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Functional Genomic and Metabolic Studies of the Adaptations of a Prominent Adult Human Gut Symbiont, Bacteroides thetaiotaomicron, to the Suckling Period

2006; Elsevier BV; Volume: 281; Issue: 47 Linguagem: Inglês

10.1074/jbc.m606509200

1083-351X

Magnus Bjursell, Eric C. Martens, Jeffrey I. Gordon,

Clostridium difficile and Clostridium perfringens research

The adult human gut microbiota is dominated by two divisions of Bacteria, the Bacteroidetes and the Firmicutes. Assembly of this community begins at birth through processes that remain largely undefined. In this report, we examine the adaptations of Bacteroides thetaiotaomicron, a prominent member of the adult distal intestinal microbiota, during the suckling and weaning periods. Germ-free NMRI mice were colonized at birth from their gnotobiotic mothers, who harbored this anaerobic Gram-negative saccharolytic bacterium. B. thetaiotaomicron was then harvested from the ceca of these hosts during the suckling period (postnatal day 17) and after weaning (postnatal day 30). Whole genome transcriptional profiles were obtained at these two time points using custom B. thetaiotaomicron GeneChips. Transcriptome-based in silico reconstructions of bacterial metabolism and gas chromatography-mass spectrometry and biochemical assays of carbohydrate utilization in vivo indicated that in the suckling gut B. thetaiotaomicron prefers host-derived polysaccharides, as well as mono- and oligosaccharides present in mother's milk. After weaning, B. thetaiotaomicron expands its metabolism to exploit abundant, plant-derived dietary polysaccharides. The bacterium's responses to postnatal alterations in its nutrient landscape involve expression of gene clusters encoding environmental sensors, outer membrane proteins involved in binding and import of glycans, and glycoside hydrolases. These expression changes are interpreted in light of a phylogenetic analysis that revealed unique expansions of related polysaccharide utilization loci in three human alimentary tract-associated Bacteroidetes, expansions that likely reflect the evolutionary adaptations of these species to different nutrient niches. The adult human gut microbiota is dominated by two divisions of Bacteria, the Bacteroidetes and the Firmicutes. Assembly of this community begins at birth through processes that remain largely undefined. In this report, we examine the adaptations of Bacteroides thetaiotaomicron, a prominent member of the adult distal intestinal microbiota, during the suckling and weaning periods. Germ-free NMRI mice were colonized at birth from their gnotobiotic mothers, who harbored this anaerobic Gram-negative saccharolytic bacterium. B. thetaiotaomicron was then harvested from the ceca of these hosts during the suckling period (postnatal day 17) and after weaning (postnatal day 30). Whole genome transcriptional profiles were obtained at these two time points using custom B. thetaiotaomicron GeneChips. Transcriptome-based in silico reconstructions of bacterial metabolism and gas chromatography-mass spectrometry and biochemical assays of carbohydrate utilization in vivo indicated that in the suckling gut B. thetaiotaomicron prefers host-derived polysaccharides, as well as mono- and oligosaccharides present in mother's milk. After weaning, B. thetaiotaomicron expands its metabolism to exploit abundant, plant-derived dietary polysaccharides. The bacterium's responses to postnatal alterations in its nutrient landscape involve expression of gene clusters encoding environmental sensors, outer membrane proteins involved in binding and import of glycans, and glycoside hydrolases. These expression changes are interpreted in light of a phylogenetic analysis that revealed unique expansions of related polysaccharide utilization loci in three human alimentary tract-associated Bacteroidetes, expansions that likely reflect the evolutionary adaptations of these species to different nutrient niches. Our adult gut is colonized with a community of 10-100 trillion microbes. This microbiota, and its collective genome (microbiome), provide us with important physiological attributes that are not encoded in our own human genome, including the ability to break down otherwise indigestible nutrients that are delivered to the distal gut, such as dietary plant polysaccharides (1Sonnenburg J.L. Xu J. Leip D.D. Chen C.H. Westover B.P. Weatherford J. Buhler J.D. Gordon J.I. Science. 