Analysis of cepA and other Bacteroides fragilis genes reveals a unique promoter structure.

There is little known about the sequences that mediate the initiation of transcription in Bacteroides fragilis, thus transcriptional start sites for 13 new genes were determined and a total of 23 promoter regions upstream of the start sites were aligned and similarities were noted. A region at about -7 contained a consensus sequence of TAnnTTTG and upstream in the region centered at about -33, another TTTG motif was found in the majority of promoters examined. Canonical, Escherichia coli, -10 and -35 consensus sequences were not readily apparent. Mutations within the -7 motif indicated the TTTG residues were essential since changes in this sequence reduced the promoter activity to that of a no promoter control in a chloramphenicol acetyl transferase transcriptional fusion model system. Additional fusion studies indicated that the -33 region was also necessary for full activity.

Effect of ferric and ferrous iron chelators on growth of Bacteroides fragilis under anaerobic conditions.

Growth of Bacteroides fragilis under anaerobic conditions in the presence of either haemin or protoporphyrin IX was inhibited by the ferrous iron chelator bipyridyl. The ferric-iron chelator desferrioxamine inhibited growth in the presence of protoporphyrin but not haemin, suggesting that even under anaerobic conditions Fe3+ is involved in uptake of non-haem iron, which is required in the absence of haemin. However, the ferric iron chelators 1,2-dimethyl-3-hydroxy-pyrid-4-one (L1) and pyridoxal isonicotinoyl hydrazone (PIH) were only weakly inhibitory. Apotransferrin, which also binds Fe3+, inhibited growth, but this was not simply due to binding of iron in the medium, as under the reducing conditions present, transferrin was unable to bind iron. This study suggests that even under anaerobic conditions, uptake of non-haem iron by B. fragilis may involve conversion of Fe2+ to Fe3+.

Growth inhibition of Bacteroides fragilis by hemopexin: proteolytic degradation of hemopexin to overcome heme limitation.

The stimulatory effect of heme on growth of Bacteroides fragilis, an anaerobic human pathogen, was strongly inhibited by hemopexin, an avid (Kd<1 pM) heme-binding plasma protein. Both rabbit and human hemopexins were bacteriostatic for a limited period of time, suggesting an adaptation by B. fragilis to heme-limited growth, and that hemopexin-bound heme can eventually be utilized by the bacteria. The inhibitory effect of hemopexin was lost when heme in the medium was replaced by protoporphyrin IX, which is bound less strongly by hemopexin (Kd approximately 1 microM). Protease activity was detected in the culture supernatant of B. fragilis grown in the presence of heme plus hemopexin but not in the presence of free heme, protoporphyrin IX or protoporphyrin IX plus hemopexin, suggesting that the enzyme(s) is induced by heme macrocycle limitation due to the scavenging effect of hemopexin. This protease activity was able to degrade rabbit hemopexin and human hemopexin, as well as human transferrin and ovalbumin, and may be a due to a serine protease since it was inhibited by phenylmethylsulfonyl fluoride (PMSF) but not by EDTA, leupeptin, pepstatin A or aprotinin. Thus, B. fragilis may overcome hemopexin-mediated heme limitation by secreting inducible protease(s), shown here to make protein-bound heme available to the microorganism.

Isolation of a ferritin from Bacteroides fragilis.

A ferritin was isolated from the obligate anaerobe Bacteroides fragilis. Estimated molecular masses were 400 kDa for the holomer and 16.7 kDa for the subunits. A 30-residue N-terminal amino acid sequence was determined and found to resemble the sequences of other ferritins (human H-chain ferritin, 43% identity; Escherichia coli gen-165 product, 37% identity) and to a lesser degree, bacterioferritins (E. coli bacterioferritin, 20% identity). The protein stained positively for iron, and incorporated 59Fe when B. fragilis was grown in the presence of [59Fe]citrate. However, the isolated protein contained only about three iron atoms per molecule, and contained no detectable haem. This represents the first isolation of a ferritin protein from bacteria. It may alleviate iron toxicity in the presence of oxygen.

Bacteriology of chronic otitis media affecting children living in Rio de Janeiro.

