungal community composition in mangrove estuaries

We investigated the role of plant host and soil variables in determining arbuscular mycorrhizal fungi (AMF) community composition in plant roots of two spatially separated mangrove estuaries on the rivers Aghanashini (14° 30' 30″ N-74° 22' 44″ E) and Gangavali (14° 35' 26″ N-74° 17' 51″ E) on the west coast of India. Both mangrove estuaries had similar plant species composition but differed in soil chemistries.We amplified a 550-bp portion of 18S small subunit (SSU) rDNA from mangrove plant roots and analysed it by restriction fragment length polymorphism (RFLP). Clones representing unique RFLP patterns were sequenced. A total of 736 clones were obtained from roots of seven and five plant species sampled at Aghanashini and Gangavali, respectively. AMF phylotype numbers in plant roots at Aghanashini (12) were higher than at Gangavali (9) indicating quantitative differences in the AMF community composition in plant roots at the two mangrove estuaries. Because both estuaries had similar plant species composition, the quantitative difference in AMF communities between the estuaries could be an attribute of the differences in rhizospheric chemistry between the two sites.Non-metric multidimensional scaling (NMDS) revealed overlap in the AMF communities of the two sites. Three and two AMF phylotypes had significant indicator value indices with specific hosts at Aghanashini and Gangavali, respectively. Environmental vector fitting to NMDS ordination did not reveal a significant effect of any soil variable on AMF composition at the two sites. However, significant effects of both plant hosts and sites were observed on rhizospheric P. Our results indicate that root AMF community composition may be an outcome of plant response to rhizospheric variables. This suggests that plant identity may have a primary role in shaping AMF communities in mangroves.

Multilocus Genotyping Reveals New Molecular Markers for Differentiating Distinct Genetic Lineages among “Candidatus Phytoplasma Solani” Strains Associated with Grapevine Bois Noir

Grapevine Bois noir (BN) is associated with infection by “Candidatus Phytoplasma solani” (CaPsol). In this study, an array of CaPsol strains was identified from 142 symptomatic grapevines in vineyards of northern, central, and southern Italy and North Macedonia. Molecular typing of the CaPsol strains was carried out by analysis of genes encoding 16S rRNA and translation elongation factor EF-Tu, as well as eight other previously uncharacterized genomic fragments. Strains of tuf-type a and b were found to be differentially distributed in the examined geographic regions in correlation with the prevalence of nettle and bindweed. Two sequence variants were identified in each of the four genomic segments harboring hlyC, cbiQ-glyA, trxA-truB-rsuA, and rplS-tyrS-csdB, respectively. Fifteen CaPsol lineages were identified based on distinct combinations of sequence variations within these genetic loci. Each CaPsol lineage exhibited a unique collective restriction fragment length polymorphism (RFLP) pattern and differed from each other in geographic distribution, probably in relation to the diverse ecological complexity of vineyards and their surroundings. This RFLP-based typing method could be a useful tool for investigating the ecology of CaPsol and the epidemiology of its associated diseases. Phylogenetic analyses highlighted that the sequence variants of the gene hlyC, which encodes a hemolysin III-like protein, separated into two clusters consistent with the separation of two distinct lineages on the basis of tufB gene sequences. Alignments of deduced full protein sequences of elongation factor-Tu (tufB gene) and hemolysin III-like protein (hlyC gene) revealed the presence of critical amino acid substitutions distinguishing CaPsol strains of tuf-type a and b. Findings from the present study provide new insights into the genetic diversity and ecology of CaPsol populations in vineyards.

Novel phytoplasma strains of X‐disease group unveil genetic markers that distinguish North American and South American geographic lineages within subgroups 16SrIII‐J and 16SrIII‐U

AbstractPhytoplasmas in the X‐disease group (16SrIII) are highly diverse in terms of geographic distributions, vectorship and plant host specificity. Such biological and ecological diversity is often correlated with distinctive genetic markers present in evolutionarily conserved genes. Based on restriction fragment length polymorphism (RFLP) markers in the 16S rRNA gene sequences, 29 subgroups have been delineated, with most of them being found in the Americas. However, it has been unknown whether distinct geographic lineages are present within a given subgroup. Prior to this study, phytoplasmas belonging to subgroups 16SrIII‐J and 16SrIII‐U were reported only in countries located in South America. In the present study, we identified new phytoplasmas strains closely related to the reference strains of the two subgroups in Mexico, a North American country. These newly identified Mexican strains possess unique RFLP, single nucleotide polymorphism, and fragmental deletion markers in 16S rRNA‐ and/or ribosomal protein‐encoding genes. Since these markers consistently distinguished the Mexican strains from their South American counterparts, they may represent emerging or previously unknown North American geographic lineages of the subgroups 16SrIII‐J and 16SrIII‐U.