2005; 307: 1955-1959Crossref PubMed Scopus (795) Google Scholar, 2Gill S.R. Pop M. Deboy R.T. Eckburg P.B. Turnbaugh P.J. Samuel B.S. Gordon J.I. Relman D.A. Fraser-Liggett C.M. Nelson K.E. Science. 2006; 312: 1355-1359Crossref PubMed Scopus (3137) Google Scholar). A recent comprehensive 16 S rRNA-based enumeration study of the distal intestinal microbiota of a small number of healthy adult humans demonstrated that >99% of detected phylogenetic types (phylotypes) belong to two of the 70 bacterial divisions (superkingdoms) currently known in nature: the Bacteroidetes and the Firmicutes (3Eckburg P.B. Bik E.M. Bernstein C.N. Purdom E. Dethlefsen L. Sargent M. Gill S.R. Nelson K.E. Relman D.A. Science. 2005; 308: 1635-1638Crossref PubMed Scopus (5278) Google Scholar, 4Ley R.E. Peterson D.A. Gordon J.I. Cell. 2006; 124: 837-848Abstract Full Text Full Text PDF PubMed Scopus (2190) Google Scholar). Within each division there is great diversity at the species and subspecies levels. Moreover, these shallow lineages show considerable variation between individual humans (3Eckburg P.B. Bik E.M. Bernstein C.N. Purdom E. Dethlefsen L. Sargent M. Gill S.R. Nelson K.E. Relman D.A. Science. 2005; 308: 1635-1638Crossref PubMed Scopus (5278) Google Scholar, 4Ley R.E. Peterson D.A. Gordon J.I. Cell. 2006; 124: 837-848Abstract Full Text Full Text PDF PubMed Scopus (2190) Google Scholar). The mechanisms controlling assembly of our microbiotas remain ill defined. This issue can be framed as follows. What is the effect of the microbial community that is available to colonize a host at the time of birth (the legacy effect)? What is the effect of the gut environment itself on shaping the available community, and is the gut selecting for properties that are common or unique to members of bacterial divisions (the host effect)? Recent reciprocal transplantation experiments have emphasized the importance of host habitat: when the gut microbiota of a conventionally raised mouse is introduced into a germ-free zebrafish recipient, and vice versa, the recipient gut acts as a biological filter to amplify members of the donor community that most closely resemble its normal (native) species. (5Rawls J.F. Mahowald M. Ley R.E. Gordon J.I. Cell. 2006; 127: 423-433Abstract Full Text Full Text PDF PubMed Scopus (660) Google Scholar). Our gut-associated Firmicutes and Bacteroidetes have not been identified outside of the intestinal habitat. Thus, they are likely acquired by transmission from our mothers or other family members soon after birth. This notion is supported by several observations. Studies using C57Bl/6J mice revealed that offspring inherit microbiotas similar to those of their mothers (6Ley R.E. Backhed F. Turnbaugh P. Lozupone C.A. Knight R.D. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 11070-11075Crossref PubMed Scopus (4137) Google Scholar). The effect of kinship was evident across generations: C57Bl/6J mothers who are sisters have gut microbiotas that are similar to one another and to those of their offspring (6Ley R.E. Backhed F. Turnbaugh P. Lozupone C.A. Knight R.D. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 11070-11075Crossref PubMed Scopus (4137) Google Scholar). Culture-based studies of humans indicate that infants, who are germ-free in utero, also acquire their initial microbiota from the vaginal and fecal microbiota of their mothers (7Gronlund M.M. Lehtonen O.P. Eerola E. Kero P. J. Pediatr. Gastroenterol. Nutr. 1999; 28: 19-25Crossref PubMed Scopus (689) Google Scholar, 8Mandar R. Mikelsaar M. Biol. Neonate. 1996; 69: 30-35Crossref PubMed Scopus (130) Google Scholar). The extent to which host genotype influences the composition of the postnatally acquired microbiota is unclear. 16 S rRNA fingerprinting studies of a small number of adult monozygotic twin pairs indicated their fecal microbiotas were more similar to one another than to their marital partners. However, the same trend was true for dizygotic twins, underscoring the importance of a shared mother (9Zoetendal E.G. Akkermans A.D.L. Vliet W.M.A.-V. Visser J.A.G.M.D. Vos W.M.D. Microbial Ecology Health Disease. 