Colonic Bacteroides Species 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 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.

Evidence for natural transfer of a tetracycline resistance gene between bacteria from the human colon and bacteria from the bovine rumen.

Previously, we demonstrated conjugal transfer of a specially constructed shuttle vector, pRDB5, from the human colonic anaerobe Bacteroides uniformis to the ruminal anaerobe Prevotella (Bacteroides) ruminicola B(1)4. We have now shown that naturally occurring gene transfer elements in Bacteroides species and Prevotella ruminicola can also be transferred between these two genera. A self-transmissible chromosomal element originally found in a clinical isolate of Bacteroides fragilis (Tcr Emr 12256) was transferred from B. uniformis 0061 to P. ruminicola B(1)4 and from P. ruminicola B(1)4 back to B. uniformis or to another human colonic species, Bacteroides thetaiotaomicron. Similarly, a conjugative plasmid (pRRI4) originally found in P. ruminicola 223 was transferred from P. ruminicola B(1)4 to B. uniformis or B. thetaiotaomicron. pRRI4 could be transferred from the colonic Bacteroides species only if the donor strain contained the Tcr Emr 12256 element in its chromosome. These results show that transfer of naturally occurring elements can be demonstrated under laboratory conditions. Evidence that such transfers may actually have occurred in nature came from our finding that the tetracycline resistance (Tcr) gene on the P. ruminicola plasmid pRRI4 hybridized on high-stringency Southern blots with the Tcr gene found on the Bacteroides Tcr elements. The presence of the same gene in such distantly related genera of bacteria is most likely to have occurred as a result of horizontal transfer.

Cloning and comparison of xylanase genes from ruminal and colonicBacteroides species

The gene encoding xylanase activity in the ruminal bacteriumBacteroides ruminicola D31d was cloned and expressed inEscherichia coli with the plasmid vector pUC18. The gene was isolated on a 4.7-kilobase pair partialPstI genomic DNA fragment. The xylanase activity expressed inE. coli was cell associated and could degrade both oatspelt xylan and Remazol Brilliant Blue-xylan. The xylanase did not have detectable activity against carboxymethylcellulose. Utilization of an endogenous promoter byE. coli was indicated by expression of xylanase activity after subcloning of the insert into pBR322 in opposite orientations. TheB. ruminicola D31d xylanase gene was compared by Southern hybridization analyses with xylanase genes cloned fromB. ruminocola 23 andB. ovatus V975, a human intestinal isolate. The D31d xylanase gene did not cross-hybridize with either of the other two genes. In addition, the 23 xylanase gene did not cross-hybridize with the other two genes according to the same technique. These results indicate that the three cloned genes do not share a high degree of genetic similarity, despite the similar enzymatic activities. This is the first study to compare cloned genes from ruminal and colonicBacteroides species.

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)

Introduction of the Bacteroides ruminicola xylanase gene into the Bacteroides thetaiotaomicron chromosome for production of xylanase activity

The xylanase gene from the ruminal bacterium Bacteroides ruminicola 23 is highly expressed in colonic Bacteroides species when carried on plasmid pVAL-RX. In order to stabilize xylanase expression in the absence of antibiotic selection, the xylanase gene was introduced into the chromosome of Bacteroides thetaiotaomicron 5482 by using suicide vector pVAL-7. Xylanase activity in the resulting strain, B. thetaiotaomicron BTX, was about 30% of that observed in B. thetaiotaomicron 5482 containing the xylanase gene on pVAL-RX. The data obtained from continuous culture experiments using antibiotic-free medium showed that expression of xylanase activity in strain BTX was extremely stable, with no demonstrated loss of the inserted xylanase gene over 60 generations, with dilution rates from 0.42 to 0.03 h-1. In contrast, the plasmid-borne xylanase gene was almost completely lost by 60 generations in the absence of antibiotic selection. Incubation of strain BTX with oatspelt xylan resulted in the degradation of more than 40% of the xylan to soluble xylooligomers. The stability of xylanase expression in B. thetaiotaomicron BTX suggests that this microorganism might be suitable for introduction into the rumen and increased xylan degradation.

