Analysis of outer membrane proteins which are associated with growth of Bacteroides thetaiotaomicron on chondroitin sulfate.

By analyzing outer membrane proteins of Bacteroides thetaiotaomicron on two-dimensional polyacrylamide gels, we were able to identify 10 protein spots that were associated with growth on chondroitin sulfate but not with growth on glucuronic acid or other monosaccharides. These proteins were distinct from the outer membrane polypeptides that were associated with growth on two other negatively charged polysaccharides, polygalacturonic acid and heparin. Of the 10 protein spots that were associated with growth on chondroitin sulfate, 4 could be detected on immunoblots with antiserum that had been raised against outer membranes from bacteria grown on chondroitin sulfate and then cross-adsorbed with membranes from bacteria grown on glucose. Synthesis of these four proteins appeared to be regulated coordinately with synthesis of the two enzymes that degrade chondroitin sulfate, chondroitin lyase I and II. Although one of the four proteins (Mr 110,000) was similar in molecular weight to the chondroitin lyases, the cross-adsorbed antiserum which detected this outer membrane protein did not cross-react with either of these two enzymes.     

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)     

Biochemical evidence that starch breakdown by Bacteroides thetaiotaomicron involves outer membrane starch-binding sites and periplasmic starch-degrading enzymes.

Bacteroides thetaiotaomicron can utilize amylose, amylopectin, and pullulan as sole sources of carbon and energy. The enzymes that degrade these polysaccharides were found to be primarily cell associated rather than extracellular. Although some activity was detected in extracellular fluid, this appeared to be the result of cell lysis. The cell-associated amylase, amylopectinase, and pullulanase activities partitioned similarly to the periplasmic marker, acid phosphatase, when cells were exposed to a cold-shock treatment. Two other enzymes associated with starch breakdown, alpha-glucosidase and maltase, appeared to be located in the cytoplasm. Intact cells of B. thetaiotaomicron were found to bind 14C-starch. Binding was probably mediated by a protein because it was saturable and was decreased by treatment of cells with proteinase K. Results of competition experiments showed that the starch-binding proteins had a preference for maltodextrins larger than maltohexaose and a low affinity for maltose and maltotriose. Both the degradative enzymes and starch binding were induced by maltose. These findings indicate that starch utilization by B. thetaiotaomicron apparently does not involve secretion of extracellular enzymes. Rather, binding of the starch molecule to the cell surface appears to be a first step to passing the molecule through the outer membrane and into the periplasmic space.     

Cloning and characterization of a Bacteroides conjugal tetracycline-erythromycin resistance element by using a shuttle cosmid vector.

The Bacteroides conjugal tetracycline resistance (Tcr) elements appear not to be plasmids. In many cases, resistance to erythromycin (Emr) is cotransferred with Tcr. Using a newly constructed shuttle cosmid, pNJR1, we cloned 44 to 50 kilobase pairs of a conjugal Tcr Emr element on overlapping cosmid clones. Cosmid libraries were made in Escherichia coli with DNA from the original clinical Bacteroides thetaiotaomicron DOT strain containing Tcr Emr-DOT or from a Bacteroides uniformis Tcr Emr-DOT transconjugant strain. The cosmid clones were mobilized from E. coli into B. uniformis in groups of 10 to 20 per filter mating, with selection for Tcr or Emr transconjugants. The Tcr and Emr genes were cloned both separately and together on 30-kilobase-pair fragments. Several of the Tcr clones also contained transfer genes that permitted self-transfer of the cosmid from B. uniformis donors to E. coli or B. uniformis recipients. Neither the Tcr nor the Emr gene conferred resistance on E. coli, and the transfer-proficient clones did not self-transfer out of E. coli. Southern blot analysis was used to compare DNA from independently isolated Bacteroides strains carrying conjugal Tcr or Tcr Emr elements and their respective B. uniformis transconjugants. Results of these analyses indicate that there are large regions of homology, including regions outside the Tcr and Emr genes, but that the elements are not identical. Some Tcr clones contained a region which hybridized to chromosomal DNA from the wild-type B. uniformis recipient strain that did not carry the Tcr Emr-DOT element. This region of homology appeared not to be a junction fragment. It was not required in a Bacteroides recipient for successful transfer of the Tcr Emr element. Although we are not sure we have cloned a junction fragment between the Tcr Emr-DOT element and the B. uniformis chromosome, the preliminary function and restriction map appears to be linear.     

