Intracellular glycosidases of human colon Bacteroides ovatus B4-11

Activity of various glycosidases in the intracellular enzyme fraction of Bacteroides ovatus B4-11 was investigated. During 120 h of incubation at 37 degrees C, ca. 30% of the crude hemicellulose was hydrolyzed by an intracellular enzyme fraction of strain B4-11. Xylose was the major sugar released from crude hemicellulose. Glycosidases (alpha-1,6-glucosidase, alpha-1,4-glucosidase, beta-1,4-glucosidase, and beta-1,4-xylosidase) were induced in B. ovatus B4-11 by crude hemicellulose and heteroxylan. When B. ovatus B4-11 was grown on either crude hemicellulose or heteroxylan, the predominant enzyme in the intracellular enzyme fraction was beta-1,4-xylosidase.     

Large transmissible clindamycin resistance plasmid in Bacteroides ovatus.

Bacteroides ovatus IB106 contained two plasmids, pBI106 (46 kilobases) and pBI136 (82 kilobases). Transmissible clindamycin-erythromycin resistance (Ccr) was mediated by pBI136 , whose Ccr determinant was closely related to the determinant on the Bacteroides R plasmids pBF4 and pBFTM10 . Hybridization studies showed that pBI106 was not involved in Ccr transfer, but it shared extensive homology to pBF4 with the exception of the pBF4 region implicated in Ccr.     

Intracellular polysaccharide of Bacteroides fragilis.

Formation of iodophilic polysaccharide (IPS) from glucose was demonstrated in 27 strains of Bacteroides fragilis. Synthesis was dependent on the glucose concentration of the medium, the pH and the growth phase. When glucose was in short supply the cellular polysaccharide was degraded rapidly at pH 4.5 to 6.5 and fatty acids accumulated in the medium. Storage of IPS was not responsible for the low carbon recoveries observed in fermentation balance studies. In electron micrographs of thin sections, the IPS was observed as cytoplasmic granules dispersed throughout the whole cell. After extraction and purification the IPS was characterized as a glycogen.     

Formation of glycosidases in batch and continuous culture of Bacteroides fragilis.

Nine strains of bacteroides fragilis were cultivated in stirred fermentors and tested for their ability to produce glycosidases. B. fragilis subsp. vulgatus B70 was used for optimizing the production of glycosidases. The highest bacterial yield was obtained in proteose peptone-yeast extract medium. The optimum pH for maximal bacterial yield was 7.0, and the optimum temperature for growth was 37 degrees C. The formation of glycosidases was optimal between pH 6.5 and 7.5, and the optimum temperature for synthesis of glycosidases was between 33 and 37 degrees C. Culture under controlled conditions in fermentors gave more reproducible production of glycosidases than static cultures in bottles. The strain was also grown in continuous culture at a dilution rate of 0.1 liter/h at pH 7.0 and 37 degrees C with a yield of 2.0 mg of dry weight per ml in the complex medium. The formation of glycosidases remained constant during the entire continuous process.     

Formation of glycosidases in batch and continuous culture of Bacteroides fragilis.

Nine strains of bacteroides fragilis were cultivated in stirred fermentors and tested for their ability to produce glycosidases. B. fragilis subsp. vulgatus B70 was used for optimizing the production of glycosidases. The highest bacterial yield was obtained in proteose peptone-yeast extract medium. The optimum pH for maximal bacterial yield was 7.0, and the optimum temperature for growth was 37 degrees C. The formation of glycosidases was optimal between pH 6.5 and 7.5, and the optimum temperature for synthesis of glycosidases was between 33 and 37 degrees C. Culture under controlled conditions in fermentors gave more reproducible production of glycosidases than static cultures in bottles. The strain was also grown in continuous culture at a dilution rate of 0.1 liter/h at pH 7.0 and 37 degrees C with a yield of 2.0 mg of dry weight per ml in the complex medium. The formation of glycosidases remained constant during the entire continuous process.     