A bacteriologic analysis was performed on the purulent exudates obtained from the middle ears of 45 children living in Rio de Janeiro with CSOM and spontaneous perforations of the ear drum. Anaerobic cultures showed anaerobic bacteria in association with aerobic or facultatively anaerobic bacteria, or both, in 30 (66.7%) specimens. Facultative anaerobic microorganisms occurred in 42 (93.3%) of the specimens analyzed, strict aerobes in 17 (37.8%), capnophilic in 6 (13.3%), and fungi in 4 (8.9%). The rate of different species of bacteria isolated per sample was 4.4:1.87 for anaerobic and 1.84 for facultative microorganisms. Multiple drug resistances in the aerobic and facultative bacteria were found, and tetracycline, erythromycin, and beta-lactam antibiotic resistances were accentuated in the anaerobic bacteria.

Antagonism among Bacteroides fragilis group strains isolated from middle ear exudates from patients with chronic suppurative otitis media.

The production and sensitivity of a bacteriocin-like substance in Bacteroides fragilis group strains isolated from middle ear exudates in children with suppurative otitis media were studied through antagonism assayed by the well method. The results of the crossed reactions showed that 10 strains (66.6%) were bacteriocinogenic, 9 were sensitive to at least 1 bacteriocin (60%), and none showed inhibitory activity against homologous strains. Different patterns of susceptibility to bacteriocin-like substances were observed among strains isolated from the same patient as well as different strains isolated from another patient. These findings indicate that bacteriocin typing of anaerobic bacteria isolated from middle ear exudates in children with otitis media might have use in epidemiologic studies.

Biochemical and genetic analyses of a catalase from the anaerobic bacterium Bacteroides fragilis.

A single catalase enzyme was produced by the anaerobic bacterium Bacteroides fragilis when cultures at late log phase were shifted to aerobic conditions. In anaerobic conditions, catalase activity was detected in stationary-phase cultures, indicating that not only oxygen exposure but also starvation may affect the production of this antioxidant enzyme. The purified enzyme showed a peroxidatic activity when pyrogallol was used as an electron donor. It is a hemoprotein containing one heme molecule per holomer and has an estimated molecular weight of 124,000 to 130,000. The catalase gene was cloned by screening a B. fragilis library for complementation of catalase activity in an Escherichia coli catalase mutant (katE katG) strain. The cloned gene, designated katB, encoded a catalase enzyme with electrophoretic mobility identical to that of the purified protein from the B. fragilis parental strain. The nucleotide sequence of katB revealed a 1,461-bp open reading frame for a protein with 486 amino acids and a predicted molecular weight of 55,905. This result was very close to the 60,000 Da determined by denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified catalase and indicates that the native enzyme is composed of two identical subunits. The N-terminal amino acid sequence of the purified catalase obtained by Edman degradation confirmed that it is a product of katB. The amino acid sequence of KatB showed high similarity to Haemophilus influenzae HktE (71.6% identity, 66% nucleotide identity), as well as to gram-positive bacterial and mammalian catalases. No similarities to bacterial catalase-peroxidase-type enzymes were found. The active-site residues, proximal and distal hemebinding ligands, and NADPH-binding residues of the bovine liver catalase-type enzyme were highly conserved in B. fragilis KatB.

Regulation of Bacteriodes fragilis katB mRNA by oxidative stress and carbon limitation.

Regulation of the katB catalase gene in the anaerobic bacterium Bacteroides fragilis was studied. Northern blot hybridization analyses revealed that katB was transcribed as an approximately 1.6-kb monocistronic mRNA. The levels of katB mRNA increased > 15-fold when anaerobic, mid-logarithmic-phase cultures were exposed to O2, O2 with paraquat, or hydrogen peroxide. Under anaerobic conditions, the low levels of katB mRNA increased in a growth-dependent manner, reaching maximum expression at late logarithmic or early stationary phase, followed by a decrease in stationary phase. Under anaerobic conditions, the expression of katB mRNA was strongly repressed by glucose and to a lesser extent by xylose. However, glucose repression was completely abolished upon exposure to oxygen. The nonfermentable carbon sources fumarate, succinate, acetate, and pyruvate did not significantly affect expression. Phosphate, nitrogen, and hemin limitation did not affect the expression of katB mRNA, suggesting that the nutritional control of katB expression is restricted to carbon and energy sources and not other forms of nutrient limitation. Primer extension analysis revealed that during both oxidative stress and carbon or energy limitation, katB utilized the same promoter region but transcription initiation occurred at two different nucleotides separated by 3 or 4 bases. Interestingly, a 6-bp inverted repeat sequence present in the katB regulatory region was also observed upstream of the B. fragilis superoxide dismutase gene sod. It is possible that this is a recognition site for a DNA binding protein involved in the regulation of oxidative stress genes in this organism.

