Anoop Mayampurath Evolution of Symbiotic Bacteria in the Distal Human Intestine Xu et. al, PLoS Biology 2007, 5 (7),

Human Microbiome Gut microbiota nutrient sources - Plant polysaccharides - Undigested plant proteins - Host glycans Turnbaugh et al. “The Human Microbiome Project” Nature 2007, Backhed et al. “Host-Bacterial Mutualism in the Human Intestine, Science 2005, 307 (5717),

Eckburg et al. “Diversity of the Human Intestinal Microbial Flora”, Science (5728) ~90% belong to Bacteroidetes and Firmicutes

Questions How has evolution shaped by the effect of intestinal environment?  “Top-Down” selection  “Bottom-up” selection Nature of adaptation in species  general (gut vs non-gut)  specialized (within a species)

Approach Sequenced B. vulgatus and B. distasonis Compare genomes to other sequenced (gut/non-gut) Bacteroidetes.  Orthologs that are shared between gut and non-gut  Orthologs unique to gut  Detect “niche” specialization Analyze functions of these special genes along with their origin (role of LGT)

orthologs among all gut species

GO Terms comparison Inference - All bacteroidetes had a share core metablome - Bacteroidetes specific to the gut have enriched genes for polysaccharide metabolism, environment sensing and membrane transport 5w – all 5 gut 7w – 5 gut plus 2 non gut (shared) 5wU – 5 gut plus 2 non gut (unique to gut) Enrichment P < 0.05 : pink P < : red Depletion P < 0.05 : light blue P < : dark blue

GO Term comparison (individual comparison) Inference - Each species has a “niched” specialization

What are these niches? (B. thetaioataomicron)  Forages polysaccharides  “Opportunistically” deploys SusC and SusD membrane proteins along with hydrolases and lysases.  Most glycoside hydrolases for plant glycans  Only sequenced species that contain lyases for animal glycans  Most enzymes for host glycan harvesting

What are these niches? (B. distasonis)  Lacks many glycosyldases  Smaller proteome  Only one candidate alpha-fucodiase  Two abundant hydrolases Family 13 (alpha-amylase proteins) Family 73 (host glycan harvesting)  Has the capacity to harvest host glycans in spite of glycan sialic acid termination  More protein degradation than B. thetaiotaomicron

What are these niches? (B. vulgatus) Intermediate between B.distasonis and B. thetaiotamicron Enzymes targeting pectin Only species containing a gene encoding xylanase

Role of LGT Phylogentic approach  Genes transferred into one lineage and genes lost in lineage except one. Genes satisfying one of the following conditions  No homologs were found in NCBI nR database  Only homologs were found in that species  Only homologs were found in non-gut  More closely related to non-gut than gut Tamamens and Moya, “Estimating the extent of horizontal gene transfer in metagenomic sequences” BMC Genomics 2008, 9 :136.

Role of LGT “lateral” genes differ from the rest of the genome in terms of GC content and codon bias. ~5% of genes in each genome as being present on account of LGT.

Distribution of LGT genes Light-Blue: Whole Genome Red: DNA Methylation Green: CPS loci Yellow: Glycosyltranferases Light-Blue: B. distasonis Red: B. vulgatus Yellow: B. thetaiotaomicron Green: B. fragills NCTC 9343 Purple : B. fragilis YHC 46 Orange: P. Gingivalis

CPS Loci Regulatory cassette Structural cassette that code for glycotransferases and carbohydrate transporters.

CPS Loci are extremely polymorphic

CPS Diversity through recombination

CPS Diversity through phage

SusC and SusD paralogs SusC are membrane proteins which along with glycan binding, also aid in transport. SusD are secreted proteins which bind to glycans using a lipid tail

SusC/SusD phylogeny

SusC/SusD diversification through gene duplication 1- Conserved hypothetical lipidated protein 2 – SusD paralog 3 – SusC paralog 4 – NHL repeat-containing protein 5 – Glutaminase A 1- Sulfatase 2- SusD 3- SusC 4 – Anti sigma factor 5- ECF sigma factor

Discussion “The B. thetaiotaomicron genome contains 261 glycoside hydrolases and polysaccharide lyases currently annotated in the Carbohydrate-Active Enzymes (CAZy) Database (24). Remarkably, this organism's genome also contains 208 homologs of susC and susD, suggesting that the molecular strategy for starch utilization has been expanded to target other nutrients”24 Martens et al. “Complex Glycan Catabolism by the Human Gut Microbiota: The Bacteroidetes Sus-like Paradigm” J Biol Chem 2009, 284(37)

How do they survive? Both “top down” and “bottom up” are present. LGT plays a role. When and How are still left to open question Importance of glycosyltransferases. Persistence from one generation to next? Personalized microbial communities

Other work Mahowald et al. “Characterizing a model human gut microbiota composed of members of its two dominant bacterial phyla”, PANS 2009, 106(14) “B. thetaiotaomicron adapts to E. rectale by up-regulating expression of a variety of polysaccharide utilization loci encoding numerous glycoside hydrolases, and by signaling the host to produce mucosal glycans that it, but not E. rectale, can access. E. rectale adapts to B. thetaiotaomicron by decreasing production of its glycan-degrading enzymes, increasing expression of selected amino acid and sugar transporters, and facilitating glycolysis by reducing levels of NADH, in part via generation of butyrate from acetate, which in turn is used by the gut epithelium. ” Brigham et al. “Sialic Acid (N-Acetyl Neuraminic Acid) Utilization by Bacteroides fragilis Requires a Novel N-Acetyl Mannosamine Epimerase”, J. Bacteriology 2009, 191(11) - Characterization of nanLET operon in Bacteroides fragilis Coyne et al. “Role of glycan synthesis in colonization of the mammalian gut by the bacterial symbiont Bacteroides fragilis”, PANS 2008, 105(13), Construct mutants defective in certain enzymes that result in inability to synthesize polysacchrides -