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PubMed2005

Analysis of bacterial diversity in acidic pond water and compost after treatment of artificial acid mine drainage for metal removal.

The microbial population of a sludge amended leaf compost material utilized for treatment of artificial acid mine drainage was studied by culture-independent molecular methods. Iron-rich and sulfurous wastewater (artificial acid mine drainage) was circulated through a column bioreactor for 16 months. After 12 months the column was inoculated with a mixed culture from an acidic pond receiving acid mine drainage from a tailings impoundment at a decommissioned site in Kristineberg, North Sweden. Hydrogen sulfide odor and the formation of black precipitates indicated that sulfate-reduction occurred in the column. 16S rDNA gene analysis by denaturing gradient gel electrophoresis, cloning, and sequencing as well as fluorescent in situ hybridization confirmed the presence of microorganisms closely related to sulfate-reducing bacteria and microorganisms from the genera Pseudoxanthmonas, Dechlorosoma, Desulfovibrio, Agrobacterium, Methylocapsa, Rhodococcus, Sulfobacillus, and some unidentified bacteria. Sulfate-reducing bacteria were found in the column bioreactor 2 weeks after inoculation, but not thereafter. This suggests they were in low abundance, even though sulfate remediation rates were significant. Instead, the population contained species similar to those previously found to utilize humic substances released from the compost material.

Bacteria↗
PubMed2001

Genetic diversity and molecular markers of cupped oysters (genera Crassostrea, Caccostrea, and Striostrea) in Thailand revealed by randomly amplified polymorphic DNA analysis.

Genetic diversity and species-diagnostic markers of 5 oysters in Thailand, Crassostrea belcheri (Sowerby, 1871), Crassostrea iredalei (Faustino, 1932), Saccostrea cucullata (Born, 1778), Saccostrea forskali (Gmelin, 1791), and Striostrea Parastriostrea) mytiloides (Lamarck, 1819), were investigated by randomly amplified polymorphic DNA (RAPD) analysis. In a total, 135, 127, and 108 genotypes were observed from primers OPA09, OPB01, and OPB08 (Operon Technologies Inc., kits A and B), and 131 and 122 genotypes from primers UBC210 and UBC220 (University of British Columbia), respectively. Two hundred fifty-four reproducible and polymorphic fragments (200-2500 bp in length) were generated across the 5 investigated species. The average number of bands per primer varied between 12.4 and 32.2. The percentage of polymorphic bands within Crassostrea (53.23%-77.67%) was lower than that within Saccostrea and Striostrea oysters (86.21%-99.36%). Nine, species-specific markers were found in C. belcheri, 4 in C. iredalei, and 2 in S. cucullata. The mean of a ratio between the number of genotypes generated by each primer and the number of investigated specimens of C. belcheri (0.58) was lower than that of the remaining species (0.90-1.00). Genetic distances between pairs of oyster samples were between 0.105 and 0.811. A neighbor-joining tree indicated distant relationships between Crassostrea and Saccostrea oysters, but closer relationships were observed between the latter and Striostrea mytiloides.

Journal Article↗
PubMed2001

Analysis of phenotypic diversity among host-independent mutants of Bdellovibrio bacteriovorus 109J.

Host-independent (H-I) mutants of the obligate bacterial parasite Bdellovibrio bacteriovorus were isolated from wild-type strain 109J. Seven H-I mutants differed in morphological features such as cell length (2-30 microm) and shape (short or long spirals or rod-like), plaque size, and pigmentation (from almost colorless to bright orange). The mutants exhibited widely different growth capabilities in rich medium, with biomass doubling times and final biomass varying by a factor of two or more. Growth was always enhanced by the addition of host cell extract or divalent cations to the growth medium, but the effect varied widely between the mutants. Analysis of the hit region, mutations in which were previously proposed to be associated with the H-I phenotype, revealed that changes in the nucleotide sequence in this region occurred only in three of the seven mutants.

Bdellovibrio↗
PubMed2002

Molecular community analysis of microbial diversity.

New technologies that avoid the need for either gene amplification (e.g. microarrays) or nucleic acid extraction (e.g. in situ PCR) have recently been implemented in microbial ecology. Together with new approaches for culturing microorganisms and an increased understanding of the biases of molecular methods, these techniques form the most exciting advances in this field during the past year.