Role of the alkyl hydroperoxide reductase (ahpCF) gene in oxidative stress defense of the obligate Anaerobe bacteroides fragilis.

In this study we report the identification and role of the alkyl hydroperoxide reductase (ahp) gene in Bacteroides fragilis. The two components of ahp, ahpC, and ahpF, are organized in an operon, and the deduced amino acid sequences revealed that B. fragilis AhpCF shares approximately 60% identity to orthologues in other gram-positive and gram-negative bacteria. Northern blot hybridization analysis of total RNA showed that the ahpCF genes were transcribed as a polycistronic 2.4-kb mRNA and that ahpC also was present as a 0.6-kb monocistronic mRNA. ahpC and ahpCF mRNAs were induced approximately 60-fold following H(2)O(2) treatment or oxygen exposure of the parent strain but were constitutive in a peroxide-resistant strain. Further investigation using an ahpCF'::beta-xylosidase gene transcriptional fusion confirmed that ahpCF had lost normal regulation in the peroxide-resistant strain compared to the parent. The ahpCF mutant was more sensitive to growth inhibition and mutagenesis by organic peroxides than the parent strain, as determined by disk inhibition assays and the frequency of mutation to fusidic acid resistance. This finding suggests that the ahp genes play an important role in peroxide resistance in B. fragilis. Under anaerobic conditions, we observed increases in the number of spontaneous fusidic acid-resistant mutants of five- and sevenfold in ahpCF and ahpF strain backgrounds, respectively, and eightfold in the ahpCF katB double mutant strain compared to the parent and katB strains. In addition, ahpCF, ahpF, and ahpCF katB mutants were slightly more sensitive to oxygen exposure than the parent strain. Moreover, the isolation of a strain with enhanced aerotolerance and high-level resistance to alkyl hydroperoxides from an ahpCF katB parent suggests that the physiological responses to peroxide toxicity and to the toxic effects of molecular oxygen are overlapping and complex in this obligate anaerobe.

Characterization of a peroxide-resistant mutant of the anaerobic Bacterium bacteroides fragilis.

A Bacteroides fragilis mutant resistant to hydrogen peroxide and alkyl peroxide was isolated by enrichment in increasing concentrations of hydrogen peroxide. The mutant strain was constitutively resistant to 100 mM H2O2 and 5 mM cumene hydroperoxide (15-min exposure). In contrast, the parent strain was protected against <10 mM H2O2 when the peroxide response was induced with a sublethal concentration of H2O2, and no protection was observed in untreated cells. In addition, catalase activity in the mutant strain was not repressed in anaerobic cultures as reported previously for the parent strain. Comparison of the protein profile of crude extracts of the B. fragilis strains revealed that at least three oxidative stress-induced proteins in the parent strain were constitutively expressed in the mutant as detected by nondenaturing polyacrylamide gel electrophoresis. N-terminal amino acid sequence of these overexpressed proteins confirmed the presence of a deregulated catalase (KatB), an alkyl hydroperoxidase reductase subunit C (AhpC), and a Dps/PexB homologue. Northern blot analysis and katB::cat transcriptional fusion studies revealed that in the mutant, katB was deregulated compared to the parent and that katB was controlled by a trans-acting regulatory mechanism. Moreover, constitutive expression of KatB and of the AhpC and Dps homologues in the H2O2-resistant mutant suggests that these proteins may share a common oxidative stress transcriptional regulator and may be involved in B. fragilis peroxide resistance.

The redox-sensitive transcriptional activator OxyR regulates the peroxide response regulon in the obligate anaerobe Bacteroides fragilis.