2001; 13: 129-134Crossref Scopus (443) Google Scholar). Although many studies have been carried out to decipher the composition of the fecal microbiota during the postnatal period, the majority have used culture-based methods and hence are subject to sampling bias and to limited sensitivity (10Pace N.R. Science. 1997; 276: 734-740Crossref PubMed Scopus (1976) Google Scholar, 11Hugenholtz P. Goebel B.M. Pace N.R. J. Bacteriol. 1998; 180: 4765-4774Crossref PubMed Google Scholar). To date, there are no reports describing the results of a comprehensive 16 S rRNA sequence-based enumeration of gut microbial communities during the postnatal period, in related or unrelated infants. Therefore, we do not know whether there is an orderly succession of colonizers leading to a climax community, or whether there is a period of open occupation followed by selection of what will become persistent (autochothonous) members of the microbiota. Culture-based enumeration studies have yielded disparate results that may reflect host and/or legacy effects. Facultative anaerobes are commonly identified among the early colonizers and are succeeded by obligate anaerobes (12Balmer S.E. Scott P.H. Wharton B.A. Arch. Dis. Child. 1989; 64: 1678-1684Crossref PubMed Scopus (50) Google Scholar, 13Roberts A.K. Chierici R. Sawatzki G. Hill M.J. Volpato S. Vigi V. Acta Paediatr. 1992; 81: 119-124Crossref PubMed Scopus (134) Google Scholar, 14Sakata H. Yoshioka H. Fujita K. Eur. J. Pediatr. 1985; 144: 186-190Crossref PubMed Scopus (162) Google Scholar, 15Yoshioka H. Iseki K. Fujita K. Pediatrics. 1983; 72: 317-321PubMed Google Scholar). However, prominent obligate anaerobes that are members of the adult microbiota have been identified as early as postnatal day 3 in vaginally delivered infants (14Sakata H. Yoshioka H. Fujita K. Eur. J. Pediatr. 1985; 144: 186-190Crossref PubMed Scopus (162) Google Scholar, 16George M. Nord K.E. Ronquist G. Hedenstierna G. Wiklund L. Ups. J. Med. Sci. 1996; 101: 233-250Crossref PubMed Scopus (15) Google Scholar), suggesting that adult-associated species establish an early presence in this environment. In the present study, we address the question of how dominant members of the adult microbiota are able to persist during the suckling period so that they can expand to levels of prominence after the transition from a diet of mother's milk to one rich in plant-derived complex polysaccharides. For this analysis, we selected Bacteroides thetaiotaomicron, a Gram-negative anaerobe that comprised 6% of all bacteria and 12% of all Bacteroidetes in the most comprehensive 16 S rRNA sequence-based enumeration of the adult human colonic microbiota published to date (3Eckburg P.B. Bik E.M. Bernstein C.N. Purdom E. Dethlefsen L. Sargent M. Gill S.R. Nelson K.E. Relman D.A. Science. 2005; 308: 1635-1638Crossref PubMed Scopus (5278) Google Scholar). One indication of B. thetaiotaomicron's adaptation to life in the adult gut is its large collection of genes dedicated to the acquisition and metabolism of plant and other dietary polysaccharides encountered in our postweaning diets (17Xu J. Bjursell M.K. Himrod J. Deng S. Carmichael L.K. Chiang H.C. Hooper L.V. Gordon J.I. Science. 2003; 299: 2074-2076Crossref PubMed Scopus (991) Google Scholar). The B. thetaiotaomicron genome is predicted to encode 226 glycoside hydrolases and 15 polysaccharide lyases, allowing it to break down polysaccharides that we cannot process on our own because our proteome lacks the requisite enzymes (e.g. B. thetaiotaomicron contains 64 arabinosidases, xylanases, and pectate lyases, whereas we have none; see Carbohydrate Active Enzymes data base (CAZy) 3The abbreviations used are: CAZy, Carbohydrate Active Enzymes; ECF, extracytoplasmic function; Pn, postnatal day n; COG, clusters of orthologous groups; GH, glycoside hydrolase; PL, polysaccharide lyase; CPS, capsular polysaccharide synthesis.3The abbreviations used are: CAZy, Carbohydrate Active Enzymes; ECF, extracytoplasmic function; Pn, postnatal day n; COG, clusters of orthologous groups; GH, glycoside hydrolase; PL, polysaccharide lyase; CPS, capsular polysaccharide synthesis. at afmb.cnrs-mrs.fr/CAZY/for a comprehensive annotation). The B. thetaiotaomicron genome also contains 209 paralogs of two cell surface proteins that bind starch (107 paralogs of SusC; 102 paralogs of SusD). SusC paralogs are predicted TonB-dependent, β-barrel-type outer membrane proteins; thus, in addition to binding polysaccharides, they likely participate in energy-dependent transport of these carbohydrates into the periplasmic space (18Reeves A.R. D'Elia J.N. Frias J. Salyers A.A. J. Bacteriol. 