Ecosystem↗
PubMed2005

Etiologic agents of acute gastroenteritis among Japanese infants and children: virus diversity and genetic analysis of sapovirus.

A total of 371 fecal specimens from infants and children with acute gastroenteritis in Maizuru, Tokyo and Osaka, Japan from July 2002 to June 2003 were tested for the presence of diarrheal viruses by reverse transcription-polymerase chain reaction (RT-PCR), reverse passive hemagglutination (PRHA), RNA-polyacrylamide gel electrophoresis (PAGE), latex agglutination and sequence analysis methods. Among diarrheal viruses detected, group A rotavirus was the most prevalent (42.2%) followed by norovirus (28.9%), group C rotavirus (8.4%), sapovirus (6.7%), adenovirus (5.3%) and astrovirus (0.9%), respectively. There was the high rate (7.6%) of viral mixed infections. Sapovirus was classified into 6 genotypes (GI/1, GI/4, GI/5, GI/6 and GII/1 and one novel tentatively called GII/5). It is noteworthy that genogroup II sapovirus can be classified into 5 genotypes. Our findings confirmed the presence of many diarrheal viruses co-circulating among Japanese infants and children and showed the great genetic diversity among sapoviruses.

Acute Disease↗
PubMed2006

[Analysis of genetic diversity of AFLP marker among populations of Oncomelania hupensis].

OBJECTIVE: To explore the degree of genetic diversity among populations of Oncomelania hupensis. METHODS: AFLP method was used to amplify the genomic DNA of thirteen snail populations from nine provinces (i. e. Yunnan, Sichuan, Guangxi, Fujian, Hunan, Hubei, Jiangxi, Anhui and Jiangsu) and the genetic diversities among snail populations were analyzed. RESULTS: The number of AFLP fragments amplified ranged from 403 to 472 for thirteen Oncomelania populations. Among the thirteen snail populations, the genetic diversity within the population from Xingzi County, Jiangxi Province, was most significant, and the percentage of polymorphic loci, Nei's genetic diversity and Shannon's information index were 93.22%, 0.345 and 0.510 respectively, while these indices for the snail population from Yizhou City, Guangxi Region, were the lowest, 55.80%, 0.191 and 0.287 respectively. The similarity between the in-group-individuals from Yizhou City, Guangxi Region, was most significant, and the average coefficient of similarity was 0. 904, and that from Dantu County, Jiangsu Province, was the lowest (0.748). The genetic diversities among snail populations were significantly different for the thirteen snail populations (P < 0.01). CONCLUSION: There is a certain genetic variation among Oncomelania snail populations from the mainland of China, and this variation is significantly different among snail populations from different areas.

Amplified Fragment Length Polymorphism Analysis↗
PubMed1990

Analysis of sequence diversity in hypervariable regions of the external glycoprotein of human immunodeficiency virus type 1.

Nucleotide sequences in three hypervariable regions of the human immunodeficiency virus type 1 (HIV-1) env gene were obtained by sequencing provirus present in peripheral blood mononuclear cells of HIV-infected individuals. Single molecules of target sequences were isolated by limiting dilution and amplified in two stages by the polymerase chain reaction, using nested primers. The product was directly sequenced to avoid errors introduced by Taq polymerase during the amplification process. There was extensive variation between sequences from the same individual as well as between sequences from different individuals. Interpatient variability was markedly less in individuals infected from a common source. A high proportion of amino acid substitutions in the hypervariable regions altered the number and positions of potential N-linked glycosylation sites. Sequences in two hypervariable regions frequently contained short (3- to 15-bp) duplications or deletions, and by amplifying peripheral blood mononuclear cell DNA containing 10(2) or 10(3) proviral molecules and analyzing the product by high-resolution electrophoresis, the total number and abundance of distinct length variants within an individual could be estimated, providing a more comprehensive analysis of the variants present than would be obtained by sequencing alone. Sequences from many individuals showed frequent amino acid substitutions at certain key positions for neutralizing-antibody and cytotoxic T-cell recognition in the immunodominant loop. The rates of synonymous and nonsynonymous nucleotide substitution in the region of this and flanking regions indicate that strong positive selection for amino acid change is operating in the generation of antigenic diversity.

