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Expression of a cloned cellulase/xylanase gene from Prevotella ruminicola in Bacteroides vulgatus, Bacteroides uniformis and Prevotella ruminicola.

A new shuttle vector, pRH3 (8.7 kb), was constructed for use in Prevotella/Bacteroides host strains. This vector combines the pRRI2 replicon from P. ruminicola, pBluescript sequences and a tetQ marker gene for selection in Prevotella/Bacteroides hosts. Following insertion of a fragment carrying an endoglucanase/xylanase gene from P. ruminicola 23 into the multiple cloning site, the resulting construct, pRH3X, was introduced into B. vulgatus 1447, B. uniformis 1100 and P. ruminicola 2202. This resulted in increases of between 4 and 50-fold in CM-cellulase and xylanase activities in cells grown with glucose. In contrast activities were barely detectable for the same construct in E. coli DH5 alpha. Most of the total xylanase activity produced was found within the cell in P. ruminicola 2202 and B. vulgatus 1447 transformed with pRH3X, and in P. ruminicola 23. An osmotic shock experiment indicated that a significant proportion of the xylanase activity in B. vulgatus 1447 cells carrying pRH3X was periplasmic.

Bacteroides↗

Bactericidal effect of pooled human serum on Bacteroides melaninogenicus, Bacteroides asaccharolyticus and Actinobacillus actinomycetemcomitans.

Bacteria belonging to the species B. melaninogenicus ss. melaninogenicus (two strains), B. melaninogenicus ss. intermedius (five strains), B. asaccharolyticus (seven strains), and Actinobacillus actinomycetemcomitans (four strains) were tested under anaerobic conditions for their sensitivity to the bactericidal effect of pooled human serum. The A. actinomycetemcomitans strains were not killed to any extent by the serum. Both B. melaninogenicus ss. melaninogenicus strains were killed by serum. The sensitivity to serum among the strains of B. asaccharolyticus and B. melaninogenicus ss. intermedius was diverse. There was no correlation between the sensitivity to serum among the strains and the levels of specific antibodies in the serum. The bactericidal activity of serum was only effective if the complement system was present in serum. All strains initiated activation of complement and binding of a C3 component to the cell surface both in normal and MgEGTA chelated serum. The possible role of serum bactericidal activity in the pathogenesis of oral infections is unclear.

Actinobacillus↗

Use of the RapID-ANA System to screen for enzyme activities that differ among species of bile-inhibited Bacteroides.

The RapID-ANA System (Innovative Diagnostics Systems, Inc., Atlanta, Ga.) was used to test 102 strains of 14 species of phenotypically similar bile-inhibited Bacteroides from humans. Bacteroides oris, Bacteroides veroralis, Bacteroides buccalis, Bacteroides melaninogenicus, Bacteroides loescheii, and Bacteroides denticola had very similar enzyme activity profiles. Clear differentiation of these six species by the RapID-ANA System was not possible, but tests for arginine aminopeptidase and beta-glucosidase were helpful. Bacteroides oralis, Bacteroides intermedius, Bacteroides corporis, Bacteroides disiens, Bacteroides bivius, Bacteroides gingivalis, Bacteroides asaccharolyticus, and Bacteroides buccae each had unique enzyme activity profiles. No consistent differences in enzyme activities were found between the two DNA homology groups within Bacteroides melaninogenicus, Bacteroides loescheii, or Bacteroides intermedius. Tests for glycine aminopeptidase, alpha-galactosidase, arginine aminopeptidase, alpha-fucosidase, N-acetylglucosaminidase, reduction of triphenyltetrazolium, and production of indole were helpful in the differentiation of the species studied.

Acetylglucosaminidase↗

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)

Bacteroides↗

Inorganic and metal-organic growth requirements of the genus Bacteroides.

The inorganic and metal-organic growth requirements of ruminal and nonruminal Bacteroides species were compared. The heme requirement of many nonruminal Bacteroides species was similar to that of Bacteroides ruminicola subsp. ruminicola and was a general tetrapyrrole requirement. Some nonruminal Bacteroides species utilized succinate or alpha-ketoglutarate, as well as tetrapyrrole-containing compounds, in place of heme. Fe(+) as well as heme was required for maximal yields of some Bacteroides species. The divalent cation requirements of Bacteroides species are complex. Mg(2+) deletion from a medium containing Mg(2+), Ca(2+), Co(2+), and Mn(2+) reduced the yields of all isolates. Ca(2+) deletion from the same medium reduced the growth yields of Bacteroides fragilis, B. fundiliformis, and one strain of B. oralis. The effects of Mg(2+) and Ca(2+) on the growth of Bacteroides isolates was influenced by other divalent cations. Relatively large quantities of Na(+) were obligately required by all of the currently recognized predominant rumen Bacteroides species. Nonruminal Bacteroides species either did not require Na(+) or required only small amounts. The Na(+) requirement of some nonruminal Bacteroides species could be partially replaced by Li(+) or Cs(+). The Na(+) requirement of rumen Bacteroides species was absolute. The inorganic and metal-organic growth requirements of Bacteroides species appear useful as aids in species differentiation.

Animals↗

Phylogeny of Bacteroides, Prevotella, and Porphyromonas spp. and related bacteria.