Characterization of an outer membrane mannanase from Bacteroides ovatus

Bacteroides ovatus utilizes guar gum, a high-molecular-weight branched galactomannanan, as a sole source of carbohydrate. No extracellular activity was detectable. Approximately 30% of the total cell-associated mannanase activity partitioned with cell membranes. When inner and outer membranes of B. ovatus were separated on sucrose gradients, the mannanase activity was associated mainly with fractions containing outer membranes. Enzyme activity was solubilized by 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) or by Triton X-100 at a detergent-to-protein ratio of 1:1. The enzyme was stable for only 4 h at 37 degrees C and for 50 to 60 h at 4 degrees C. Analysis of the products of the CHAPS-solubilized mannanase on Bio-Gel A-5M and Bio-Gel P-10 gel filtration columns indicated that the enzyme breaks guar gum into high-molecular-weight fragments. The CHAPS-solubilized mannanase was partially purified by chromatography on a FPLC Mono Q column. The partially purified mannanase preparation contained three major polypeptides (Mr 94,500, 61,000, and 43,000) and several minor ones. High mannanase activity was seen only when B. ovatus was grown on guar gum. Cross-absorbed antiserum detected two other guar gum-associated outer membrane proteins: a CHAPS-extractable 49,000-dalton polypeptide and a 120,000-dalton polypeptide that was not solubilized by CHAPS. Neither of these polypeptides was detectable in the partially purified mannanase preparation. These results indicate that there are at least two guar gum-associated outer membrane polypeptides other than the mannanase.     

Laminarinase (beta-glucanase) activity in Bacteroides from the human colon.

Laminarin, a beta(1 leads to 3)-glucan similar to those found in plant cell walls, is fermented by some species of anaerobic bacteria from the human colon. Laminarinase (EC 3.2.1.6) and beta-glucosidase (EC 3.2.1.21) activities were determined in strains representing Bacteroides thetaiotaomicron, Bacteroides distasonis, and an unnamed deoxyribonucleic acid homology group of Bacteroides fragilis. In all three species, laminarinase activity was inducible by laminarin and was predominantly cell bound. The products of laminarinase activity varied with each species. In the case of B. thetaiotaomicron, the major product of laminarin hydrolysis was glucose (70 to 90%), and there were small amounts of laminaribiose (G2) and oligomers of glucose as high as G4. In the case of group '0061-1,' glucose (40 to 50%) and oligomers of glucose as high as G6 were found. The laminarinase of B. distasonis differed from the laminarinases of the other two species in that it mainly produced oligomers of glucose (G2-G5). beta-Glucosidase activity was also found in all three species. beta-Glucosidase was induced by glucose-containing disaccharides as well as by laminarin. The beta-glucosidases of the three Bacteroides species differed with respect to level of activity, induction pattern, and sensitivity to inhibition by D-glucono-1,5-lactone.     

Intracellular activity and secretion of various lysosomal glycosidases in cultured human skin fibroblasts

The intracellular activities of four lysosomal glycosidases (alpha-L-fucosidase, beta-D-hexosaminidase, beta-D-galactosidase and beta-D-glucuronidase) in human skin fibroblasts cultured in a medium with 0.1% serum increased in a greater degree than that in a medium with 10% serum. Only two glycosidases (alpha-L-fucosidase and beta-D-hexosaminidase) were secreted by fibroblasts in the culture medium. The extracellular activity of alpha-L-fucosidase and beta-D-hexosaminidase was equivalent to 80 and 25% of their intracellular activity in serum-sufficient fibroblasts and 40 and 15%--in serum-restricted fibroblasts. These results suggest that the observer phenomena are controlled by the levels of autophagy, endocytosis and membrane recycling.     

Purification and characterization of a cell-associated, soluble mannanase from Bacteroides ovatus.

Bacteroides ovatus, a human colonic anaerobe, utilizes the galactomannan guar gum as a sole source of carbohydrate. Previously, we found that none of the galactomannan-degrading enzymes were extracellular, and we characterized an outer membrane mannanase which hydrolyzes the backbone of guar gum to produce large fragments. We report here the purification and characterization of a second mannanase from B. ovatus. This enzyme is cell-associated and soluble. Using ion-exchange chromatography, gel filtration, and chromatofocusing steps, we have purified the soluble mannanase to apparent homogeneity. The enzyme has a native molecular weight of 190,000 and a monomeric molecular weight of 61,000. It is distinct from the membrane mannanase not only with respect to cellular location but also with respect to stability and isoelectric point (pI of 6.9 for the membrane mannanase and pI of 4.8 for the soluble mannanase). The soluble mannanase, like the membrane mannanase, hydrolyzed guar gum to produce large fragments rather than monosaccharides. However, if galactosyl side chains were removed from the galactomannan fragments by alpha-galactosidase, both the soluble mannanase and the membrane mannanase could degrade guar gum to monosaccharides. Thus either or both of these two enzymes, working together with alpha-galactosidase, appear to be sufficient for the breakdown of guar gum to the level of monosaccharides.     