Amino Acid Sequence↗
PubMed2006

[Analysis of bacterial diversity of kefir grains by denaturing gradient gel electrophoresis and 16S rDNA sequencing].

Kefir is an acidic, mildly alcoholic dairy beverage produced by the fermentation of milk with a grain-like starter culture. These grains usually contain a relatively stable and specific balance of microbes that exist in a complex symbiotic relationship. Kefir grains can be considered a probiotic source as it presents anti-bacterial, anti-mycotic, anti-neoplasic and immunomodulatory properties. The microorganisms in Kefir grains are currently identified by traditional methods such as growth on selective media, morphological and biochemical characteristics. However, the microorganisms that isolate by these methods can not revert to Kefir grains which indicate that there are some other bacteria that are not isolate from it. In this study, PCR-based Denaturing gradient gel electrophoresis(DGGE) and sequence analysis of 16S ribosomal RNA gene (16S rDNA) clone libraries was used for the rapid and accurate identification of microorganisms from Kefir grains. The PCR primers were designed from conserved nucleotide sequences on region V3 of 16S rDNA with GC rich clamp at the 5'-end. PCR was performed using the primers and genomic DNAs of Kefir grains bacteria. The generated region V3 of 16S rDNA fragments were separated by denaturing gel, and the dominant 16S rDNA bands were cloned, sequenced and subjected to an online similarity search. Research has shown that regions V3 of 16S rDNAs have eight evident bands on the DGGE gel. The sequence analysis of these eight bands has indicated that they belong to different four genera, among them three sequences are similar to Sphingobacterium sp. whose similarities with database sequences are over 98%, three sequences are similar to Lactobacillus sp. whose similarities with database sequences are over 96%, the other two sequence are similar to Enterobacter sp., and Acinetobacter sp. whose similarities with database sequences are over 99% respectively. Although the DGGE method may have a lower sensitivity than the ordinary PCR methods, because when universal bacterial PCR primers are used, only the dominant microbiota of an ecosystem will be visualized on a DGGE gel, producing complex banding patterns. However, it could visualize the bacterial qualitative compositions and reveal the major species of the Kefir grains. Among them Sphingobacterium can be found in Kefir grains as the predominant flora which is reported for the first time. PCR-based DGGE and sequence analysis of 16S rDNA proved to be a valuable culture-independent approach for the rapid and specific identification of the microbial species present in microecosystem and probiotic products.

Bacteria↗
PubMed2001

Phylogenetic Analysis of Microbial Diversity in the Rhizoplane of Oilseed Rape (Brassica napus cv. Westar) Employing Cultivation-Dependent and Cultivation-Independent Approaches.

The structure of the microbial rhizoplane community of the important crop plant oilseed rape was studied by using a culture-dependent as well as a culture-independent approach based on 16S rDNA amplification. After isolation of the microbial community from the rhizoplane of oilseed rape (Brassica napus cv. Westar), the collected suspension was divided into two parts. One part was used for cultivation of bacteria onto three different growth media to establish a culture collection. From the other part of the rhizoplane suspension, genomic DNA was isolated and purified. Thereafter, 16S rDNA was amplified by PCR and cloned to obtain a library of 16S rDNA genes representative for the bacterial communities of this habitat. Phylogenetic 16S rDNA sequence analysis of 103 clones of this library revealed considerable differences from the corresponding nucleotide sequences of 111 cultured bacteria. Whereas the 16S rDNA clone library was dominated by a-Proteobacteria and bacteria of the Cytophaga-Flavobacterium-Bacteroides (CFB) phylum (51% and 30%, respectively), less than 17% of the cultured bacteria belonged to these two groups. More than 64% of the cultivated isolates were allocated to the b- and g-subclasses of the Proteobacteria, which were present in the clone library at about 14%. Most of the clones of the a-Proteobacteria of the library showed highest similarity to Bradyrhizobium sp. No such bacteria were found in the culture collection. Similarly, the second dominant group of the clone library comprising members of the CFB phylum was represented in the culture collection by a single isolate. The phylogenetic analysis of isolates of the culture collection clearly emphasized the need to use different growth media for recovery of rhizoplane bacteria. Whereas most of the a-Proteobacteria were recovered on complex medium, most of the b-Proteobacteria were isolated onto minimal media. Our results demonstrate that the combined approach pursued in this paper is necessary to explore the biodiversity of bacterial rhizoplane communities.