1996; 178: 823-830Crossref PubMed Scopus (117) Google Scholar). SusD paralogs are predicted to be secreted proteins with an N-terminal lipid tail that allows them to associate with the outer membrane (19Shipman J.A. Berleman J.E. Salyers A.A. J. Bacteriol. 2000; 182: 5365-5372Crossref PubMed Scopus (158) Google Scholar). B. thetaiotaomicron also contains an expanded set of environmental sensors and regulators that includes 50 extracytoplasmic function (ECF) σ factors and 26 anti-σ factors, 79 members of classic two-component systems, plus 32 novel “hybrid” two-component systems that incorporate all of the domains encountered in a two-component system into a single polypeptide (17Xu J. Bjursell M.K. Himrod J. Deng S. Carmichael L.K. Chiang H.C. Hooper L.V. Gordon J.I. Science. 2003; 299: 2074-2076Crossref PubMed Scopus (991) Google Scholar). At least some of the latter proteins are used by B. thetaiotaomicron to sense polysaccharides and coordinately regulate genes involved their metabolism (20Sonnenburg E.D. Sonnenburg J.L. Manchester J.K. Hansen E.E. Chiang H.C. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 8834-8839Crossref PubMed Scopus (110) Google Scholar). Genes encoding SusC and SusD paralogs are typically positioned adjacent to one another in the B. thetaiotaomicron genome (102 of 107 loci containing susC paralogs) and are often part of larger gene clusters encoding enzymes involved in carbohydrate metabolism (62 of 107 loci) (17Xu J. Bjursell M.K. Himrod J. Deng S. Carmichael L.K. Chiang H.C. Hooper L.V. Gordon J.I. Science. 2003; 299: 2074-2076Crossref PubMed Scopus (991) Google Scholar). At least 18 of the 62 clusters that include glycoside hydrolases also contain ECF-type σ factors and adjacent anti-σ factors. This arrangement suggests that the polysaccharide binding, import, and catabolism functions represented in these clusters are co-regulated to allow for efficient, prioritized, and adaptive foraging of glycans based on their availability. Our analysis, described below, of the adaptations of B. thetaiotaomicron to the suckling period in a simplified gnotobiotic mouse model supports this view: it emphasizes the importance of host mucosal polysaccharides in supporting B. thetaiotaomicron during the suckling period (habitat effect) and links the evolution of these polysaccharide utilization clusters to their coordinate regulation. Mother-to-Pup Transmission of B. thetaiotaomicron—All of the experimental manipulations involving mice used protocols approved by the Washington University Animal Studies Committee. Mice belonging to the NMRI-KI inbred line were reared in gnotobiotic isolators (21Hooper L.V. Midtvedt T. Gordon J.I. Annu. Rev. Nutr. 2002; 22: 283-307Crossref PubMed Scopus (1147) Google Scholar) under a strict 12-h light cycle (lights on at 6:00 a.m.) and given unlimited access to autoclaved water and an autoclaved standard polysaccharide-rich chow diet (B&K Universal, Grimston, UK). In a given experiment, one male and two pregnant female NMRI mice were gavaged with 100 μl of an overnight culture of B. thetaiotaomicron strain VPI-5482 (ATCC 29148) containing 108 colony-forming units in TYG medium (20Sonnenburg E.D. Sonnenburg J.L. Manchester J.K. Hansen E.E. Chiang H.C. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 8834-8839Crossref PubMed Scopus (110) Google Scholar). Littermates were sacrificed at postnatal day 17 (P17) and P30. The stomachs of all P17 animals were surveyed to verify that there were no chow particles present. The density of bacterial colonization of the cecum and colon was determined based on colony-forming units and quantitative real time PCR assays (20Sonnenburg E.D. Sonnenburg J.L. Manchester J.K. Hansen E.E. Chiang H.C. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 8834-8839Crossref PubMed Scopus (110) Google Scholar, 22Samuel B.S. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 10011-10016Crossref PubMed Scopus (461) Google Scholar). Whole Genome Transcriptional Profiling of B. thetaiotaomicron in Vivo—Ceca from P17 mice were snap frozen in liquid nitrogen. After thawing in RNAprotect solution (1 ml/sample; Qiagen), each cecum was cut open and vortexed for 10 s at 25 °C to separate luminal contents from the mucosa, and the mixture was centrifuged (1,200 × g for 10 s) to remove tissue fragments. The resulting supernatants were subjected to a bacterial cell lysis protocol described below. The contents were removed from P30 ceca by manual extrusion from their distal ends and snap frozen in liquid nitrogen. The samples were later thawed in 1 ml of RNAprotect prior to lysis. Cecal samples from P17 mice were lysed in TE buffer (10 mm Tris, 1 mm EDTA, pH 8.0) containing 1 mg/ml of lysozyme (specific activity 50,000 units/mg; Sigma; 10 min of incubation at room temperature). Cecal samples from P30 mice were combined with 500 μl of 200 mm NaCl/20 mm EDTA, and the cells were lysed by bead beating (6Ley R.E. Backhed F. Turnbaugh P. Lozupone C.A. Knight R.D. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 11070-11075Crossref PubMed Scopus (4137) Google Scholar). Total RNA was isolated and prepared for hybridization to custom B. thetaiotaomicron GeneChips according to Sonnenburg et al. (1Sonnenburg J.L. Xu J. Leip D.D. Chen C.H. Westover B.P. Weatherford J. Buhler J.D. Gordon J.I. Science. 2005; 307: 1955-1959Crossref PubMed Scopus (795) Google Scholar). 4All of the GeneChip data used in this study are available from the Gene Expression Omnibus data base (www.ncbi.nlm.nih.gov/projects/geo/) under accession number GSE279. Microarray Suite 5 software (Affymetrix) was used for initial data analysis. Selection criteria for identifying genes that were differentially expressed between P17 and P30 ceca were 100% present calls for the condition where the average signal for a given probe set was higher, and a false discovery rate cut-off of 1% based on significance analysis of microarrays (v2.20) analysis of the data set (23Tusher V.G. Tibshirani R. Chu G. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5116-5121Crossref PubMed Scopus (9648) Google Scholar). Genes satisfying these criteria were categorized using COGs (clusters of orthologous groups) (23Tusher V.G. Tibshirani R. Chu G. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5116-5121Crossref PubMed Scopus (9648) Google Scholar, 24Tatusov R.L. Koonin E.V. Lipman D.J. Science. 1997; 278: 631-637Crossref PubMed Scopus (2715) Google Scholar). Hypergeometric distribution was used to identify significantly overrepresented COGs. Bacterial genes whose expression changed at P17 compared with P30 or vice versa were also searched against (i) the CAZy data base to identify enzymatic activities related to polysaccharide metabolism and (ii) MetaCyc (25Caspi R. Foerster H. Fulcher C.A. Hopkinson R. Ingraham J. Kaipa P. Krummenacker M. Paley S. Pick J. Rhee S.Y. Tissier C. Zhang P. Karp P.D. Nucleic Acids Res. 2006; 34: 511-516Crossref PubMed Scopus (283) Google Scholar) to place genes onto known metabolic and signaling pathways. GeneChip Profiling of B. thetaiotaomicron during Growth in Vitro—An overnight 10-ml culture of B. thetaiotaomicron, grown in an aqueous solution of 10% tryptone (Becton Dickinson Co.) and 1.9 μm hematin (Sigma), was introduced into the 1.2-liter reaction vessel of a Bioflo 110 batch culture fermentor (New Brunswick Scientific) containing 800 ml of the same 10% tryptone medium. The bacteria were then incubated at 37 °C with agitation at 100 rpm under an atmosphere of 20% CO2, 80% N2 (sparged at 0.1 liter/min). During mid-log phase (A600 = 0.3), lactose, glucose, or galactose was added to a final concentration of 0.2%. Aliquots of the culture (10 ml) were removed immediately before and 60 min after carbohydrate addition and placed in RNAprotect. Bacterial cells were collected by centrifugation (3,000 × g for 15 min at 4 °C), and the samples were prepared for hybridization to B. thetaiotaomicron GeneChips as above (1Sonnenburg J.L. Xu J. Leip D.D. Chen C.H. Westover B.P. Weatherford J. Buhler J.D. Gordon J.I. Science. 2005; 307: 1955-1959Crossref PubMed Scopus (795) Google Scholar). Biochemical Assays of Cecal Contents—Gas chromatography-mass spectrometry analysis of cecal contents was carried out as detailed in Ref. 1Sonnenburg J.L. Xu J. Leip D.D. Chen C.H. Westover B.P. Weatherford J. Buhler J.D. Gordon J.I. Science. 