Production of mucin degrading sulphatase and glycosidases by Bacteroides thetaiotaomicron

Bacteroides thetaiotaomicron NCTC 10582 grown in media containing pig gastric mucin was found to be capable of producing all the glycosidases required to degrade the carbohydrate moieties of human colonic mucin. These are α-fucosidase, β -galactosidase, α- N -acetylgalactosaminidase, β-N -acetylglucosaminidase and neuraminidase. Moreover, a novel glycosulphatase was identified using glucose-6-sulphate as substrate. This enzyme has a Km of 43·4 mmol/l and a pH optimum of 5·0. The bacteria, when cultured for 24 h in broth, were capable of removing 18% of [³⁵S]-sulphate from [³⁵S]-labelled mucin and of removing 15% of [³H]-glucosamine from [³H]-glucosamine-labelled human colonic mucin. The results suggest that this bacterium is likely to play an important role in mucus degradation in the human colon.     

Characterization of a Metal-independent CAZy Family 6 Glycosyltransferase from Bacteroides ovatus

The myriad functions of complex carbohydrates include modulating interactions between bacteria and their eukaryotic hosts. In humans and other vertebrates, variations in the activity of glycosyltransferases of CAZy family 6 generate antigenic variation between individuals and species that facilitates resistance to pathogens. The well characterized vertebrate glycosyltransferases of this family are multidomain membrane proteins with C-terminal catalytic domains. Genes for proteins homologous with their catalytic domains are found in at least nine species of anaerobic commensal bacteria and a cyanophage. Although the bacterial proteins are strikingly similar in sequence to the catalytic domains of their eukaryotic relatives, a metal-binding Asp-X-Asp sequence, present in a wide array of metal ion-dependent glycosyltransferases, is replaced by Asn-X-Asn. We have cloned and expressed one of these proteins from Bacteroides ovatus, a bacterium that is linked to inflammatory bowel disease. Functional characterization shows it to be a metal-independent glycosyltransferase with a 200-fold preference for UDP-GalNAc as substrate relative to UDP-Gal. It efficiently catalyzes the synthesis of oligosaccharides similar to human blood group A and may participate in the synthesis of the bacterial O-antigen. The kinetics for GalNAc transfer to 2′-fucosyl lactose are characteristic of a sequential mechanism, as observed previously for this family. Mutational studies indicate that despite the lack of a metal cofactor, there are pronounced similarities in structure-function relationships between the bacterial and vertebrate family 6 glycosyltransferases. These two groups appear to provide an example of horizontal gene transfer involving vertebrates and prokaryotes.The structures of complex glycans are determined by the specificities of the glycosyltransferases (GTs)² that catalyze their biosynthesis. GTs fall into two groups that differ in mechanism, based on whether the anomeric configuration of the donor substrate (α for most UDP-sugars) is retained or inverted in the product (1–3). They are classified into 90 different families in the CAZy data base based on sequence similarities (4, 5), but the majority of those that have been structurally characterized fall into one of two fold types, designated GT-A and GT-B (2). The retaining GTs of CAZy family 6 (GT6) have a GT-A fold and catalyze the transfer of either galactose or GalNAc into an α-linkage with the 3-OH group of β-linked galactose or GalNAc. GT6 includes the histo-blood group A and B GTs (GTA and GTB), the α-galactosyltransferase (α3GT) that catalyzes the synthesis of the xenoantigen or α-gal epitope, Forssman glycolipid synthase, isogloboside 3 synthase, and their homologues from other vertebrates (6). GT6 enzymes from vertebrates are type-2 membrane proteins with N-terminal cytosolic domains, a transmembrane helix, a spacer, and a C-terminal catalytic domain (6). Crystallographic studies of recombinant catalytic domains of GTA, GTB, and α3GT have provided detailed information about their interactions with substrates, metal cofactor, and inhibitors (7–9). Most GT-A fold GTs, including those in the GT6 family, require divalent metal ions, such as Mn²⁺, for catalytic activity; their metal dependence is linked to a shared DXD sequence motif. Residues of this motif interact with the metal ion and both the ribose and phosphates of the donor substrate to produce an appropriate substrate orientation and conformation for catalysis and to stabilize the UDP leaving group (3, 7–10).Mammalian members of GT6 are responsible for variations in glycan structures between different species and individuals as the result of selective enzyme inactivation in certain species (α3GT, Forssman glycolipid synthase, and isogloboside 3 synthase) or the inheritance of multiple alleles at one locus that encode enzymes with different substrate specificity (GTA and GTB) or are inactive (11–14). The presence of circulating antibodies against glycan structures that are subject to interspecies and individual variability has been linked