New subgroup 16SrXI-F phytoplasma strain associated with sugarcane grassy shoot (SCGS) disease in India.

A novel phytoplasma subgroup strain SC-Phy385 (GenBank accession number HF586648) was found associated with Sugarcane Grassy Shoot (SCGS) disease in India. Sugarcane (Saccharum officinarum L.) leaf samples with 'grassy shoot' symptoms were collected from the western region of Maharashtra state. The analysis based on restriction fragment length polymorphism (RFLP) profiles and 16S rRNA gene sequences indicated that most of the phytoplasma strains belong to the 16SrXI-B group. However, SCGS phytoplasma strain SC-Phy385 showed unique RFLP profiles with a similarity coefficient of 0.87 using online interactive phytoplasma classification tool, iPhyClassifier. The actual and virtual RFLP profiles of SC-Phy385 using amplified products of the 16S rRNA partial gene and its multiple sequence alignment with other RYD group phytoplasma sequences revealed that this strain was different from phytoplasma groups described earlier, representing new subgroup 16SrXI-F. The 16S rRNA gene-based phylogenetic analysis of reported RYD and BGWL group phytoplasmas showed that they are a genetically diverse group of phytoplasmas.

Characteristics of ciprofloxacin resistant Campylobacter spp. isolated from chicken meat in Turkey

The objective of the current study was to investigate mutations in the gyrA and 23S rRNA gene fragments the high level ciprofloxacin resistant (HL-CipR) Campylobacter isolates (C. jejuni n = 6 and and C. coli n = 9) obtained from chicken meat samples, in Turkey. PCR-based restriction fragment length polymorphism (RFLP) was used to characterize these isolates. In addition, the presence of several virulence traits among these isolates was also examined. Of the 15 HL-CipR Campylobacter strains, there were eight unique RFLP banding patterns. All HL-CipR Campylobacter strains had mutations in codon 86 (Thr-86 to Ile) in the gyrA gene. Four C. jejuni isolates had missense mutation of Asp-203 to Ser, whereas one C. jejuni strain also presented a change at Ala-40 to Ser. A2075G substitution in the 23S rRNA gene was identified in five isolates (C. coli n = 3 and C. jejuni n = 2), whereas none of the isolates had A2074G substitution. The tetO gene conferring resistance to tetracycline were observed among five Campylobacter isolates. Of Campylobacter strains, 13 (86.6 %) were found to be positive for one or more virulence factors, the cdt genes being the most detected. The results of the current study extends the current knowledge about molecular mechanisms for erythromycin and ciprofloxacin resistance as well as virulence traits by investigation of HL-CipR Campylobacter isolates from chicken meat sold in Turkey.

IS6110 Restriction Fragment Length Polymorphism Typing of Drug-resistant Mycobacterium tuberculosis Strains from Northeast South Africa