Journal Article↗
PubMed2006

[AFLP analysis on genetic diversity of wild Dactylis glomerata L. germplasm resources].

Thirty-seven accessions of wild Dactylis glomerata L. were used in assessment of genetic diversity by means of amplified fragment length polymorphism (AFLP). Nine primer pairs were selected from 64 preparations and then used to analyze the plant materials. A total of 400 DNA fragments were amplified, among which 336 (84.0%) were polymorphic. Genetic distance, from 0.0692-0.4214 among accessions, was calculated by the NTSYS-pc software. A dendrogram was constructed using UPGMA based on genetic distance matrix. Eight clusters were defined at the genetic similarity of 0.81. A two-dimensional PCO plot representing relationships among accessions was also produced by EIGEN, demonstrating the same distribution pattern as from the cluster analysis. Based on molecular analysis, the genetic variation of D. glomerata germplasms was closely associated with ploidy levels and geographical distributions.

Amplified Fragment Length Polymorphism Analysis↗
PubMed2001

Analysis of genetic diversity and structure in Ethiopian populations of Phytolacca dodecandra using RAPD.

The genetic diversity and structure in 17 wild populations (249 individuals) of Phytolacca dodecandra (endod) sampled along altitudinal gradients of 1600-3000 meters above sea level (m.a.s.l.) in Ethiopia was studied using random amplified polymorphic DNA (RAPD). A total of 70 polymorphic loci (P) scored from 12 RAPD primers were used to calculate different diversity indices within and between populations, habitats, geographical regions, climatic zones and altitude groups. The number of polymorphic loci and overall Shannon information measure (H) in the populations varied from 30 to 55 and from 0.228 to 0.418, respectively. In general, differences in population variability were found significantly correlated to effective population size. Both P and H were significantly higher in an undisturbed than in a disturbed habitat, and in the lowland and central-highland than in the highland altitude group. However, for both parameters the differences were not statistically significant between regions and climatic zones. Genetic distance between populations varied from 0.301 to 0.628. Cluster analysis performed using the genetic distance matrix revealed a clear separation of the highland populations (2501-3000 m.a.s.l.) from those of the lowland/central-highlands (1600-2500 m.a.s.l.) irrespective of their geographical regions and climatic zones. Analysis of molecular variance (AMOVA) indicated that differences in habitat, geographical regions and climatic zones explained 4.6%, 2.5% and 4.6%, respectively. But none of these differences were significant. Altitude explained 17.2% of the total variance and was highly significant. The data, therefore, clearly indicated the association of genetic structure in endod with altitude. The proportion of RAPD variation found among populations (21.2-35.0%) was somewhat intermediate between values reported for selfing and outcrossing species. The fixation index (FST) values (0.350 to 0.384) indicated very high genetic differentiation among populations.

Ethiopia↗
PubMed2006

Analysis of nucleotide diversity of NAT2 coding region reveals homogeneity across Native American populations and high intra-population diversity.

N-acetyltransferase 2 (NAT2), an important enzyme in clinical pharmacology, metabolizes antibiotics such as isoniazid and sulfamethoxazole, and catalyzes the transformation of aromatic and heterocyclic amines from the environment and diet into carcinogenic intermediates. Polymorphisms in NAT2 account for variability in the acetylator phenotype and the pharmacokinetics of metabolized drugs. Native Americans, settled in rural areas and large cities of Latin America, are under-represented in pharmacogenetics studies; therefore, we sequenced the coding region of NAT2 in 456 chromosomes from 13 populations from the Americas, and two from Siberia, detecting nine substitutions and 11 haplotypes. Variants *4 (37%), *5B (23%) and *7B (24%) showed high frequencies. Average frequencies of fast, intermediate and slow acetylators across Native Americans were 18, 56 and 25%, respectively. NAT2 intra-population genetic diversity for Native Americans is higher than East Asians and similar to the rest of the world, and NAT2 variants are homogeneously distributed across native populations of the continent.

Acetylation↗
PubMed1999

Analysis of genomic diversity within the Xr-region of the protein A gene in clinical isolates of Staphylococcus aureus.