Heterologous expression of the Bacteroides ruminicola xylanase gene in Bacteroides fragilis and Bacteroides uniformis

A cloned xylanase gene from the ruminal bacterium Bacteroides ruminicola 23 was transferred by conjugation into the colonic species Bacteroides fragilis and Bacteroides uniformis by using the Escherichia coli-Bacteroides shuttle vector pVAL-1. The cloned gene was expressed in both species, and xynalase specific activity in crude extracts was found to be at least 1400-fold greater than that found in the B. ruminicola strain. Analysis of crude extract proteins from the recombinant B. fragilis by SDS-PAGE demonstrated a new 60 000 molecular weight protein. The xynalase activity expressed in both E. coli and B. fragilis was capable of degrading xylan to xylooligosaccharides in vitro. This is the first demonstration that colonic Bacteroides species can express a gene from a ruminal Bacteroides species.

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.

Mobilization of Bacteroides plasmids by Bacteroides conjugal elements.

A 4.2-kilobase cryptic Bacteroides plasmid, pB8-51, is found in several colonic Bacteroides species. To determine whether pB8-51 is mobilized by any of the known Bacteroides conjugal elements, we constructed an Escherichia coli-Bacteroides shuttle vector, pVAL-1, which contains pB8-51. We constructed Bacteroides uniformis 0061 derivatives which carry pVAL-1 and various Bacteroides conjugal elements. The Bacteroides conjugal elements tested were six conjugal tetracycline resistance (Tcr) elements (which appear to be chromosomal), i.e., Tcr ERL, Tcr V479, Tcr Emr ERL, Tcr Emr 12256, Tcr Emr DOT, and Tcr Emr CEST, and the conjugal erythromycin resistance (Emr) plasmid pBF4. These Tcr conjugal elements have not been extensively characterized, except for Tcr ERL. All six Tcr elements tested mobilized pVAL-1 at high frequency (10(-3) to 10(-5)) from one Bacteroides strain to another or from a Bacteroides strain to E. coli. Pregrowth of the donors (containing one of the Tcr elements and pVAL-1) in 1 microgram of tetracycline per ml enhanced the transfer of pVAL-1 by 20- to 10,000-fold, depending on which Tcr element was present in the donor. An Ems derivative of pBF4 (pBF4 delta E2) mobilized pVAL-1 from one Bacteroides strain to another at a frequency of 10(-4) but did not mobilize pVAL-1 from a Bacteroides strain to E. coli as efficiently. Thus the Tcr conjugal elements and pBF4 recognize a mobilization region on pB8-51.

Evidence that polygalacturonic acid may not be a major source of carbon and energy for some colonic Bacteroides species.

Five Bacteroides species that are found in the human colon can utilize polygalacturonic acid (PGA) when they are grown in laboratory media: Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bacteroides ovatus, Bacteroides fragilis subsp. a, and Bacteroides sp. strain 3452A (an unnamed DNA-DNA homology group). PGA-degrading enzymes from B. thetaiotaomicron have been isolated and characterized previously. To determine whether a PGA lyase activity in human feces could be attributed to any of these species, we first determined the properties of PGA lyases from the other four Bacteroides species. PGA lyases from all the Bacteroides species were soluble, cell associated, and inducible by PGA. All had similar pH optima (8.4 to 8.8) and similar molecular weights (50,000). All activities were enhanced by calcium. The PGA lyases from the five species differed with respect to isoelectric point: B. thetaiotaomicron (pI 7.5), B. vulgatus (pI 7.7), B. ovatus (pI 5.8, 7.2), B. fragilis subsp. a (pI 6.1), and Bacteroides sp. strain 3452A (pI 7.7). The PGA lyase activity in human feces resembled those of the Bacteroides PGA lyases in that it had a pH optimum of 8.4 to 8.8 and was enhanced by calcium. However, it differed from the Bacteroides PGA lyases both with respect to isoelectric point (pI 4.2 to 4.4) and molecular weight (100,000). On the basis of these findings, it appears that the PGA lyase activity in human feces is not produced by any of the Bacteroides species surveyed in this survey. Moreover, there was no detectable PGA lyase activity in feces that had the same properties as the Bacteroides enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of proteins involved in chondroitin sulfate utilization by three colonic Bacteroides species

