IN-VITRO RECONSTITUTION OF SULFITE REDUCTASE FROM PSEUDOMONAS AERUGINOSA

Recent work has established a link between a ferredoxin:NAD(P)H oxidoreductase (FprA) and sulfite assimilation in members of the genus Pseudomonas.  This suggested that FprA is a component of a novel sulfite reductase enzyme.  That hypothesis is consistent with the fact that only one component of the well-characterized E. coli a8?4 sulfite reductase has been identified in Pseudomonas genomes; i.e the ? siroheme subunit CysI is present but not the a flavoprotein subunit CysJ.  This led to the hypothesis that FprA is a component of a novel sulfite reductase enzyme.  Our aim is to test that hypothesis by in-vitro reconstitution using the purified proteins CysI and FprA.  We have successfully overexpressed and purified FprA from Pseudomonas aeruginosa.  The strategy for production of purified CysI has been complicated by the requirement for concomitant expression of CysG (siroheme synthase).  We are also investigating the possibility that a downstream, overlapping reading frame (PA1837) may also be necessary for functional CysI production

Inherent bacterial DNA contamination of extraction and sequencing reagents may affect interpretation of microbiota in low bacterial biomass samples

Additional file 1: Table S1. Taxonomic listing of all bacterial genera and species detected as contaminants of DNA extraction and processing kits in the present study and previously reported

Comparison of the relative abundance of bacterial Families (A) and Genera (B) within the submucosa of the diseased ileum in Crohn's disease as compared to the ileal submucosa in nIBD controls.

<p>There were major changes in 12 families and 20 genera. <i>Legend</i>: Bacteria present in less than 50% of patients are excluded. All data presented are statistically significant (p = <0.05).</p

Microbial Population Differentials between Mucosal and Submucosal Intestinal Tissues in Advanced Crohn's Disease of the Ileum

<div><p>Since Crohn's disease is a transmural disease, we hypothesized that examination of deep submucosal tissues directly involved in the inflammatory disease process may provide unique insights into bacterial populations transgressing intestinal barriers and bacterial populations more representative of the causes and agents of the disease. We performed deep 16s microbiota sequencing on isolated ilea mucosal and submucosal tissues on 20 patients with Crohn's disease and 15 non-inflammatory bowel disease controls with a depth of coverage averaging 81,500 sequences in each of the 70 DNA samples yielding an overall resolution down to 0.0001% of the bacterial population. Of the 4,802,328 total sequences generated, 98.9% or 4,749,183 sequences aligned with the Kingdom Bacteria that clustered into 8545 unique sequences with <3% divergence or operational taxonomic units enabling the identification of 401 genera and 698 tentative bacterial species. There were significant differences in all taxonomic levels between the submucosal microbiota in Crohn's disease compared to controls, including organisms of the Order <i>Desulfovibrionales</i> that were present within the submucosal tissues of most Crohn's disease patients but absent in the control group. A variety of organisms of the Phylum <i>Firmicutes</i> were increased in the subjacent submucosa as compared to the parallel mucosal tissue including <i>Ruminococcus</i> spp., <i>Oscillospira</i> spp., <i>Pseudobutyrivibrio</i> spp., and <i>Tumebacillus</i> spp. In addition, <i>Propionibacterium</i> spp. and <i>Cloacibacterium</i> spp. were increased as well as large increases in <i>Proteobacteria</i> including <i>Parasutterella</i> spp. and <i>Methylobacterium</i> spp. This is the first study to examine the microbial populations within submucosal tissues of patients with Crohn's disease and to compare microbial communities found deep within the submucosal tissues with those present on mucosal surfaces. Our data demonstrate the existence of a distinct submucosal microbiome and ecosystem that is not well reflected in the mucosa and/or downstream fecal material.</p></div

Differences in the number of OTU’s detected between mucosal and submucosal DNA samples suggesting that inadvertent influence of the submucosa with microbes from the mucosa was minimal.^*

<p>*Although some contamination of submucosal tissues with mucosal microbes may occur (e.g., <i>Campylobacter</i> spp.), the high prevalence of certain genera (e.g., <i>Tumebacillus</i> spp. and others) within subjacent submucosal tissues as opposed to the parallel mucosa strongly support the notion that mucosal contamination is not a major factor contributing to the submucosal microbiota.</p><p>Differences in the number of OTU’s detected between mucosal and submucosal DNA samples suggesting that inadvertent influence of the submucosa with microbes from the mucosa was minimal.^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134382#t001fn001" target="_blank">*</a>}</p

Bacterial populations of the diseased submucosa and mucosa at the center of the diseased tissues in Crohn's disease.

<p>The values in parentheses represent the average relative frequency in mucosa/submucosa. An asterisk (*) denotes that the difference between the mucosa and submucosa was statistically significant (p = <0.05) while a plus (+) represents a trend toward statistical significance (p = >0.05 but <0.065).</p

Comparison of the relative abundance of bacterial Families (A) and Genera (B) within the submucosa of the diseased ileum in Crohn's disease as compared to the ileal submucosa in nIBD controls.

<p>There were major changes in 12 families and 20 genera. <i>Legend</i>: Bacteria present in less than 50% of patients are excluded. All data presented are statistically significant (p = <0.05).</p