Data_Sheet_1.docx

<p>Background: The human gut microbiota is a dynamic community of microorganisms that mediate important biochemical processes. Differences in the gut microbial composition have been associated with inflammatory bowel diseases (IBD) and other intestinal disorders. In this study, we quantified and compared the frequencies of eight genotoxic and/or pro-inflammatory bacterial genes found in metagenomic Whole Genome Sequences (mWGSs) of samples from individuals with IBD vs. a cohort of healthy human subjects.</p><p>Methods: The eight selected gene sequences were clbN, clbB, cif, cnf-1, usp, tcpC from Escherichia coli, gelE from Enterococcus faecalis and murB from Akkermansia muciniphila. We also included the sequences for the conserved murB genes from E. coli and E. faecalis as markers for the presence of Enterobacteriaceae or Enterococci in the samples. The gene sequences were chosen based on their previously reported ability to disrupt normal cellular processes to either promote inflammation or to cause DNA damage in cultured cells or animal models, which could be linked to a role in IBD. The selected sequences were searched in three different mWGS datasets accessed through the Human Microbiome Project (HMP): a healthy cohort (N = 251), a Crohn’s disease cohort (N = 60) and an ulcerative colitis cohort (N = 17).</p><p>Results: Firstly, the sequences for the murB housekeeping genes from Enterobacteriaceae and Enterococci were more frequently found in the IBD cohorts (32% E. coli in IBD vs. 12% in healthy; 13% E. faecalis in IBD vs. 3% in healthy) than in the healthy cohort, confirming earlier reports of a higher presence of both of these taxa in IBD. For some of the sequences in our study, especially usp and gelE, their frequency was even more sharply increased in the IBD cohorts than in the healthy cohort, suggesting an association with IBD that is not easily explained by the increased presence of E. coli or E. faecalis in those samples.</p><p>Conclusion: Our results suggest a significant association between the presence of some of these genotoxic or pro-inflammatory gene sequences and IBDs. In addition, these results illustrate the power and limitations of the HMP database in the detection of possible clinical correlations for individual bacterial genes.</p

Multiple sequence alignment of the five ACP domains.

<p>The ACP domains from the <i>Photobacterium profundum</i> PUFA synthase were aligned using ClustalW. The black bars denote the stretches of protein sequence predicted to be α-helices.</p

Expected and Measured Molecular Weights of ACP Constructs in Solution.

<p>Expected and Measured Molecular Weights of ACP Constructs in Solution.</p

Full CD spectrum for the tandem ACP fragment obtained (A) before (B) during and (C) after thermal denaturation at 90°<b>C.</b>

<p>Full CD spectrum for the tandem ACP fragment obtained (A) before (B) during and (C) after thermal denaturation at 90°<b>C.</b></p

Domain structure prediction.

<p>The UMA method was employed to identify the ACP domains. The UMA score, a measurement of the likelihood that an amino acid is located within a domain (as opposed to within an unstructured linker) was plotted as a function of the position in the amino acid sequence. Five areas of high UMA score were identified as potential ACP fragments.</p

Oligonucleotides used for the amplification of the different proteins in this study.

<p>Oligonucleotides used for the amplification of the different proteins in this study.</p

Solution structure of tandem ACP.

<p>(A) The three-dimensional bead model constructed by ‘dammif’ reveals a molecular volume of 96,600 ų. (B) Simulation of the scattering data based on structural models reveals that an extended and flexible overall configuration can sufficiently account for the observed data. Figures A and B are on a different scale.</p

The polyunsaturated fatty acid synthase domain structure.

<p>A total of five genes are required for the production of PUFAs: pfaA contains a beta-ketoacyl synthase (KS), an acyltransferase (AT), five tandem acyl carrier proteins (ACP) and a ketoreductase (KR) domain. pfaB consists of a single AT. pfaC contains two KS domains and two tandem dehydratase (DH) domains, pfaD consists of a single enoyl reductase (ER) domain. pfaE consists of a phosphopantetheinyl transferase (PPTase).</p

Characterization of ACP domains by mass spectrometry.

<p>(A) An ESI-MS spectrum was generated for the tandem ACP (MW  = 59,116) before modification by attachment of the phosphopantetheine moiety of CoA. (B) After incubation with PPTase and CoA the protein was modified in four attachment sites, as evidenced by the ESI-MS mass spectrum.</p

Input parameters for the UMA calculations.

<p>Input parameters for the UMA calculations.</p