Human Colonic Bacteroides Research Articles

Dietary pectic glycans are degraded by coordinated enzyme pathways in human colonic Bacteroides.

The major nutrients available to human colonic Bacteroides species are glycans exemplified by pectins, a network of covalently linked plant cell wall polysaccharides containing galacturonic acid (GalA). Metabolism of complex carbohydrates by the Bacteroides genus is orchestrated by polysaccharide utilisation loci or PULs. In Bacteroides thetaiotaomicron, a human colonic bacterium, the PULs activated by the different pectin domains have been identified, however, the mechanism by which these loci contribute to the degradation of these GalA-containing polysaccharides is poorly understood. Here we show that each PUL orchestrates the metabolism of specific pectin molecules, recruiting enzymes from two previously unknown glycoside hydrolase (GH) families. The apparatus that depolymerizes the backbone of rhamnogalacturonan-I (RGI) is particularly complex. This system contains several GHs that trim the remnants of other pectin domains attached to RGI, while nine enzymes contribute to the degradation of the backbone comprising a rhamnose-GalA repeating unit. The catalytic properties of the pectin degrading enzymes are optimized to protect the glycan cues that activate the specific PULs ensuring a continuous supply of inducing molecules throughout growth. The contribution of Bacteroides spp. to the metabolism of the pectic network is illustrated by cross-feeding between organisms.

The excision proteins of CTnDOT positively regulate the transfer operon

The Bacteroides conjugative transposon, CTnDOT, is an integrated conjugative element (ICE), found in many human colonic Bacteroides spp. strains. It has a complex regulatory system for both excision from the chromosome and transfer and mobilization into a new host. It was previously shown that a cloned DNA segment encoding the xis2c, xis2d, orf3, and exc genes was required for tetracycline dependent activation of the Ptra promoter. The Xis2c and Xis2d proteins are required for excision while the Exc protein stimulates excision. We report here that neither the Orf3 nor the Exc proteins are involved in activation of the Ptra promoter. Deletion analysis and electromobility shift assays showed that the Xis2c and Xis2d proteins bind to the Ptra promoter to activate the tra operon. Thus, the recombination directionality factors of CTnDOT excision also function as activator proteins of the Ptra promoter.

A newly discovered Bacteroides conjugative transposon, CTnGERM1, contains genes also found in gram-positive bacteria.

Results of a recent study of antibiotic resistance genes in human colonic Bacteroides strains suggested that gene transfer events between members of this genus are fairly common. The identification of Bacteroides isolates that carried an erythromycin resistance gene, ermG, whose DNA sequence was 99% identical to that of an ermG gene found previously only in gram-positive bacteria raised the further possibility that conjugal elements were moving into Bacteroides species from other genera. Six of seven ermG-containing Bacteroides strains tested were able to transfer ermG by conjugation. One of these strains was chosen for further investigation. Results of pulsed-field gel electrophoresis experiments showed that the conjugal element carrying ermG in this strain is an integrated element about 75 kb in size. Thus, the element appears to be a conjugative transposon (CTn) and was designated CTnGERM1. CTnGERM1 proved to be unrelated to the predominant type of CTn found in Bacteroides isolates-CTns of the CTnERL/CTnDOT family-which sometimes carry another type of erm gene, ermF. A 19-kbp segment of DNA from CTnGERM1 was cloned and sequenced. A 10-kbp portion of this segment hybridized not only to DNA from all the ermG-containing strains but also to DNA from strains that did not carry ermG. Thus, CTnGERM1 seems to be part of a family of CTns, some of which have acquired ermG. The percentage of G+C content of the ermG region was significantly lower than that of the chromosome of Bacteroides species-an indication that CTnGERM1 may have entered Bacteroides strains from some other bacterial genus. A survey of strains isolated before 1970 and after 1990 suggests that the CTnGERM1 type of CTn entered Bacteroides species relatively recently. One of the genes located upstream of ermG encoded a protein that had 85% amino acid sequence identity with a macrolide efflux pump, MefA, from Streptococcus pyogenes. Our having found >90% sequence identity of two upstream genes, including mefA, and the remnants of two transposon-carried genes downstream of ermG with genes found previously only in gram-positive bacteria raises the possibility that gram-positive bacteria could have been the origin of CTnGERM1.