Tuberculosis (TB) remains a deadly infectious disease affecting millions of people worldwide; 95% of TB cases, with 98% of death occur in developing countries. The situation in South Africa merits special attention. A total of 21,913 sputum specimens of suspected TB patients from three provinces of South Africa routinely submitted to the TB laboratory of Dr. George Mukhari (DGM) Hospital were assayed for Mycobacterium tuberculosis (MTB) growth and antibiotic susceptibility. The genetic diversity of 338 resistant strains were also studied. DNA isolated from the strains were restricted with Pvu II, transferred on to a nylon membrane and hybridized with a PCR-amplified horseradish peroxidase 245 bp IS6110 probe. Of the 338 resistant strains, 2.09% had less than 5 bands of IS6110, and 98% had 5 or more bands. Unique restriction fragment length polymorphism (RFLP) patterns were observed in 84.3% of the strains, showing their epidemiological independence, and 15.7% were grouped into 22 clusters. Thirty-two strains (61.5%) from the 52 that clustered were from Mpumalanga, 16/52 (30.8%) from Gauteng, and 4/52 (9.6%) from Limpopo province. Clustering was not associated with age. However, strains from male patients in Mpumalanga were more likely to be clustered than strains from male patients in Limpopo and/or Gauteng province. The minimum estimate for the proportion of resistant TB that was due to transmission is 9.06% (52-22 = 30/331). Our results indicate that transmission of drug-resistant strains may contribute substantially to the emergence of drug-resistant tuberculosis in South Africa.

A Method for Molecular Analysis of Catalase Gene Diversity in Seawater

Catalase plays an important role in the metabolism of marine bacteria and has potential impact on the marine environment. Four PCR primers were designed to amplify the catalase gene fragments in marine bacteria by applying metagenomic DNA from Yellow Sea surface water as the template. Of the four reproducible target PCR products, the longest one with 900bp were chosen for catalase gene library construction by the T-vector and the white Escherichia coli colonies in the library was screened through restriction-digesting the reamplified insert fragments by the selected restriction endonuclease MboI, and then the bands of the resulting products were displayed in the agarose gel by electrophoresis. The unique restriction fragment length polymorphism (RFLP) pattern was selected and the corresponding catalase gene fragments were sequenced, which verified that every unique RFLP pattern represented one type of catalase. This PCR-RFLP method above was established to investigate the bacterial catalase diversity in seawater.

Phosphate Solubilizing Ability and Phylogenetic Diversity of Bacteria from P-Rich Soils Around Dianchi Lake Drainage Area of China

The phylogenetic diversity of phosphate solubilizing bacteria (PSB) distributed in P-rich soils in the Dianchi Lake drainage area of China was characterized, and the tricalcium phosphate (TCP) solubilizing activities of isolated PSB were determined. Among 1 328 bacteria isolated from 100 P-rich soil samples, 377 isolates (28.39% of the total) that exhibited TCP solubilization activity were taken as PSB. These PSB showed different abilities to solubilize TCP, with the concentrations of solubilized P in bacterial cultures varying from 33.48 to 69.63 mg L−1. A total of 123 PSB isolates, with relatively high TCP solubilization activity (> 54.00 mg L−1), were submitted for restriction fragment length polymorphism (RFLP) analysis, which revealed 32 unique RFLP patterns. Based on these patterns, 62 representative isolates, one to three from each RFLP pattern, were selected for 16S rRNA sequencing. Phylogenetic analysis placed the 123 PSB into three bacterial phyla, namely Proteobacteria, Actinobacteria and Firmicutes. Members of Proteobacteria were the dominant PSB, where 107 isolates represented by 26 RFLP patterns were associated with the genera of Burkholderia, Pseudomonas, Acinetobacter, Enterobacter, Pantoea, Serratia, Klebsiella, Leclercia, Raoultella and Cedecea. Firmicutes were the subdominant group, in which 13 isolates were affiliated with the genera of Bacillus and Brevibacterium. The remaining 3 isolates were identified as three species of the genus Arthrobacter. This research extends the knowledge on PSB in P-rich soils and broadens the spectrum of PSB for the development of environmentally friendly biophosphate fertilizers.

Phylogenetic analysis of the gut bacterial microflora of the fungus-growing termite Macrotermes barneyi