to resistance to pathogens that also carry the glycans; these antibodies are thought to arise from exposure to potential pathogens, including enveloped viruses and bacteria that carry structurally similar glycans (11).In addition to the well characterized enzymes discussed previously, atypical members of the GT6 family have been identified in mammals that have sequence changes in highly conserved regions of the active site, including the DXD motif (6). However, no glycosyltransferase activity was detected in recombinant forms of two of these, and their functions are unclear (6). Although GT6 members are widely distributed among vertebrates, no homologues have been found in other eukaryotes (6). However, GT6 members have been identified in several bacterial species (15–17). GT6 enzymes from Escherichia coli O86, and Helicobacter mustelae that appear to function in the biosynthesis of the lipopolysaccharide O-antigen have been cloned and expressed by Wang and co-workers (16, 17) and found to have specificities similar to those of human GTB and GTA, respectively. These enzymes have been applied in the enzymatic synthesis of oligosaccharides. Other homologues are encoded by Hemophilus somnus, Psychroacter sp., PRwf-1 (15), Francisella philomiragia, and three Bacteroides species, Bacteroides ovatus, Bacteroides caccae, and Bacteroides stercoris, as well as a cyanophage, PSSM-2 (15). Genes for other homologues from unidentified species are present in the marine metagenome (18, 19) and human gut metagenome (20, 21). The phage and bacterial enzymes are substantially truncated at the N terminus relative to the catalytic domains of vertebrate GT6 representatives and are smaller than the reported minimal functional unit of a primate α3GT (22). When bacterial and vertebrate GT6 amino acid sequences are aligned (Fig. 1 and supplemental Figs. S1 and S2), it can be seen that the metal-binding DXD of the eukaryotic GTs is replaced by NXN (where X is Ala, Gly, or Ser) in the bacterial homologues. The cyanophage GT6 member and related proteins in the marine metagenome, however, retain the DXD motif. This conspicuous difference in the bacterial proteins is particularly interesting, since, in the mammalian enzymes, the aspartates of the DXD and adjacent residues are crucial for catalytic activity (10, 23).Open in a separate windowFIGURE 1.An alignment of selected bacterial, cyanophage and mammalian GT6 amino acid sequences. Abbreviations and Interpro sequence IDs (in parentheses) are as follows. HuA, human histo-blood group A synthase (A1EAJ6); Bova, bovine α1,3-galactosyltransferase ({"type":"entrez-protein","attrs":{"text":"P14769","term_id":"120970","term_text":"P14769"}}P14769); PSSM2, cyanophage PSSM-2 ({"type":"entrez-protein","attrs":{"text":"Q58M87","term_id":"75096044","term_text":"Q58M87"}}Q58M87); Bs, B. stercoris (B0NSM3); Bo1, B. ovatus GT1 (A7LVT2); Bo2, B. ovatus GT2 (A7M0P3); Bc, B. caccae (A5ZC71). The boxed regions in the alignment identify regions that have been shown to be involved in interactions with substrates and cofactor and in catalysis in bovine α1,3-galactosyltransferase and histo-blood group A and B enzymes. These are labeled (below) as follows. A, interactions with uracil; B, interactions with the galactose moiety of UDP-Gal; C, interactions with Mn²⁺, phosphates, and galactose; D, interactions with acceptor substrate; E, interactions with Gal or GalNAc of donor substrate; F, interactions with monosaccharide of donor substrate and acceptor and catalysis; The arrow (above) denotes the intron/exon boundary in vertebrate GT6s, and the asterisks indicate the residues in BoGT6a that were subjected to mutagenesis.B. ovatus is a Gram-negative commensal bacterium that inhabits the distal mammalian gut and has been implicated in the pathology of inflammatory bowel disease in humans (24). The B. ovatus genome contains two genes that encode GT6 representatives (Fig. 1). We selected one of these for initial investigation, and designate it BoGT6a (family 6 glycosyltransferase 1 of Bacteroides). The gene for this protein was amplified by PCR and cloned and expressed in His-tagged form in E. coli BL21(DE3). Assays with a variety of substrates show that its substrate specificity is similar to that of human GTA. Previous studies of the activities of bacterial enzymes were conducted in the presence of Mn²⁺ (16, 17), but we find that the B. ovatus enzyme does not require divalent metal ions for activity and is fully active in EDTA. Despite this striking difference, BoGT6a is similar to its metal-dependent relatives in catalytic properties; also, the effects of amino acid substitutions for residues corresponding to several that act in substrate binding and catalysis in vertebrate GT6 glycosyltransferases suggest that they have similar structure-function relationships. These results indicate that the metal cofactor is not a conserved feature in the GT6 family. They also raise questions about the catalytic mechanism of prokaryotic GT6 members and the evolutionary relationship between bacterial, phage, and vertebrate enzymes.     