Use of inducible disaccharidases to assess the importance of different carbohydrate sources for Bacteroides ovatus growing in the intestinal tracts of germfree mice.

Patterns of disaccharidase expression were used to determine which polysaccharides were the major sources of carbohydrate for Bacteroides ovatus growing in the intestinal tracts of monocolonized germfree mice. Results indicate that B. ovatus grows on a variety of different carbohydrates, which are present in low concentrations, rather than relying on one type of carbohydrate as the major carbohydrate source.     

Contribution of a neopullulanase, a pullulanase, and an alpha-glucosidase to growth of Bacteroides thetaiotaomicron on starch.

Bacteroides thetaiotaomicron, a gram-negative colonic anaerobe, can utilize three forms of starch: amylose, amylopectin, and pullulan. Previously, a neopullulanase, a pullulanase, and an alpha-glucosidase from B. thetaiotaomicron had been purified and characterized biochemically. The neopullulanase and alpha-glucosidase appeared to be the main enzymes involved in the breakdown of starch, because they were responsible for most of the starch-degrading activity detected in B. thetaiotaomicron cell extracts. To determine the importance of these enzymes in the starch utilization pathway, we cloned the genes encoding the neopullulanase and alpha-glucosidase. The gene encoding the neopullulanase (susA) was located upstream of the gene encoding the alpha-glucosidase (susB). Both genes were closely linked to another starch utilization gene, susC, which encodes a 115-kDa outer membrane protein that is essential for growth on starch. The gene encoding the pullulanase, pulI, was not located in this region in the chromosome. Disruption of the neopullulanase gene, susA, reduced the rate of growth on starch by about 30%. Elimination of susA in this strain allowed us to detect a low residual level of enzyme activity, which was localized to the membrane fraction. Previously, we had shown that a disruption in the pulI gene did not affect the rate of growth on pullulan. We have now shown that a double mutant, with a disruption in susA and in the pullulanase gene, pulI, was also able to grow on pullulan. Thus, there is at least one other starch-degrading enzyme besides the neopullulanase and the pullulanase. Disruption of the alpha-glucosidase gene, susB, reduced the rate of growth on starch only slightly. No residual alpha-glucosidase activity was detectable in extracts from this strain. Since this strain could still grow on maltose, maltotriose, and starch, there must be at least one other enzyme capable of degrading the small oligomers produced by the starch-degrading enzymes. Our results show that the starch utilization system of B. thetaiotaomicron is quite complex and contains a number of apparently redundant degradative enzymes.     

Evidence for natural horizontal transfer of tetQ between bacteria that normally colonize humans and bacteria that normally colonize livestock.

Though numerous studies have shown that gene transfer occurs between distantly related bacterial genera under laboratory conditions, the frequency and breadth of horizontal transfer events in nature remain unknown. Previous evidence for natural intergeneric transfers came from studies of genes in human pathogens, bacteria that colonize the same host. We present evidence that natural transfer of a tetracycline resistance gene, tetQ, has occurred between bacterial genera that normally colonize different hosts. A DNA sequence comparative approach was taken to examine the extent of horizontal tetQ dissemination between species of Bacteroides, the predominant genus of the human colonic microflora, and between species of Bacteroides and of the distantly related genus Prevotella, a predominant genus of the microflora of the rumens and intestinal tracts of farm animals. Virtually identical tetQ sequences were found in a number of isolate pairs differing in taxonomy and geographic origin, indicating that extensive natural gene transmission has occurred. Among the exchange events indicated by the evidence was the very recent transfer of an allele of tetQ usually found in Prevotella spp. to a Bacteroides fragilis strain.     