Termites are an extremely successful group of wood-degrading organisms and are therefore important both for their roles in carbon turnover in the environment and as potential sources of biochemical catalysts for efforts aimed at converting wood into biofuels. To contribute to the evolutional study of termite digestive symbiosis, a bacterial 16S rRNA gene clone library from the gut microbial community of the fungus-growing termite Macrotermes barneyi was constructed. After screening by restriction fragment length polymorphism (RFLP) analysis, 25 out of 105 clones with unique RFLP patters were sequenced and phylogenetically analyzed. Many of the clones (95%) were derived from three phyla within the domain bacteria: Bacteroidetes, Firmicutes andProteobacteria. In addition, a few clones derived from Deferribacteres, Actinobacteria andPlanctomycetes were also found. No one clone affiliated with the phylum Spirochaeteswas identified, in contrast to the case of wood-feeding termites. The phylogenetic analysis revealed that nearly half of the representative clones (11 phylotypes) formed monophyletic clusters with clones obtained from other termite species, especially with the sequences retrieved from fungus-growing termites. These results indicate that the presence of termite-specific bacterial lineages implies a coevolutional relationship of gut microbes and host termites. The remaining 14 clones formed a cluster, and there was very low sequence similarity (30 to 40%) to known 16S rRNA sequences. The 16S rRNA gene sequence data showed that the majority of the intestinal microflora of M. barneyiconsisted of new, uncultured species previously unknown to microbiologists. Key words: Termite, bacteria, symbiosis, restriction, fragment, length, polymorphism.

A survey of the bacterial composition of kurut from Tibet using a culture-independent approach

Kurut (fermented yak milk) made by natural fermentation is a very important dairy food for the local people in Tibet (China). It is important to fully understand the bacterial composition of kurut for quality improvement and industrial production. Because more than 99% of prokaryotes cannot be cultured and identified by methods currently used in taxonomy, we applied a culture-independent approach to explore the microbial biodiversity of this traditional food. In this study, a bacterial 16S rRNA gene clone library, including 460 clones, was constructed using total DNA extracted from 30 samples of kurut. After screening by restriction fragment length polymorphism (RFLP) analysis, 56 operational taxonomic units (OTU) with unique RFLP patterns were obtained. Then, 1 representative sequence of every OTU was sequenced and phylogenetically analyzed. The representative phylotypes were affiliated with 5 groups, including Lactococcus lactis ssp. lactis, Lactobacillus helveticus, Streptococcus thermophilus, Lactobacillus delbrueckii ssp. bulgaricus, and Acetobacter. In addition, nearly one-third of the representative clones (132 clones) had low similarity to species in GenBank (<97%), and these phylotypes were regarded as unknown bacteria. The characteristics of kurut are determined not only by lactic acid bacteria well known by the culture-dependent approach but also by bacteria that have not yet been identified.

Molecular Characterization of Cronobacter Lipopolysaccharide O-Antigen Gene Clusters and Development of Serotype-Specific PCR Assays

Cronobacter (formerly Enterobacter sakazakii) is a recently defined genus consisting of six species, C. sakazakii, C. malonaticus, C. dublinensis, C. muytjensii, C. turicensis, and Cronobacter genomospecies 1. In this study, MboII restriction fragment length polymorphism (RFLP) patterns of O-antigen gene clusters, located between galF and gnd, were used to identify serotypes in Cronobacter spp. Seven O-antigen RFLP clusters were generated, including three C. sakazakii clusters, previously identified as serotypes O1, O2, and O3. The O-antigen regions of six strains with unique RFLP patterns, including two C. sakazakii strains, two C. malonaticus strains, one C. turicensis strain, and one C. muytjensii strain, revealed three O-antigen gene clusters shared among Cronobacter species. PCR assays were developed, targeting the wzx O-antigen polymerase gene, and used to screen 231 Cronobacter strains to determine the frequency of these newly identified serotypes.

Genetic Diversity ofBorrelia burgdorferiSensu Lato Isolates from Northeastern China

Thirty-two strains of Borrelia burgdorferi sensu lato were isolated from Ixodes persulcatus ticks collected from northeastern China from May to June in 2004 and 2005. Restriction fragment length polymorphism (RFLP) analysis and sequence analysis of 5S-23S rRNA intergenic spacer revealed that 29 (90.6%) belonged to Borrelia garinii, demonstrating B, C, and a unique pattern. The remaining three isolates (9.4%) were Borrelia afzelii with pattern D. The phylogenetic analysis based on 5S-23S rRNA intergenic spacer showed that B. garinii and B. afzelii genospecies clustered into two separate lineages. B. garinii strains were classified into three different branches: All the strains with RFLP pattern C were in the same branch, strain VH10 with a unique RFLP pattern clustered with strains VH9 and MDH2 with pattern B, and the rest of the strains with pattern B constitute another branch. These findings demonstrate the genetic diversity of B. burgdorferi sensu lato isolates from northeastern China.