The NAD(P)H-utilizing glutamate dehydrogenase of Bacteroides thetaiotaomicron belongs to enzyme family I, and its activity is affected by trans-acting gene(s) positioned downstream of gdhA

Previous studies have suggested that regulation of the enzymes of ammonia assimilation in human colonic Bacteroides species is coordinated differently than in other eubacteria. The gene encoding an NAD(P)H-dependent glutamate dehydrogenase (gdhA) in Bacteroides thetaiotaomicron was cloned and expressed in Escherichia coli by mutant complementation from the recombinant plasmid pANS100. Examination of the predicted GdhA amino acid sequence revealed that this enzyme possesses motifs typical of the family I-type hexameric GDH proteins. Northern blot analysis with a gdhA-specific probe indicated that a single transcript with an electrophoretic mobility of approximately 1.6 kb was produced in both B. thetaiotaomicron and E. coli gdhA+ transformants. Although gdhA transcription was unaffected, no GdhA enzyme activity could be detected in E. coli transformants when smaller DNA fragments from pANS100, which contained the entire gdhA gene, were analyzed. Enzyme activity was restored if these E. coli strains were cotransformed with a second plasmid, which contained a 3-kb segment of DNA located downstream of the gdhA coding region. Frameshift mutagenesis within the DNA downstream of gdhA in pANS100 also resulted in the loss of GdhA enzyme activity. Collectively, these results are interpreted as evidence for the role of an additional gene product(s) in modulating the activity of GDH enzyme activity. Insertional mutagenesis experiments which led to disruption of the gdhA gene on the B. thetaiotaomicron chromosome indicated that gdhA mutants were not glutamate auxotrophs, but attempts to isolate similar mutants with insertion mutations in the region downstream of the gdhA gene were unsuccessful.

Characterization of a new type of Bacteroides conjugative transposon, Tcr Emr 7853

Results of previous investigations suggested that the conjugative transposons found in human colonic Bacteroides species were all members of a closely related family of elements, exemplified by Tcr Emr DOT. We have now found a new type of conjugative transposon, Tcr Emr 7853, that does not belong to this family. Tcr Emr 7853 has approximately the same size as the Tcr Emr DOT-type elements (70 to 80 kbp) and also carries genes encoding resistance to tetracycline (Tcr) and erythromycin (Emr); however, it differs from previously described conjugative transposons in a number of ways. Its transfer is not regulated by tetracycline and its transfer genes are not controlled by the regulatory genes rteA and rteB, which are found on Tcr Emr DOT and related conjugative transposons. Its ends do not cross-hybridize with the ends of Tcr Emr DOT-type conjugative transposons, and the Emr gene it carries does not cross-hybridize with ermF, the Emr gene found on all previously studied Bacteroides conjugative transposons. There is only one region with high sequence similarity between Tcr Emr 7853 and previously characterized elements, the region that contains the Tcr gene, tetQ. This sequence similarity ends 145 bp upstream of the start codon and 288 bp downstream from the stop codon. A 2-kbp region upstream of tetQ on Tcr Emr 7853 cross-hybridized with four additional EcoRV fragments of Bacteroides thetaiotaomicron 7853 DNA other than the one that contained tetQ. These additional cross-hybridizing bands were not part of Tcr Emr 7853, but one of them cotransferred with Tcr Emr 7853 in some matings. Thus, at least one of the additional cross-hybridizing bands may be associated with another conjugative element or with an element that is mobilized by Tcr Emr 7853. DNA that cross-hybridized with the upstream region was found in one clinical isolate of Bacteroides ovatus and four Tcr isolates of Prevotella ruminicola.

Tetracycline regulation of genes on Bacteroides conjugative transposons.

Human colonic Bacteroides species harbor a family of large conjugative transposons, called tetracycline resistance (Tcr) elements. Activities of these elements are enhanced by pregrowth of bacteria in medium containing tetracycline, indicating that at least some Tcr element genes are regulated by tetracycline. Previously, we identified a central regulatory locus on the Tcr elements that contained two genes, rteA and rteB, which appeared to encode a two-component regulatory system (A. M. Stevens, J. M. Sanders, N. B. Shoemaker, and A. A. Salyers, J. Bacteriol. 174:2935-2942, 1992). In the present study, we describe a gene which is located downstream of rteB in a separate transcriptional unit and which requires RteB for expression. Sequence analysis of this gene showed that it encoded a 217-amino-acid protein, which had no significant sequence similarity to any proteins in the GenBank or EMBL data base. An insertional disruption in the gene abolished self-transfer of the Tcr element to Bacteroides recipients, indicating that the gene was essential for self-transfer. The disruption also affected mobilization of coresident plasmids. Mobilization frequency was reduced 100- to 1,000-fold if the recipient was Escherichia coli but was not affected to the same extent if the recipient was an isogenic Bacteroides strain. The complex phenotype of the disruption mutant suggested that the newly identified gene, like rteA and rteB, had a regulatory function. Accordingly, it has been designated rteC. Our results indicate that regulation of Tc(r) element functions is unexpectedly complex and may involve a cascade of regulators, with RteA and RteB exerting central control over secondary regulators like RteC, which in turn control subsets of Tcr element structural genes.