Characterization of Bacteroides ovatus plasmid pBI136 and structure of its clindamycin resistance region.

Genetic and physical analyses were used to characterize the Bacteroides ovatus R plasmid pBI136. Results from restriction endonuclease cleavage studies were used to construct a physical map of the plasmid for the enzymes EcoRI, BamHI, ClaI, XbaI, SalI, and SmaI. Based on the sizes of restriction fragments generated in these studies, the plasmid was estimated to be 80.6 kilobase pairs (kb). A 7.2-kb region of the plasmid required for resistance to lincosamide and macrolide (LM) antibiotics was mapped by analysis of spontaneously occurring LM-sensitive deletion derivatives. Hybridization studies showed that this region and an adjoining 2.9-kb EcoRI fragment were responsible for the previously reported homology among Bacteroides plasmids pBF4, pBFTM10, and pBI136. Within this region of homology, 0.5 kb was attributed to a directly repeated sequence thought to bound the LM resistance determinant on pBF4 and pBFTM10. Two pBI136 EcoRI fragments spanning the putative LM resistance region were cloned in Escherichia coli, and heteroduplex analysis of these recombinant plasmids revealed the presence of a 1.2-kb directly repeated sequence. These results suggested that the pBI136 LM resistance determinant resides on an 8.4-kb segment of DNA containing 6.0 kb of intervening DNA sequences bounded by a 1.2-kb directly repeated sequence.     

Evidence for extensive resistance gene transfer among Bacteroides spp. and among Bacteroides and other genera in the human colon

Transfer of antibiotic resistance genes by conjugation is thought to play an important role in the spread of resistance. Yet virtually no information is available about the extent to which such horizontal transfers occur in natural settings. In this paper, we show that conjugal gene transfer has made a major contribution to increased antibiotic resistance in Bacteroides species, a numerically predominant group of human colonic bacteria. Over the past 3 decades, carriage of the tetracycline resistance gene, tetQ, has increased from about 30% to more than 80% of strains. Alleles of tetQ in different Bacteroides species, with one exception, were 96 to 100% identical at the DNA sequence level, as expected if horizontal gene transfer was responsible for their spread. Southern blot analyses showed further that transfer of tetQ was mediated by a conjugative transposon (CTn) of the CTnDOT type. Carriage of two erythromycin resistance genes, ermF and ermG, rose from <2 to 23% and accounted for about 70% of the total erythromycin resistances observed. Carriage of tetQ and the erm genes was the same in isolates taken from healthy people with no recent history of antibiotic use as in isolates obtained from patients with Bacteroides infections. This finding indicates that resistance transfer is occurring in the community and not just in clinical environments. The high percentage of strains that are carrying these resistance genes in people who are not taking antibiotics is consistent with the hypothesis that once acquired, these resistance genes are stably maintained in the absence of antibiotic selection. Six recently isolated strains carried ermB genes. Two were identical to erm(B)-P from Clostridium perfringens, and the other four had only one to three mismatches. The nine strains with ermG genes had DNA sequences that were more than 99% identical to the ermG of Bacillus sphaericus. Evidently, there is a genetic conduit open between gram-positive bacteria, including bacteria that only pass through the human colon, and the gram-negative Bacteroides species. Our results support the hypothesis that extensive gene transfer occurs among bacteria in the human colon, both within the genus Bacteroides and among Bacteroides species and gram-positive bacteria.     