The phylogenetic structure of the bacteroides subgroup of the cytophaga-flavobacter-bacteroides (CFB) phylum was examined by 16S rRNA sequence comparative analysis. Approximately 95% of the 16S rRNA sequence was determined for 36 representative strains of species of Prevotella, Bacteroides, and Porphyromonas and related species by a modified Sanger sequencing method. A phylogenetic tree was constructed from a corrected distance matrix by the neighbor-joining method, and the reliability of tree branching was established by bootstrap analysis. The bacteroides subgroup was divided primarily into three major phylogenetic clusters which contained most of the species examined. The first cluster, termed the prevotella cluster, was composed of 16 species of Prevotella, including P. melaninogenica, P. intermedia, P. nigrescens, and the ruminal species P. ruminicola. Two oral species, P. zoogleoformans and P. heparinolytica, which had been recently placed in the genus Prevotella, did not fall within the prevotella cluster. These two species and six species of Bacteroides, including the type species B. fragilis, formed the second cluster, termed the bacteroides cluster. The third cluster, termed the porphyromonas cluster, was divided into two subclusters. The first contained Porphyromonas gingivalis, P. endodontalis, P. asaccharolytica, P. circumdentaria, P. salivosa, [Bacteroides] levii (the brackets around genus are used to indicate that the species does not belong to the genus by the sensu stricto definition), and [Bacteroides] macacae, and the second subcluster contained [Bacteroides] forsythus and [Bacteroides] distasonis. [Bacteroides] splanchnicus fell just outside the three major clusters but still belonged within the bacteroides subgroup. With few exceptions, the 16 S rRNA data were in overall agreement with previously proposed reclassifications of species of Bacteroides, Prevotella, and Porphyromonas. Suggestions are made to accommodate those species which do not fit previous reclassification schemes.

Bacteroides↗

Purification and characterization of a ferredoxin from Rhizobium japonicum bacteroids.

An eight-iron, eight-sulfur ferredoxin from Rhizobium japonicum bacteroids of soybean root nodules has been purified to apparent homogeneity as judged by disc gel electrophoresis. The purification procedure included chromatography on DEAE-cellulose, Bio-Gel P-60, and hydroxylapatite. Specific activities of several purified preparations of bacteroid ferredoxin ranged from 1700 to 1900 nmol of C2H4 produced . min-1 . mg-1 in the reaction mediating electron transfer between illuminated chloroplasts and bacteroid nitrogenase. A molecular weight of 6740 for the protein was determined by low speed sedimentation equilibrium and a molecular weight of 6500 was estimated from the mobility of bacteroid ferredoxin relative to the mobility of standard proteins during sodium dodecyl sulfate disc gel electrophoresis. All of the common amino acids were present except arginine, methionine, and tryptophan. The absorbance spectrum of the oxidized protein exhibited maxima at 285 nm and 380 nm with a shoulder near 305 nm. The A380/A285 ratio was 0.76 and the extinction coefficient at 380 nm for the oxidized protein was found to be 30,800 M-1. Equilibration of bacteroid ferredoxin with methyl viologen at various potentials revealed a midpoint oxidation-reduction potential of -484 mV. Spectrophotometric examination of iron-sulfur clusters extruded from bacteroid ferredoxin with benzenethiol and the transfer of its iron-sulfur clusters to other ferredoxins established the presence of two [4Fe-4S] clusters in a molecule of bacteroid ferredoxin. The EPR spectrum of oxidized ferredoxin consisted of a small signal at g = 2.02 integrating to 0.19 spin/molecule. The EPR spectrum of ferredoxin reduced with 5-deazaflavin exhibited a signal with features at g values of 1.88, 1.94, 2.01, and 2.07, and integrated to 1.7 spins/molecule. The EPR properties of bacteroid ferredoxin are characteristic of a ferredoxin operating between the 1+ and 2+ oxidation levels. Bacteroid ferredoxin mediated electron transfer to clostridial hydrogenase, but was not reduced by the clostridial phosphoroclastic system in the presence of pyruvate. Bacteroid ferredoxin reduced by illuminated 5-deazariboflavin also supported a high rate of C2H2 reduction by bacteroid nitrogenase which was free of Na2S2O4. It was concluded, on this basis, that bacteroid ferredoxin has the capability of functioning as the electron donor for nitrogenase in R. japonicum.

Amino Acids↗

An electron microscope survey of the surface structures and hydrophobicity of oral and non-oral species of the bacterial genus Bacteroides.

Seventeen strains of Bacteroides representing 10 species were examined by negative staining; the majority were from the mouth but a few non-oral strains were included. Seven species had peritrichously-arranged, non-flagellar appendages which could be divided by morphology and ultrastructure into two subgroups, fibrils and fimbriae. Bacteroides asaccharolyticus strains B536 and B537 and Bacteroides gingivalis strains W50, W83, WPH15 and WPH35 had fimbriae with mean width of 4.4 nm and 0.5 to 6.0 microns long depending on the strain. The fimbrial length within each strain also varied. Fibrils were present on two fresh oral isolates of Bacteroides melaninogenicus, Bacteroides intermedius strains T588 and W09, Bacteroides corporis ATCC 33547, Bacteroides oralis ATCC 33269 and Bacteroides buccae ATCC 33574. Fibrils consistently clumped into bundles of variable thickness and formed a fringe around the cell periphery, ranging from 0.27 to 1.2 microns long depending on the strain. Fibril lengths of each strain were uniform. Fibrils had no measurable width and the clumps tapered towards the free ends. Bacteroides loeschii VPI 9085, Bacteroides pentosaceus strains NP333 and J1 and Bacteroides capillosus 29799 had no detectable surface appendages. Fimbriate strains had a layer outside the outer membrane, with a mean thickness of between 17.8 and 28.6 nm. Both fibrillar and fimbriate strains produced many small membranous vesicles budding from the outer membrane. There were two morphological forms of vesicles, ones with either fimbriae or fibrils (species-dependent) and ones with no attached appendages. Of eleven strains tested for cell-surface hydrophobicity by partitioning between hexadecane and buffer, all but one was non-hydrophobic.

Bacteroides↗