Project description:Analysis of gingival crevicular fluid (GCF) samples may give information of the identity of unattached (planktonic) subgingival bacteria, the 35 forefront candidates for systemic dispersal via ulcerated periodontal pocket epithelium. Our study represents the first one targeting the identity of bacteria in gingival crevicular fluid. Methodology/Principal findings: We determined bacterial species diversity in GCF samples of a group of periodontitis patients and delineated contributing bacterial and host-associated factors. Subgingival paper point (PP) samples from the same sites were taken for comparison. After DNA extraction, 16S rRNA genes were PCR amplified and DNA-DNA hybridization was performed using a microarray for over 300 bacterial species or groups. Altogether 133 species from 41 genera and 8 phyla 45 were detected with 9 to 62 and 18 to 64 species in GCF and PP samples, respectively, 46 per patient. Projection to latent structures by means of partial least squares (PLS) was applied to the multivariate data analysis. PLS regression analysis showed that species of genera including Campylobacter, Selenomonas, Porphyromonas, Catonella, Tannerella, Dialister, Peptostreptococcus, Streptococcus and Eubacterium had significant positive correlations and the number of teeth with low-grade attachment loss a significant negative correlation to species diversity in GCF samples. OPLS/O2PLS discriminant analysis revealed significant positive correlations to GCF sample group membership for species of genera Campylobacter, Leptotrichia, Prevotella, Dialister, Tannerella, Haemophilus, Fusobacterium, Eubacterium, and Actinomyces. Conclusions/Significance: Among a variety of detected species those traditionally classified as Gram-negative anaerobes growing in mature subgingival biofilms were the main predictors for species diversity in GCF samples as well as responsible for distinguishing GCF samples from PP samples. GCF bacteria may provide new prospects for studying dynamic properties of subgingival biofilms. The microbial profiles of GCF and subgingival plaque were analyzed from 17 subjects with periodontal disease.

Project description:Microtoming Coupled with Microarray Analysis to Evaluate Potential Differences in the Metabolic Status of Geobacter sulfurreducens at Different Depths in Anode Biofilms Differences in the Metabolic Status of Geobacter sulfurreducens at Different Depths in A Current Producing Biofilm Further insight into the metabolic status of cells within anode biofilms is essential for understanding the functioning of microbial fuel cells and developing strategies to optimize their power output. In order to further compare the metabolic status of cells growing close to the anode versus cells in the outer portion of the anode biofilm, mature anode biofilms were treated to stop turnover over of mRNA and then encased in resin which was sectioned into 100 nm shavings with a diamond knife and pooled into inner (0-20 µm from anode surface) and outer (30-60 µm) fractions. Whole genome DNA microarray analysis of RNA extracted from the shavings revealed that, at a 2-fold lower threshold, there were 146 genes that had significant (p<0.05), differences in transcript abundance between the inner and outer portions of the biofilm. Only 1 gene, GSU0093, a hypothetical ABC transporter, had significantly higher transcript abundances in the outer biofilm. Genes with lower transcript abundance in the outer biofilm included genes for ribosomal proteins and NADH dehydrogenase, suggesting that cells in the outer biofilm had lower metabolic rates. However, the differences in transcript abundance were relatively low (<3-fold) and the outer biofilm did not have significantly lower expression of the genes for TCA cycle enzymes which previous studies have demonstrated are sensitive indicators of changes in rates of metabolism in G. sulfurreducens. There also was no significant difference in the transcript levels for outer-surface cell components thought to be important in electron transfer in anode biofilms. Lower expression of genes involved in stress responses in the outer biofilm may reflect the development of low pH near the surface of the anode. The results of the metabolic staining and gene expression studies suggest that cells throughout the biofilm are metabolically active and can potentially contribute to current production. The microtoming/microarray strategy described here may be useful for evaluating gene expression with depth in a diversity of microbial biofilms. Three biological replicates were hybridized in triplicate on a coustom affimetrix tilling array using prokaryotic protocol (p69Affy, p75 Adobe) for labeling, hybridization and scanning.

Project description:To effectively monitor microbial populations in acidic environments and bioleaching systems, a comprehensive 50-mer-based oligonucleotide microarray was developed based on most of the known genes associated with the acidophiles. This array contained 1,072 probes in which there were 571 related to 16S rRNA and 501 related to functional genes. Acid mine drainage (AMD) presents numerous problems to the aquatic life and surrounding ecosystems. However, little is known about the geographic distribution, diversity, composition, structure and function of AMD microbial communities. In this study, we analyzed the geographic distribution of AMD microbial communities from twenty sites using restriction fragment length polymorphism (RFLP) analysis of 16S rRNA genes, and the results showed that AMD microbial communities were geographically distributed and had high variations among different sites. Then an AMD-specific microarray was used to further analyze nine AMD microbial communities, and showed that those nine AMD microbial communities had high variations measured by the number of detected genes, overlapping genes between samples, unique genes, and diversity indices. Statistical analyses indicated that the concentrations of Fe, S, Ca, Mg, Zn, Cu and pH had strong impacts on both phylogenetic and functional diversity, composition, and structure of AMD microbial communities. This study provides insights into our understanding of the geographic distribution, diversity, composition, structure and functional potential of AMD microbial communities and key environmental factors shaping them. This study investigated the geographic distribution of Acid Mine Drainages microbial communities using a 16S rRNA gene-based RFLP method and the diversity, composition and structure of AMD microbial communities phylogenetically and functionally using an AMD-specific microarray which contained 1,072 probes ( 571 related to 16S rRNA and 501 related to functional genes). The functional genes in the microarray were involved in carbon metabolism (158), nitrogen metabolism (72), sulfur metabolism (39), iron metabolism (68), DNA replication and repair (97), metal-resistance (27), membrane-relate gene (16), transposon (13) and IST sequence (11).

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