Role of starch as a substrate for Bacteroides vulgatus growing in the human colon.

Bacteroides vulgatus is the numerically predominant Bacteroides species in the human colonic microflora. Unlike other colonic Bacteroides species, B. vulgatus is not a versatile utilizer of polysaccharides. The only types of polysaccharide that support rapid growth and high growth yields by all strains are the starches amylose and amylopectin. Amylase and alpha-glucosidase activities are among the highest found in a bacterial fraction obtained from human feces. This observation raised the question of whether B. vulgatus was the source of the fecal enzymes. Both alpha-glucosidase and amylase were produced at 20- to 40-fold-higher levels when B. vulgatus was grown on maltose, amylose, or amylopectin than when B. vulgatus was grown on glucose or other monosaccharides. Both enzymes had the same pI (4.6 to 5.0) and undenatured molecular weight (150,000). The pIs and molecular weights of the B. vulgatus amylase and alpha-glucosidase were the same as those of the fecal enzymes. To determine whether the B. vulgatus alpha-glucosidase was identical to the fecal alpha-glucosidase, we partially purified the B. vulgatus enzyme and raised an antiserum against it. Using this antiserum, we showed that all strains of B. vulgatus produced the same enzyme. The antiserum did not detect the B. vulgatus alpha-glucosidase in the bacterial fraction from human feces, even when a partially purified preparation of the fecal enzyme was used. Thus the alpha-glucosidase activity in the bacterial fraction from human feces is not the B. vulgatus enzyme.     

Fermentation of mucin and plant polysaccharides by strains of Bacteroides from the human colon.

Ten Bacteroides species found in the human colon were surveyed for their ability to ferment mucins and plant polysaccharides ("dietary fiber"). A number of strains fermented mucopolysaccharides (heparin, hyaluronate, and chondroitin sulfate) and ovomucoid. Only 3 of the 188 strains tested fermented beef submaxillary mucin, and none fermented porcine gastric mucin. Many of the Bacteroides strains tested were also able to ferment a variety of plant polysaccharides, including amylose, dextran, pectin, gum tragacanth, gum guar, larch arabinogalactan, alginate, and laminarin. Some plant polysaccharides such as gum arabic, gum karaya, gum ghatti and fucoidan, were not utilized by any of the strains tested. The ability to utilize mucins and plant polysaccharides varied considerably among the Bacteroides species tested.     

Analysis of proteins associated with growth of Bacteroides ovatus on the branched galactomannan guar gum.

Bacteroides ovatus, a gram-negative obligate anaerobe from the human colon, can ferment the branched galactomannan guar gum. Previously, three enzymes involved in guar gum breakdown were characterized. The expression of these enzymes appeared to be regulated; i.e., specific activities were higher in extracts from bacteria grown on guar gum than in extracts from bacteria grown on the monosaccharide constituents of guar gum, mannose and galactose. In the present study, we used two-dimensional gel analysis to determine the total number of B. ovatus proteins enhanced during growth on guar gum. Twelve soluble proteins and 20 membrane proteins were expressed at higher levels in guar gum-grown cells than in galactose-grown cells. An unexpected finding was that the expression of the two galactomannanases was induced by glucose as well as guar gum. Three other proteins, one membrane protein and two soluble proteins, had this same expression pattern. The remainder of the guar gum-associated proteins seen on two-dimensional gels and the guar gum-associated alpha-galactosidase were induced in cells grown on guar gum but not in cells grown on glucose. Two transposon-generated mutants (M-5 and M-7) that could not grow on guar gum were isolated. Both mutants still expressed the galactomannanases and the alpha-galactosidase. They also still expressed all of the guar gum-associated proteins that could be detected in two-dimensional gels of glucose-grown or galactose-grown cells. A second transposon insertion that suppressed the guar gum-negative phenotype of M-5 was isolated and characterized. The characteristics of this suppressor mutant indicated that the original transposon insertion was probably in a regulatory locus.     

Analysis of proteins associated with growth of Bacteroides ovatus on the branched galactomannan guar gum.