Silent information regulator 2 proteins encoded by Cryptosporidium parasites

Screening in a database has revealed that Cryptosporidium hominis encodes a silent information regulator 2 (Sir2), a nicotinamide adenine dinucleotide (NAD)-dependent protein deacetylase. Cellular localization of the protein, ChSir2, was analyzed by the use of the social amoeba Dictyostelium discoideum as a model system. Fluorescent microscopic analysis showed that ChSir2 fused with green fluorescent protein was localized in the D. discoideum nucleus. D. discoideum expressing ChSir2 grew faster and reached higher cell density than did D. discoideum harboring a control vector. These results suggest that ChSir2 is a nucleus-localizing protein that plays an important role in the growth of C. hominis. We cloned and sequenced the genes for Sir2 orthologs encoded by three isolates of C. hominis, two isolates of Cryptosporidium parvum and one isolate of Cryptosporidium meleagridis. The orthologs conserve critical catalytic or NAD-binding residues but do not have similarity with human Sir2 proteins (SIRTs). Cryptosporidium Sir2 orthologs would therefore be attractive therapeutic targets. The Cryptosporidium orthologs were classified into four variants based on their nucleotide sequences. Each of the four variants produces its own unique restriction fragment length polymorphism pattern by digestion with TfiI.

Genotyping of Mycobacterium avium subsp. avium isolates from domestic animals in Slovenia by IS901 RFLP

ABSTRACT: Apart from birds, &lt;I&gt;Mycobacterium avium&lt;/I&gt; subsp. &lt;I&gt;avium (MAA) &lt;/I&gt; is often isolated from granulomatous lesions in pigs and occasionally from cattle and other animals. The objectives of this study were the detection of IS&lt;I&gt;901&lt;/I&gt; restriction fragment length polymorphism (RFLP) types of &lt;I&gt;MAA&lt;/I&gt; isolates from different species of domestic animals between the years 1998 and 2004 and the comparison of the detected RFLP types with previously described RFLP types collected in the database of the OIE Reference Laboratory for Avian Tuberculosis (Brno, Czech Republic). Furthermore, the RFLP types of the isolates obtained from &lt;I&gt;MAA&lt;/I&gt; outbreaks on one of the largest pig farms in Slovenia were also investigated. A total of 62 isolates (56 from pigs, five from poultry and one from cattle) were identified with IS&lt;I&gt;901&lt;/I&gt; PCR and IS&lt;I&gt;901&lt;/I&gt; RFLP typed using restriction endonucleases &lt;I&gt;Pvu&lt;/I&gt;II and &lt;I&gt;Pst&lt;/I&gt;I. Seven &lt;I&gt;Pvu&lt;/I&gt;II RFLP and 11 &lt;I&gt;Pst&lt;/I&gt;I RFLP types resulted in 12 combined &lt;I&gt;Pvu&lt;/I&gt;II &lt;I&gt;Pst&lt;/I&gt;I types; none of these matched the combined RFLP types described in previous studies. Our contributions to the database were two new &lt;I&gt;Pvu&lt;/I&gt;II and eight new &lt;I&gt;Pst&lt;/I&gt;I RFLP types. Identical RFLP types were found among isolates of animals originating from individual farms. Finding of identical RFLP types within a farm is not surprising because the animals were epidemiologically related and infected with one strain. A unique RFLP type F-A17 was detected in isolates from different pig herds and also in isolates from poultry. Detection of identical RFLP types on different farms may reflect one &lt;I&gt;MAA&lt;/I&gt; source. The other combined &lt;I&gt;Pvu&lt;/I&gt;II &lt;I&gt;Pst&lt;/I&gt;I RFLP types were identified only once which indicates considerable variety of &lt;I&gt;MAA&lt;/I&gt; RFLP types in Slovenia.

Borrelia carolinensis sp. nov., a new (14th) member of the Borrelia burgdorferi Sensu Lato complex from the southeastern region of the United States.