In vitro evaluation of calcium pectinate: a potential colon-specific drug delivery carrier.

Calcium pectinate (CaP)--the insoluble salt of pectin--can potentially be used as a colon-specific drug delivery system. The use of CaP as a carrier was based on the assumption that, like pectin, it can be decomposed by specific pectinolytic enzymes in the colon but that it retains its integrity in the physiological environment of the small bowel. The biodegradation of the carrier was characterized by monitoring the percent cumulative release of the insoluble drug indomethacin, incorporated into pectin or CaP matrices. Compressed tablets of pectin and indomethacin were analyzed for degradation in the presence of Pectinex 3XL, a typical pectinolytic enzyme mixture, and in the presence of the human colonic bacterium Bacteroides ovatus. The degradation of CaP-indomethacin tablets was assessed in the presence of Pectinex 3XL and in rat cecal contents. The release of indomethacin was significantly increased (end-time percentage cumulative release vs control) in the presence of Pectinex 3XL (89 +/- 20 vs 16 +/- 2 for CaP tablets), Bacteroides ovatus (12 and 22 vs 5.2 for pectin tablets), and rat cecal contents (61 +/- 16 vs 4.9 +/- 1.1 for CaP tablets). The weight loss of tablet mass was significantly higher (end-time dry weight vs control) in the presence of Pectinex 3XL (0 vs 75 +/- 6% of initial weight for CaP tablets). These findings indicate the potential of CaP, compressed into tablets with insoluble drug, to serve as a specific drug delivery system to the colon.

Chromosomal gene transfer elements of the Bacteroides group.

Many human colonic Bacteroides strains carry large ( > 60 kbp) chromosomal elements that can transfer themselves from the chromosome of the donor to the chromosome of the recipient. Most of these elements carry a tetracycline resistance gene (tetQ) and many also carry an erythromycin resistance gene (ermF), but at least one cryptic member of the family has been identified. Molecular analysis of excision and integration events has shown that the self-transmissible Bacteroides elements are not transposons but may represent a new class of integrating elements. The Bacteroides elements are most similar to the streptococcal conjugative transposons, such as Tn916. The Bacteroides Tcr/TcrEmr elements can mobilize DNA that is not contained within the elements themselves. They not only mobilize co-resident plasmids but also cause the excision, circularization and mobilization of discrete unlinked 10-11 kbp segments of chromosomal DNA. Self-transfer and other activities of the Tcr/TcrEmr elements are regulated by tetracycline. Thus, tetracycline not only selects for acquisition of an element but also stimulates element transfer in the first place.

Utilization of pancreatic trypsin and chymotrypsin by proteolytic and nonproteolyticBacteroides fragilis-type bacteria

The ability of five human colonic bacteroides (B. fragilis, B. splanchnicus, B. thetaiotaomicron, B. vulgatus, andB. ovatus) to grow on pancreatic trypsin and chymotrypsin, as well as other organic nitrogen sources, was studied in batch culture. All isolates grew on trypsin and, with the exception ofB. thetaiotaomicron, on chymotrypsin. The proteolytic activities of these bacteria varied considerably. However, the ability to utilize either trypsin or chymotrypsin was not related to the proteolytic potential of the bacteria and probably depended upon their ability to assimilate low-molecular-weight peptides generated during autolysis of the enzymes.

A Bacteroides ovatus chromosomal locus which contains an alpha-galactosidase gene may be important for colonization of the gastrointestinal tract