Protein A of Staphylococcus aureus contains a polymorphic Xr-region characterized by a tandem repeat of eight amino acid units. In this study, the diversity of genes encoding the repeat regions and their relatedness among S. aureus strains was analyzed. Ten different protein-A types characterized by repeat numbers 4-13 were identified in a total of 293 clinical isolates. The protein-A type with 10 repeat units (10 repeats) in the Xr-region was most frequently detected in methicillin-resistant S. aureus, whereas the majority of methicillin-susceptible strains were distributed almost evenly into protein-A types with 7-11 repeats. Strains that belonged to a single coagulase type were classified into multiple protein-A types, e.g. strains with the common coagulase types II and VII were differentiated into 7 and 8 protein-A types, respectively. Nucleotide sequence analysis of the Xr-region of 42 representative strains revealed the presence of 37 different genotypes (spa types), which were constituted by a combination of several of 24 different repeat unit genotypes. Based on the similarity in arrangement of repeat unit genotypes, 34 strains with different repeat numbers were classified into 5 genetic clusters (C1-C5). The clusters C1, C2 and C3 consisted exclusively of strains with identical coagulase types II, III, and IV, respectively. These findings suggested that the protein-A gene of S. aureus has evolved from a common ancestral clone in individual clusters independently.

Amino Acid Sequence↗
PubMed2005

A biochemical and structural analysis of genetic diversity within the HLA-A*11 subtype.

The HLA-A*11 subtype includes 17 naturally occurring variants (-A*1101 to -A*1117) distributed among different ethnic groups worldwide. At present, only HLA-A*1101 has been characterized at the molecular, structural, and immunological level. Developing similar knowledge on other HLA-A*11 alleles is highly important for bone marrow and graft transplantation. This is also important to better understand disease linkages within the HLA-A*11 subtype given that HLA-A*11 molecules are associated with resistance to acquisition of HIV-1 infection and various autoimmune diseases. To broaden our understanding of HLA-A*11 molecules, we have determined the impact of natural polymorphism on the peptide-binding properties of several HLA-A*11 molecules: -A*1103, -A*1106, -A*1108, -A*1110, -A*1111, and -A*1114. We used an approach that combines data from thermal stability studies of recombinant, soluble forms of these molecules in complex with HIV-1 peptides, together with a detailed structural analysis of the resulting HLA-A*11 molecule/peptide complexes based on crystal and molecular model structures. Our analysis shows that natural polymorphism within the HLA-A*11 subtype is distributed along the alpha1 and alpha2 helices of the peptide-binding groove, in marked contrast to the pattern of polymorphism in HLA-A*2 and HLA-B*27 subtypes. Natural polymorphism greatly altered the abilities of individual -A*11 molecules to form stable complexes with HIV-1 peptides. In comparison to -A*1101, natural polymorphism altered the peptide-presenting properties of -A*1103, -A*1108, and -A*1114 and has the potential to affect the peptide-selecting properties of -A*1106, -A*1110, and -A*1111 as well. Overall, our findings suggest that HLA-A*11 molecules may stimulate alloreactive CD8+ cytotoxic T-cell responses.

Binding Sites↗
PubMed2003

Multidimensional chemical genetic analysis of diversity-oriented synthesis-derived deacetylase inhibitors using cell-based assays.

Systematic chemical genetics aims to explore the space representing interactions between small molecules and biological systems. Beyond measuring binding interactions and enzyme inhibition, measuring changes in the activity of proteins in intact signaling networks is necessary. Toward this end, we are partitioning chemical space into regions with different biological activities using a panel of cell-based assays and small molecule "chemical genetic modifiers." Herein, we report on the use of this methodology for the discovery of 617 small molecule inhibitors of histone deacetylases from a multidimensional screen of an encoded, diversity-oriented synthesis library. Following decoding of chemical tags and resynthesis, we demonstrate the selectivity of one inhibitory molecule (tubacin) toward alpha-tubulin deacetylation and another (histacin) toward histone deacetylation. These small molecules will facilitate dissecting the role of acetylation in a variety of cell biological processes.

Acetylation↗
PubMed2025

Comparative virulence analysis of seven diverse strains of Orientia tsutsugamushi reveals a multifaceted and complex interplay of virulence factors responsible for disease.