Approximately 118 Borrelia isolates were cultured from a variety of rodents, birds, and ticks collected in the southern United States. In addition to a highly diverse group of Borrelia bissettii strains and a homogenous group of Borrelia burgdorferi sensu stricto strains, a group of 16 isolates with unusual characteristics was found. The isolates were cultured from ear biopsy samples of the rodents Peromyscus gossypinus and Neotoma floridana trapped at five localities in South Carolina. A multilocus sequence analysis of the rrf-rrl intergenic spacer, 16S rRNA, fla, ospA, and p66 genes were used to clarify the taxonomic status of the new group of B. burgdorferi sensu lato isolates. Thirteen species of the B. burgdorferi sensu lato complex were used as controls. Unique restriction fragment length polymorphism patterns of the rrf-rrl intergenic spacer region and fla gene were recognized. Unique signature nucleotides were also found in the 16S rRNA gene. A phylogenetic analysis shows that the 16 new isolates cluster together but separately from the other species in the B. burgdorferi sensu lato complex. Our data strongly support the recognition of the 16 isolates as a new B. burgdorferi sensu lato species. We propose to name this genospecies "Borrelia carolinensis" with respect to the place of its currently known geographic location.

Factors Associated with Differences between Conventional Contact Tracing and Molecular Epidemiology in Study of Tuberculosis Transmission and Analysis in the City of Barcelona, Spain

The aim of this study was to analyze the factors associated with conventional contact tracing (CCT) and molecular epidemiology (ME) methods in assessing tuberculosis (TB) transmission, comparing the populations studied and the epidemiological links established by both methods. Data were obtained from TB case and CCT registries, and ME was performed using IS6110-based restriction fragment length polymorphism (RFLP) analysis and mycobacterial interspersed repetitive unit 12 (MIRU12) typing as a secondary typing method. During two years (2003 and 2004), 892 cases of TB were reported, of which 687 (77%) were confirmed by culture. RFLP analysis was performed with 463 (67.4%) of the 687 isolated strains, and MIRU12 types in 75 strains were evaluated; 280 strains (60.5%) had a unique RFLP pattern, and 183 (39.5%) shared patterns, grouping into 65 clusters. CCT of 613 (68.7%) of 892 cases detected 44 clusters involving 101 patients. The results of both CCT and ME methods yielded 96 clusters involving 255 patients. The household link was the one most frequently identified by CCT (corresponding to 80.7% of the cases clustered by this method), whereas nonhousehold and unknown links were associated with 94.1% of the strains clustered by ME. When both methods were used in 351 cases (39.3%), they showed the same results in 214 cases (61%). Of the remainder, 106 (30.2%) were clustered only by ME, 19 (5.5%) were clustered only by CCT, and 12 (3.4%) were clustered by both methods but into different clusters. Patients with factors potentially associated with social problems were less frequently studied by CCT (P = 0.002), whereas patients of <15 years of age, most with negative cultures, were less frequently studied by ME (P = 0.005). Significant differences in the populations studied by ME versus CCT were observed, possibly explaining the scarce correlation found between the results of these methods. Moreover, ME allowed the detection of nonhousehold contact relationships, whereas CCT was more useful for tracing transmission chains involving patients of <15 years of age. In conclusion, the two methods are complementary, suggesting the need to improve the methodology of contact study protocols.

Human methanogen diversity and incidence in healthy and diseased colonic groups using mcrA gene analysis

BackgroundThe incidence and diversity of human methanogens are insufficiently characterised in the gastrointestinal tract of both health and disease. A PCR and clone library methodology targeting the mcrA gene was adopted to facilitate the two-fold aim of surveying the relative incidence of methanogens in health and disease groups and also to provide an overview of methanogen diversity in the human gastrointestinal tract.ResultsDNA faecal extracts (207 in total) from a group of healthy controls and five gastrointestinal disease groups were investigated. Colorectal cancer, polypectomised, irritable bowel syndrome and the control group had largely equivalent numbers of individuals positive for methanogens (range 45–50%). Methanogen incidence in the inflammatory bowel disease groups was reduced, 24% for ulcerative colitis and 30% for Crohn's disease. Four unique mcrA gene restriction fragment length polymorphism profiles were identified and bioinformatic analyses revealed that the majority of all sequences (94%) retrieved from libraries were 100% identical to Methanobrevibacter smithii mcrA gene. In addition, mcrA gene sequences most closely related to Methanobrevibacter oralis and members of the order Methanosarcinales were also recovered.ConclusionThe mcrA gene serves as a useful biomarker for methanogen detection in the human gut and the varying trends of methanogen incidence in the human gut could serve as important indicators of intestinal function. Although Methanobrevibacter smithii is the dominant methanogen in both the distal colon of individuals in health and disease, the diversity of methanogens is greater than previously reported. In conclusion, the low incidence of methanogens in Inflammatory Bowel Disease, the functionality of the methanogens and impact of methane production in addition to competitive interactions between methanogens and other microbial groups in the human gastrointestinal tract warrants further investigation.