Bacteroides ovatus, a gram-negative obligate anaerobe from the human colon, can ferment the branched galactomannan guar gum. Previously, three enzymes involved in guar gum breakdown were characterized. The expression of these enzymes appeared to be regulated; i.e., specific activities were higher in extracts from bacteria grown on guar gum than in extracts from bacteria grown on the monosaccharide constituents of guar gum, mannose and galactose. In the present study, we used two-dimensional gel analysis to determine the total number of B. ovatus proteins enhanced during growth on guar gum. Twelve soluble proteins and 20 membrane proteins were expressed at higher levels in guar gum-grown cells than in galactose-grown cells. An unexpected finding was that the expression of the two galactomannanases was induced by glucose as well as guar gum. Three other proteins, one membrane protein and two soluble proteins, had this same expression pattern. The remainder of the guar gum-associated proteins seen on two-dimensional gels and the guar gum-associated alpha-galactosidase were induced in cells grown on guar gum but not in cells grown on glucose. Two transposon-generated mutants (M-5 and M-7) that could not grow on guar gum were isolated. Both mutants still expressed the galactomannanases and the alpha-galactosidase. They also still expressed all of the guar gum-associated proteins that could be detected in two-dimensional gels of glucose-grown or galactose-grown cells. A second transposon insertion that suppressed the guar gum-negative phenotype of M-5 was isolated and characterized. The characteristics of this suppressor mutant indicated that the original transposon insertion was probably in a regulatory locus.     

Co-utilization of polymerized carbon sources by Bacteroides ovatus grown in a two-stage continuous culture system.

Bacteroides ovatus NCTC 11153 was grown in a two-stage continuous culture system at various growth rates (vessel 1, D = 0.06 to 0.19 h-1; vessel 2, D = 0.03 to 0.09 h-1) on media containing mixtures of starch and arabinogalactan as carbon sources. The cell-associated enzyme activities needed to hydrolyze both substrates (amylase, arabinogalactanase, alpha-glucosidase, beta-galactosidase, and alpha-arabinofuranosidase) were variously influenced by growth rate and polysaccharide availability but were detected under all growth conditions tested. Measurements of residual carbohydrate in spent culture media showed that both polysaccharides were co-utilized during growth under putative C-limited conditions. The arabinogalactan was partly depolymerized in N-limited chemostats, and significant amounts of arabinose- and galactose-containing oligosaccharides accumulated in the cultures, indicating that starch was being preferentially utilized. Acetate, propionate, and succinate were the major fermentation products formed by C-limited bacteria, but under N limitation, lactate was also produced. Molar ratios of succinate increased concomitantly with the dilution rate in C-limited chemostats, whereas molar ratios of propionate decreased. During N-limited growth, however, decarboxylation of succinate to propionate was relatively independent of growth rate. Cell viability was higher in C-limited cultures compared with those grown under N limitation and was greatest at high dilution rates, irrespective of nutrient limitation.     

Genetic analysis of a locus on the Bacteroides ovatus chromosome which contains xylan utilization genes.

Bacteroides ovatus, a gram-negative obligate anaerobe found in the human colon, can utilize xylan as a sole source of carbohydrate. Previously, a 3.8-kbp segment of B. ovatus chromosomal DNA, which contained genes encoding a xylanase (xylI) and a bifunctional xylosidase-arabinosidase (xsa), was cloned, and expression of the two genes was studied in Escherichia coli (T. Whitehead and R. Hespell, J. Bacteriol. 172:2408-2412, 1990). In the present study, we have used segments of the cloned region to construct insertional disruptions in the B. ovatus chromosomal locus containing these two genes. Analysis of these insertional mutants demonstrated that (i) xylI and xsa are probably part of the same operon, with xylI upstream of xsa, (ii) the true B. ovatus promoter was not cloned on the 3.5-kbp DNA fragment which expressed xylanase and xylosidase in E. coli, (iii) there is at least one gene upstream of xylI which could encode an arabinosidase, and (iv) xylosidase rather than xylanase may be a rate-limiting step in xylan utilization. Insertional mutations in the xylI-xsa locus reduced the rate of growth on xylan, but the concentration of residual sugars at the end of growth was the same as that with the wild type. Thus, a slower rate of growth on xylan was not accompanied by less extensive digestion of xylan. Mutants in which xylI had been disrupted still expressed some xylanase activity. This second activity was associated with membranes and produced xylose from xylan, whereas the xylI gene product partitioned primarily with the soluble fraction and produced xylobiose from xylan.