Three species of colonic bacteria can ferment the mucopolysaccharide chondroitin sulfate: Bacteroides ovatus, Bacteroides sp. strain 3452A (an unnamed DNA homology group), and B. thetaiotaomicron. Proteins associated with the utilization of chondroitin sulfate by B. thetaiotaomicron have been characterized previously. In this report we compare chondroitin lyases and chondroitin sulfate-associated outer membrane polypeptides of B. ovatus and Bacteroides sp. strain 3452A with those of B. thetaiotaomicron. All three species produce two soluble cell-associated chondroitin lyases, chondroitin lyase I and II. Purified enzymes from the three species have similar pH optima, Km values, and molecular weights. However, peptide mapping experiments show that the chondroitin lyases from B. ovatus and Bacteroides sp. strain 3452A are not identical to those of B. thetaiotaomicron. A cloned gene that codes for the chondroitin lyase II from B. thetaiotaomicron hybridized on a Southern blot with DNA from B. ovatus or Bacteroides sp. strain 3452A only when low-stringency conditions were used. Antibody to chondroitin lyase II from B. thetaiotaomicron did not cross-react with chondroitin lyase II from B. ovatus or Bacteroides sp. strain 3452A. Chondroitin lyase activity in all three species was inducible by chondroitin sulfate. B. ovatus and Bacteroides sp. strain 3452A, like B. thetaiotaomicron, have outer membrane polypeptides that appear to be regulated by chondroitin sulfate, but the chondroitin sulfate-associated outer membrane polypeptides differ in molecular weight. Despite these differences, the ability of intact bacteria to utilize chondroitin sulfate, as indicated by growth yields in carbohydrate-limited continuous culture and the rate at which the chondroitin lyases were induced, was the same for all three species.

Use of a species-specific DNA hybridization probe for enumerating Bacteroides vulgatus in human feces.

pBV-1, a recombinant plasmid that contains a chromosomal DNA fragment from Bacteroides vulgatus, hybridized to DNA from B. vulgatus but not to DNA from other colonic Bacteroides species. This plasmid was used as a DNA probe to detect and enumerate B. vulgatus in pure culture, in mixed cultures, and in a bacterial fraction from human feces. Bacteria in a pure or mixed culture were lysed by heating the culture in NaOH. The DNA in the disrupted cell suspension was then trapped on nitrocellulose paper by vacuum filtration. If fecal samples were used instead of pure or mixed cultures, it was first necessary to partially purify the DNA by low-speed centrifugation (2,000 X g) and phenol-chloroform extraction before filtering. When 32P-labeled pBV-1 was incubated with filters containg B. vulgatus DNA, the amount of radioactivity that bound to the filters was proportional to the number of B. vulgatus filtered as long as the filtering capacity of the nitrocellulose was not exceeded. Using this procedure, we obtained a value for the concentration of B. vulgatus in human feces (2 X 10(10) to 3 X 10(10) per g of dry weight) that is similar to values obtained by other investigators using conventional bacteriological techniques (3 X 10(10) to 6 X 10(10) per g of dry weight). The advantage of the DNA hybridization method over conventional techniques is that it is not necessary to isolate pure cultures of bacteria from complex specimens such as feces. Furthermore, our method bypasses the cumbersome set of biochemical tests normally used to identify anaerobic bacteria. The major limitation of our method is its sensitivity.(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.