Diversity and Abundance of Nitrate Assimilation Genes in the Northern South China Sea

Marine heterotrophic microorganisms that assimilate nitrate play an important role in nitrogen and carbon cycling in the water column. The nasA gene, encoding the nitrate assimilation enzyme, was selected as a functional marker to examine the nitrate assimilation community in the South China Sea (SCS). PCR amplification, restriction fragment length polymorphism (RFLP) screening, and phylogenetic analysis of nasA gene sequences were performed to characterize in situ nitrate assimilatory bacteria. Furthermore, the effects of nutrients and other environmental factors on the genetic heterogeneity of nasA fragments from the SCS were evaluated at the surface in three stations, and at two other depths in one of these stations. The diversity indices and rarefaction curves indicated that the nasA gene was more diverse in offshore waters than in the Pearl River estuary. The phylotype rank abundance curve showed an abundant and unique RFLP pattern in all five libraries, indicating that a high diversity but low abundance of nasA existed in the study areas. Phylogenetic analysis of environmental nasA gene sequences further revealed that the nasA gene fragments came from several common aquatic microbial groups, including the Proteobacteria, Cytophaga-Flavobacteria (CF), and Cyanobacteria. In addition to the direct PCR/sequence analysis of environmental samples, we also cultured a number of nitrate assimilatory bacteria isolated from the field. Comparison of nasA genes from these isolates and from the field samples indicated the existence of horizontal nasA gene transfer. Application of real-time quantitative PCR to these nasA genes revealed a great variation in their abundance at different investigation sites and water depths.

Bacterial phylogenetic diversity in a constructed wetland system treating acid coal mine drainage

Microorganisms in acid mine drainage are typically acidophiles that mediate the oxidation of reduced compounds of iron and sulfur. However, microbial populations in wetland systems constructed to treat acid mine drainage are not well characterized. The purpose of this study was to analyze bacterial diversity, using cultivation-independent molecular ecological techniques, in a constructed wetland that received acid drainage from an abandoned underground coal mine. DNA was purified from Fe(III)-precipitates from the oxidized surface zone of wetland sediments and 16S rRNA gene sequences were amplified and cloned. A total of 200 clones were analyzed by restriction fragment length polymorphism (RFLP) and 77 unique RFLP patterns were obtained with four restriction enzymes. Of these patterns, 30 most dominant unique clones were selected for sequencing of their 16S rRNA genes. Half of these 30 clones could be matched with autotrophic iron- and sulfur-oxidizing bacteria ( Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans). Several clones also formed a clade with heterotrophic iron-oxidizing bacteria (TRA2-10, TRA3-20, and TRA5-3) and heterotrophic bacteria ( Stenotrophomas maltophilia, Bordetella spp., Alcaligenes sp., Alcaligenes faecalis, and Alcaligenes xylosoxidans). Approximately 40% and 35% of the analyzed RFLP restriction patterns were consistent with A. ferrooxidans and A. thiooxidans, respectively. The relatively high frequency of acidithiobacilli is consistent with the chemical and physical characteristics of this site—i.e., continuous, abundant supply of reduced iron and sulfur compounds, pH 3–4, ambient temperature, and limited organics originating from the coal seam and from vegetation or soil surrounding the inlet channel to the wetland. The RFLP results were consistent with our previous culture-independent PCR-DGGE and FISH study, showing relatively low bacterial diversity and predominance of mesophilic acidithiobacilli in oxic wetland sediments.