2005; 307: 1955-1959Crossref PubMed Scopus (795) Google Scholar. Other biochemical assays were conducted on cecal contents that had been freeze-dried and resuspended in 400 μl of ice-cold buffer containing 0.2 m NaOH and 1mm EDTA. Aliquots (80 μl) of this mixture were removed, and an alkaline extract was prepared by incubation at 80 °C for 20 min, followed by placement on ice and neutralization (80 μl of a solution containing 0.25 m HCl and 100 mm Tris base). An acid extract was prepared by taking another 60-μl aliquot, adding 20 μl of 0.7 m HCl, incubating the material for 20 min at 80 °C, placing the solution back on ice, and adding 40 μl of Tris Base. All of the extracts were stored at -80 °C. The levels of ATP and NADH were measured in the alkaline extracts, whereas glucose, galactose, lactose, and NAD+ were assayed in acid extracts using well established pyridine nucleotide-based enzyme cycling methods (26Passoneau J.V. Lowry O.H. Enzymatic Analysis: A Practical Guide. Humana Press, Totowa, NJ1993: 85-228Crossref Google Scholar). The data are expressed relative to the protein content of the cecal sample (Bradford assay; Bio-Rad). ClustalW Analysis of SusC/SusD Paralogs—Pairs of genes encoding SusC and SusD paralogs were identified in the genome sequences of B. thetaiotaomicron VPI-5482 (17Xu J. Bjursell M.K. Himrod J. Deng S. Carmichael L.K. Chiang H.C. Hooper L.V. Gordon J.I. Science. 2003; 299: 2074-2076Crossref PubMed Scopus (991) Google Scholar), Bacteroides fragilis NCTC 9343 (27Cerdeno-Tarraga A.M. Patrick S. Crossman L.C. Blakely G. Abratt V. Lennard N. Poxton I. Duerden B. Harris B. Quail M.A. Barron A. Clark L. Corton C. Doggett J. Holden M.T. Larke N. Line A. Lord A. Norbertczak H. Ormond D. Price C. Rabbinowitsch E. Woodward J. Barrell B. Parkhill J. Science. 2005; 307: 1463-1465Crossref PubMed Scopus (220) Google Scholar), B. fragilis YCH46 (28Kuwahara T. Yamashita A. Hirakawa H. Nakayama H. Toh H. Okada N. Kuhara S. Hattori M. Hayashi T. Ohnishi Y. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 14919-14924Crossref PubMed Scopus (180) Google Scholar), and Porphyromonas gingivalis (29Hall L.M. Fawell S.C. Shi X. Faray-Kele M.C. Aduse-Opoku J. Whiley R.A. Curtis M.A. Infect. Immun. 2005; 73: 4253-4262Crossref PubMed Scopus (37) Google Scholar) by performing individual BLAST searches against each genome using amino acid sequences of previously annotated SusC and SusD paralogs as queries (30Reeves A.R. Wang G.R. Salyers A.A. J. Bacteriol. 1997; 179: 643-649Crossref PubMed Scopus (147) Google Scholar). The low scoring hits from each search (E values between 10-4 and 10-10) were themselves used as BLAST queries to reveal more divergent putative paralogs in each genome; this process was repeated until no new paralogs were identified. Lists of putative SusC and SusD paralogs were compared for each species. Paralogs were included in a subsequent clustalW analysis based on the requirement that each had a separately predicted, adjacent partner (this process was instrumental in excluding related TonB-dependent hemin, vitamin B12, and iron-siderophore receptors from the list of putative SusC paralogs). The resulting data set included 240 paralog pairs: 102 pairs in B. thetaiotaomicron, 69 pairs in B. fragilis NCTC 9343, 65 pairs in B. fragilis YCH46, and four pairs in P. gingivalis. Multiple sequence alignments were generated for SusC and for SusD paralogs using clustalW (31Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (54908) Google Scholar). Tree determination (neighbor-joining method) and bootstrapping were performed using Paup 4.0b10 (32Swofford D.L. PAUP: Phylogenetic Analysis Using Parsimony (and Other Methods). Sinauer Associates, Sunderland, MA2002Google Scholar), based on the clustalW alignment. Cladogram trees were drawn from the consensus of 100 trees that were generated for each alignment, with P. gingivalis RagA/RagB (homologs of SusC/SusD) proteins selected as arbitrary roots. Branches with bootstrap values ≥70% were retained. Comparison of the SusC and the SusD cladograms suggested that these two protein families resolve into nearly identical branches (supplemental Table S1), supporting the notion that they diverged in parallel with one another during their expansion in these genomes: 141/240 SusC paralogs (58.8%) were