Orientia tsutsugamushi is an obligate intracellular bacterium found in Leptotrombidium mites that causes the human disease scrub typhus. A distinguishing feature of O. tsutsugamushi is its extensive strain diversity, yet differences in virulence between strains are not well defined nor well understood. We sought to determine the bacterial drivers of pathogenicity by comparing seven strains using murine infections combined with epidemiological human data to rank each strain in terms of relative virulence. Murine cytokine expression data revealed that the two most virulent strains, Ikeda and Kato, induced higher levels of IL-6, IL-10, IFN-&#x3b3; and MCP-1 than other strains, consistent with increased levels of these cytokines in patients with severe scrub typhus. We sought to identify the mechanistic basis of the observed differential virulence between strains by comparing their genomes, in vitro growth properties and cytokine/chemokine induction in host cells. We found that there was no single gene or gene group that correlated with virulence, and no clear pattern of in vitro growth rate that predicted disease. However, microscopy-based analysis of the intracellular infection cycle revealed that the only fully avirulent strain in our study, TA686, differed from all the virulent strains in its subcellular localisation and expression of its surface protein ScaC. This leads us to a model whereby drivers of pathogenicity in Orientia tsutsugamushi are distributed throughout the genome, likely in the large and varying arsenal of effector proteins encoded by different strains, and that these interact in complex ways to induce differing immune responses and thus differing disease outcomes in mammalian hosts.

Orientia tsutsugamushi↗
PubMed2005

Improvements for comparative analysis of changes in diversity of microbial communities using internal standards in PCR-DGGE.

The use of internal standards both during DNA extraction and PCR-DGGE procedure gives the opportunity to analyse the relative abundance of individual species back to the original sample, thereby facilitating relative comparative analysis of diversity. Internal standards were used throughout the DNA extraction and PCR-DGGE to compensate for experimental variability. Such variability causes decreased reproducibility among replicate samples as well as compromise comparisons between samples, since experimental errors cannot be differentiated from actual changes in the community abundance and structure. The use of internal standards during DNA extraction and PCR-DGGE is suitable for ecological and ecotoxicological experiments with microbial communities, where relative changes in the community abundance and structure are studied. We have developed a protocol Internal Standards in Molecular Analysis of Diversity (ISMAD) that is simple to use, inexpensive, rapid to perform and it does not require additional samples to be processed. The internal standard for DNA extraction (ExtrIS) is a fluorescent 510-basepair PCR product which is added to the samples prior to DNA extraction, recovered together with the extracted DNA from the samples and analysed with fluorescence spectrophotometry. The use of ExtrIS during isolation of sample DNA significantly reduced variation among replicate samples. The PCR internal standard (PCR(IS)) originates from the Drosophila melanogaster genome and is a 140-basepair long PCR product, which is amplified by non-competitive primers in the same PCR reaction tubes as the target DNA and analysed together with the target PCR product on the same DGGE gel. The use of PCR(IS) during PCR significantly reduced variation among replicate samples both when assessing total PCR product and when comparing bands representing species on a DGGE gel. The entire ISMAD protocol was shown to accurately describe changes in relative abundance in an environmental sample using PCR-DGGE. It should, however, be mentioned that despite the use of ISMAD some inherent biases still exist in DNA extraction and PCR-DGGE and these should be taken into consideration when interpreting the diversity in a sample based on a DGGE gel.

Bacteria↗
PubMed2002

Complexities in ETS-domain transcription factor function and regulation: lessons from the TCF (ternary complex factor) subfamily. The Colworth Medal Lecture.

The ETS-domain transcription factor family can be divided into a series of subfamilies. Elk-1 represents the founding member of the ternary complex factor (TCF) subfamily. By focusing on the TCF subfamily, we can demonstrate the complexities that exist in the function and regulation of ETS-domain transcription factors. This article focuses on Elk-1 in detail and summarizes the functions of other TCFs. The key themes covered include the domain structure of the TCFs, the mechanisms of complex formation with serum response factor, regulation of TCFs by mitogen-activated protein kinase cascades, and transcriptional regulatory properties of the TCFs. Finally, the emerging role of the TCFs in vivo is discussed. A picture is developing indicating that, while these proteins exhibit significant sequence and functional conservation, key differences in their structure and regulation are being identified which may relate to unique functions of these proteins in vivo.

Amino Acid Sequence↗