Solving the problem of how to eat something as big as yourself: Diverse bacterial strategies for degrading polysaccharides

Polysaccharide digestion by bacteria is an important activity in many ecosystems, and a number of bacterial genera can perform this function. Although many papers have been published about the properties of isolated polysaccharide-degrading enzymes, relatively little is known about how intact bacteria degrade polysaccharides. This review summarizes recent findings suggesting that there are at least three different strategies. the most familiar one is the excretion of extracellular polysaccharidases, which diffuse to and degrade nearby polysaccharides. An example of this type of strategy is provided by the plant pathogen,Erwinia spp. A second strategy is to have the enzyme exposed to the extracellular medium but attached to the surface of the cell. Examples of this strategy are provided by the pullulanase system ofKlebsiella oxytoca and the cellulosomes ofClostridium thermocellum. A strategy that could be seen as a combination of the extracellular enzyme strategy and the surface organelle strategy is provided byVibrio harveyi, which attaches to its substrate, chitin, via proteins that appear to be specialized for attachment and produces extracellular enzymes that attack the chitin. A third strategy is to import the polysaccharide, as appears to be done byBacteroides spp. In this instance, the polysaccharide is bound to an outer membrane receptor, then passes into the periplasm where the degradative enzymes are located. The ecological advantages and disadvantages of these systems are discussed, and areas where further research is needed are defined.     

Insertion and excision of Bacteroides conjugative chromosomal elements.

Many strains of Bacteroides harbor large chromosomal elements that can transfer themselves from the chromosome of the donor to the chromosome of the recipient. Most of them carry a tetracycline resistance (Tcr) gene and have thus been designated Tcr elements. In the present study, we have used transverse alternating field electrophoresis to show that all but one of the Tcr elements screened were approximately 70 to 80 kbp in size. The exception (Tcr Emr 12256) was 150 to 200 kbp in size and may be a hybrid element. All of the Tcr elements inserted in more than one site, but insertion was not random. The Tcr elements sometimes cotransfer unlinked chromosomal segments, or nonreplicating Bacteroides units (NBUs). Transverse alternating field electrophoresis analysis showed that insertion of NBUs was not random and that the NBUs did not insert near the Tcr element. Although attempts to clone one or both ends of a Tcr element have not been successful, ends of a cryptic element (XBU4422) were cloned previously and shown to be homologous to the ends of Tcr elements. We have obtained DNA sequences of junction regions between XBU4422 and its target from several different insertions. Comparison of junction sequences with target sequences showed that no target site duplication occurred during insertion and that XBU4422 carried 4 to 5 bp of adjacent chromosomal DNA when it excised from the chromosome and inserted in a plasmid. We identified a short region of sequence similarity between one of the ends of XBU4422 and its target site that may be important for insertion. This sequence contained an 8-bp segment that was identical to the recombinational hot spot sequence on Tn21. XBU4422 could exise itself from plasmids into which it inserted. In most cases, the excision left a single additional A behind in the target site, but precise excision was seen in one case.     

Location and characterization of genes involved in binding of starch to the surface of Bacteroides thetaiotaomicron.

Previous studies of starch utilization by the gram-negative anaerobe Bacteroides thetaiotaomicron have demonstrated that the starch-degrading enzymes are cell associated rather than extracellular, indicating that the first step in starch utilization is binding of the polysaccharide to the bacterial surface. Five transposon-generated mutants of B. thetaiotaomicron which were defective in starch binding (Ms-1 through Ms-5) had been isolated, but initial attempts to identify membrane proteins missing in these mutants were not successful. We report here the use of an immunological approach to identify four maltose-inducible membrane proteins, which were missing in one or more of the starch-binding mutants of B. thetaiotaomicron. Three of the maltose-inducible proteins were outer membrane proteins (115, 65, and 43 kDa), and one was a cytoplasmic membrane protein (80 kDa). The genes encoding these proteins were shown to be clustered in an 8.5-kbp segment of the B. thetaiotaomicron chromosome. Two other loci defined by transposon insertions, which appeared to contain regulatory genes, were located within 7 kbp of the cluster of membrane protein genes. The 115-kDa outer membrane protein was essential for utilization of maltoheptaose (G7), whereas loss of the other proteins affected growth on starch but not on G7. Not all of the proteins missing in the mutants were maltose regulated. We also detected two constitutively produced proteins (32 and 50 kDa) that were less prominent in all of the mutants than in the wild type. Both of these were outer membrane proteins.