resolved into 24 different clades exhibiting 100% bootstrap values; 139 of these (98.6%) had SusD partners that were resolved into similar (2 of 24) or identical (22 of 24) clades in a SusD cladogram (data not shown). Because of this observation and to increase the resolving power of our cladogram analysis, the predicted amino acid sequences of each protein encoded by a given susC/susD pair were joined in silico to generate a single amino acid sequence for each pair and then entered into clustalW for neighbor-joining alignment as described above. B. thetaiotaomicron Transfer from Gnotobiotic Mothers to Their Offspring—Pregnant 10-14-week-old germ-free mice belonging to the NMRI inbred strain were inoculated with B. thetaiotaomicron on the 10th day of gestation. After they gave birth, a subset of suckling pups in each litter was sacrificed at P17. Another subset was killed at P30, after weaning to a standard autoclaved polysaccharide-rich chow diet (initiated at P20). Levels of colonization were quantified in the cecum, an anatomically distinct structure interposed between the distal small intestine and colon that allows for ready and reliable harvest of luminal contents, and in the colon. B. thetaiotaomicron colonized all of the pups surveyed. P17 pups contained 7.03 ± 1.49 × 1011 and 1.39 ± 0.38 × 1012 colony-forming units/ml of cecal and colonic contents, respectively. The corresponding values for P30 mice were 1.42 ± 0.17 × 1012 and 1.78 ± 0.43 × 1012 colony-forming units/ml (means ± S.E., n = 11 litters, 2-10 mice/litter; p < 0.01 for P17 versus P30 cecum; no significant difference (p > 0.05) for colon)). Thus, in the absence of competitors, B. thetaiotaomicron can reliably colonize the suckling mouse intestine at densities that are only slightly lower in the cecum compared with the colon. The levels of colonization in these regions of the distal gut are similar to what we have observed in adult mice who were gavaged once with the same size inoculum at 6-10 weeks of age and then sacrificed 2 or 4 weeks later (1Sonnenburg J.L. Xu J. Leip D.D. Chen C.H. Westover B.P. Weatherford J. Buhler J.D. Gordon J.I. Science. 2005; 307: 1955-1959Crossref PubMed Scopus (795) Google Scholar). In Vivo Transcriptional Profiling of B. thetaiotaomicron at P17 and P30—To gain insight into how B. thetaiotaomicron adapts to the cecal habitats of suckling and weaned mice, we performed whole genome transcriptional profiling of the bacterium using GeneChips containing probe sets representing 98.6% of its 4,779 predicted protein-coding genes (1Sonnenburg J.L. Xu J. Leip D.D. Chen C.H. Westover B.P. Weatherford J. Buhler J.D. Gordon J.I. Science. 2005; 307: 1955-1959Crossref PubMed Scopus (795) Google Scholar). RNA was isolated from cecal contents and then pooled from three to four P17 pups from each litter (pooling was necessary to obtain sufficient material) or from cecal contents harvested from individual littermates that had been allowed to live until P30. Individual RNA samples from six P30 animals, representing three different litters, and six pools of RNA from twenty-three P17 animals, representing five litters, were used as templates for synthesis of cDNA targets; each of the 12 targets was hybridized to a separate B. thetaiotaomicron GeneChip. Analysis of the resulting GeneChip data sets using significance analysis of microarrays software (23Tusher V.G. Tibshirani R. Chu G. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5116-5121Crossref PubMed Scopus (9648) Google Scholar) and the selection criteria described under “Experimental Procedures” identified 1,266 genes whose expression was defined as significantly different between the two ages; 583 were higher in B. thetaiotaomicron colonizing the ceca of P17 compared with P30 mice, whereas 683 were expressed at higher levels at P30 (for a list, see supplemental Table S2). Differentially expressed genes were then grouped according to their COG assignments (supplemental Fig. S1). By comparing the percentage representation of the groups of regulated genes in a given COG, with the percentage representation all B. thetaiotaomicron genes in that COG, we determined that four COGs were significantly “overrepresented” among genes that were differentially expressed in either condition (p < 0.05). “Translation” (COG J) was the most ov