An alpha-galactosidase gene has been cloned from the human colonic Bacteroides species Bacteroides ovatus 0038. This alpha-galactosidase appears to be distinct from two previously characterized alpha-galactosidases, I and II, from the same strain and has been designated alpha-galactosidase III. Partially purified alpha-galactosidase III from Escherichia coli EM24 containing pFG61 delta SE had a pI of 7.6, as compared with the reported pI values for the known alpha-galactosidases of 5.6 for I and 6.9 for II. Its molecular weight as estimated on sodium dodecyl sulfate-polyacrylamide gels was 78,000, whereas the molecular weights of alpha-galactosidases I and II were 85,000 and 80,500, respectively. The only substrate hydrolyzed by alpha-galactosidase III was melibiose, whereas the other two alpha-galactosidases were able to degrade melibiose, raffinose, and stachyose and partially degraded guar gum. alpha-Galactosidase III had a pH optimum of 6.7 to 7.2. Finally, a single crossover insertion which disrupted the gene in the B. ovatus chromosome had no effect on expression of alpha-galactosidases I and II. Although this insertion had no effect on the ability of B. ovatus to grow in laboratory medium on any of the galactoside-containing carbohydrates tested, the insertion mutant was outcompeted by wild type when a combination of mutant and wild type was used to colonize germfree mice. Insertions on either side of the gene had the same effect. Thus, the locus which contains alpha-galactosidase III may be important for colonization in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

The region of a Bacteroides conjugal chromosomal tetracycline resistance element which is responsible for production of plasmidlike forms from unlinked chromosomal DNA might also be involved in transfer of the element

Large (greater than 50 kilobases) conjugal chromosomal tetracycline resistance (Tcr) elements have been found in many human colonic Bacteroides strains. Recently, N. B. Shoemaker and A. A. Salyers (J. Bacteriol, 170:1651-1657, 1988) reported that some of these Tcr elements appeared to mediate production of plasmidlike forms, NBU1 and NBU2, from an unlinked region of the chromosome of Bacteroides uniformis 0061. Production of the plasmidlike forms and the transfer frequency of the Tcr elements were both enhanced by preexposure to tetracycline. Thus it appeared that genes involved in production of plasmidlike forms (Plf activity) might be coregulated with transfer genes and that Plf activity might have a role in transfer of the Tcr elements. By screening subclones of a Tcr element, Tcr Emr DOT, we have shown that the genes necessary for Plf activity on the Tcr element are within a 10-kilobase region adjacent to the Tcr gene. Subclones of this region were then used to construct insertional gene disruptions in a Tcr element, Tcr ERL, which is closely related to the Tcr Emr DOT element. Two of the disruption mutants were Plf-. Both had reduced transfer frequencies, one (omega RDB2) 10(2)-fold lower than that of the wild-type element and the other (omega RDBT) 10(4)-fold lower. omega RDB2 was also deficient in the ability to mobilize coresident plasmids, whereas omega RDBT exhibited nearly wild-type mobilization activity. The phenotypes of the mutants indicate that there are at least two genes necessary for Plf activity and that both may be involved in transfer of the element. The third disruption mutant (omegaRDB1), which expressed Plf constitutively, also had a transfer frequency 10(2) -fold lower than that of the wild-type element and was deficient in mobilization of coresident plasmids. The relationship between Plf genes and transfer, therefore, appears to be a complex one.

Assessing the importance of host-derived polysaccharides as carbon sources for bacteria growing in the human colon

Although dietary polysaccharides undoubtedly serve as an important source of carbon and energy for bacteria that reside in the human colon, polysaccharides of host origin also enter the colon and might serve as carbon sources for some bacteria that can utilize them. In this paper, we review what is known about the characteristics of the polysaccharide utilization systems of Bacteroides, a genus of obligate anaerobes which is probably responsible for most of the polysaccharide fermentation that occurs in the colon. We then describe two approaches, based on this information, which are being used to determine which polysaccharides are actually being fermented by Bacteroides in the human colon. The first approach involves detecting and characterizing enzyme activities in extracts of a bacterial fraction from human feces. The results of these studies indicate that polygalacturonic acid and starch, two readily fermented dietary polysaccharides, are probably not major sources of carbon and energy for Bacteroides in the colon. A second approach involves assessing the ability of mutants that are deficient in their utilization of particular polysaccharides to colonize the intestinal tracts of germ-free mice. The results of these studies indicate that although host polysaccharides may serve as carbon sources in vivo, they are not major ones. Thus, neither of these approaches provides evidence for a single primary source of carbohydrate in vivo. It appears likely that human colonic Bacteroides may be using a variety of polysaccharides rather than relying on a single source of carbohydrate.

Tetracycline-dependent appearance of plasmidlike forms in Bacteroides uniformis 0061 mediated by conjugal Bacteroides tetracycline resistance elements.

Some human colonic Bacteroides strains carry conjugal tetracycline resistance (Tcr) elements, which are thought to be chromosomal. We have found that some of these Tcr elements can mediate the appearance of plasmidlike forms in Bacteroides uniformis 0061. When B. uniformis 0061, containing a conjugal Tcr element designated Tcr ERL, was grown in medium containing tetracycline (1 microgram/ml), two circular DNA forms were found in the alkaline plasmid preparations: NBU1 (10.3 +/- 0.5 kilobases) and NBU2 (11.5 +/- 0.5 kilobases). Restriction analysis of NBU1 and NBU2 showed that they were not identical, although Southern blot analysis indicated that they did contain some region(s) of homology. Results of Southern blot analysis also demonstrated that both NBU1 and NBU2 were normally integrated in the chromosome of B. uniformis or in some undetected large plasmid. Although we were unable to determine the exact structure and location of the integrated forms of NBU1 and NBU2 in B. uniformis, they appear to be in close proximity to each other. Neither NBU1 or NBU2 could be detected as a plasmidlike form in cells exposed to UV light, thymidine starvation, mitomycin C, or autoclaved chlortetracycline (50 micrograms/ml). Four conjugal Tcr elements other than the Tcr ERL element were able to mediate the appearance of NBU1 alone, and two Tcr elements did not mediate the excision of either NBU1 or NBU2. Three strains from different Bacteroides species contained some DNA sequences which had homology to NBU1 and NBU2.

Enumeration of polysaccharide-degrading Bacteroides species in human feces by using species-specific DNA probes.

DNA probes that are specific for each of five predominant species of human colonic Bacteroides (B. thetaiotaomicron, B. uniformis, B. distasonis, "Bacteroides group 3452-A", and B. ovatus) were used to detect and enumerate these species in fecal samples from two adult volunteers. These five species are capable of fermenting many of the complex polysaccharides that are thought to be sources of carbon and energy for bacteria in the colon. Estimates for the concentrations of some of these species in feces have not been previously available because of the difficulties in differentiating colonic Bacteroides spp. by conventional biochemical tests. Our results indicate that all the species except B. ovatus were present in high numbers (greater than 10(9)/g [dry weight]) in the feces of both volunteers. However, the concentrations of the more versatile polysaccharide-degrading species within this group of organisms (7.6 X 10(9) to 12.0 X 10(9)/g [dry weight] for B. thetaiotaomicron; 2.9 X 10(9) to 6.3 X 10(9)/g [dry weight] for "Bacteroides group 3452-A") did not differ significantly from the concentrations of less versatile polysaccharide-degrading species (1.2 X 10(10) to 2.0 X 10(10)/g [dry weight] for B. uniformis; 5.8 X 10(9) to 8.4 X 10(9)/g [dry weight] for B. distasonis). B. ovatus was not detectable by our method. Since our lower limit of detection is approximately 1 X 10(9) to 2 X 10(9)/g (dry weight) of feces, this is consistent with earlier estimates that indicated that the concentration of B. ovatus in feces is near or below this value.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of randomly cloned DNA fragments for identification of Bacteroides thetaiotaomicron.

Randomly cloned fragments of DNA from Bacteroides thetaiotaomicron were used as hybridization probes for differentiation of B. thetaiotaomicron from closely related Bacteroides species. HindIII digestion fragments of DNA from B. thetaiotaomicron (type strain) were inserted into plasmid pBR322 and labeled with [alpha-32P]dCTP by nick translation. These labeled plasmids were screened for hybridization to HindIII digests of chromosomal DNA from type strains of the following human colonic Bacteroides species: B. thetaiotaomicron, Bacteroides ovatus, reference strain 3452-A (formerly part of B. distasonis), Bacteroides uniformis, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides distasonis, Bacteroides eggerthii, and reference strain B5-21 (formerly B. fragilis subsp. a). Two of the five cloned fragments hybridized only to DNA from B. thetaiotaomicron. Each of these two fragments hybridized to the same DNA restriction fragment in five strains of B. thetaiotaomicron other than the strain from which the DNA was cloned. One of the cloned fragments (pBT2) was further tested for specificity by determining its ability to hybridize to DNA from 65 additional strains of colonic Bacteroides.

Digestion of larch arabinogalactan by a strain of human colonic Bacteroides growing in continuous culture.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTDigestion of larch arabinogalactan by a strain of human colonic Bacteroides growing in continuous cultureAbigail A. Salyers, Roger Arthur, and Alex KuritzaCite this: J. Agric. Food Chem. 1981, 29, 3, 475–480Publication Date (Print):May 1, 1981Publication History Published online1 May 2002Published inissue 1 May 1981https://doi.org/10.1021/jf00105a009RIGHTS & PERMISSIONSArticle Views142Altmetric-Citations34LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (870 KB) Get e-Alerts Get e-Alerts