The peroxide response-inducible genes ahpCF, dps, and katB in the obligate anaerobe Bacteroides fragilis are controlled by the redox-sensitive transcriptional activator OxyR. This is the first functional oxidative stress regulator identified and characterized in anaerobic bacteria. oxyR and dps were found to be divergently transcribed, with an overlap in their respective promoter regulatory regions. B. fragilis OxyR and Dps proteins showed high identity to homologues from a closely related anaerobe, Porphyromonas gingivalis. Northern blot analysis revealed that oxyR was expressed as a monocistronic 1-kb mRNA and that dps mRNA was approximately 500 bases in length. dps mRNA was induced over 500-fold by oxidative stress in the parent strain and was constitutively induced in the peroxide-resistant mutant IB263. The constitutive peroxide response in strain IB263 was shown to have resulted from a missense mutation at codon 202 (GAT to GGT) of the oxyR gene [oxyR(Con)] with a predicted D202G substitution in the OxyR protein. Transcriptional fusion analysis revealed that deletion of oxyR abolished the induction of ahpC and katB following treatment with hydrogen peroxide or oxygen exposure. However, dps expression was induced approximately fourfold by oxygen exposure in DeltaoxyR strains but not by hydrogen peroxide. This indicates that dps expression is also under the control of an oxygen-dependent OxyR-independent mechanism. Complementation of DeltaoxyR mutant strains with wild-type oxyR and oxyR(Con) restored the inducible peroxide response and the constitutive response of the ahpCF, katB, and dps genes, respectively. However, overexpression of OxyR abolished the catalase activity but not katB expression, suggesting that higher levels of intracellular OxyR may be involved in other physiological processes. Analysis of oxyR expression in the parents and in DeltaoxyR and overexpressing oxyR strains by Northern blotting and oxyR'::xylB fusions revealed that B. fragilis OxyR does not control its own expression.

Oxidative stress response in an anaerobe, Bacteroides fragilis: a role for catalase in protection against hydrogen peroxide.

Survival of Bacteroides fragilis in the presence of oxygen was dependent on the ability of bacteria to synthesize new proteins, as determined by the inhibition of protein synthesis after oxygen exposure. The B. fragilis protein profile was significantly altered after either a shift from anaerobic to aerobic conditions with or without paraquat or the addition of exogenous hydrogen peroxide. As determined by autoradiography after two-dimensional gel electrophoresis, approximately 28 newly synthesized proteins were detected in response to oxidative conditions. These proteins were found to have a broad range of pI values (from 5.1 to 7.2) and molecular weights (from 12,000 to 79,000). The hydrogen peroxide- and paraquat-inducible responses were similar but not identical to that induced by oxygen as seen by two-dimensional gel protein profile. Eleven of the oxidative response proteins were closely related, with pI values and molecular weights from 5.1 to 5.8 and from 17,000 to 23,000, respectively. As a first step to understanding the resistance to oxygen, a catalase-deficient mutant was constructed by allelic gene exchange. The katB mutant was found to be more sensitive to the lethal effects of hydrogen peroxide than was the parent strain when the ferrous iron chelator bipyridyl was added to culture media. This suggests that the presence of ferrous iron in anaerobic culture media exacerbates the toxicity of hydrogen peroxide and that the presence of a functional catalase is important for survival in the presence of hydrogen peroxide. Further, the treatment of cultures with a sublethal concentration of hydrogen peroxide was necessary to induce resistance to higher concentrations of hydrogen peroxide in the parent strain, suggesting that this was an inducible response. This was confirmed when the bacterial culture, treated with chloramphenicol before the cells were exposed to a sublethal concentration of peroxide, completely lost viability. In contrast, cell viability was greatly preserved when protein synthesis inhibition occurred after peroxide induction. Complementation of catalase activity in the mutant restored the ability of the mutant strain to survive in the presence of hydrogen peroxide, showing that the catalase (KatB) may play a role in oxidative stress resistance in aerotolerant anaerobic bacteria.

The complex oxidative stress response of Bacteroides fragilis: the role of OxyR in control of gene expression.

Gram-negative anaerobes in the genus Bacteroides are the predominant members of the GI-tract microflora where they play an important role in normal intestinal physiology. Bacteroides spp. also are significant opportunistic pathogens responsible for an array of intra-abdominal and other infections. Bacteroides fragilis is the most common anaerobic pathogen and it possesses virulence factors such as a capsule and neuraminidase that contribute to its success as a pathogen. Infection occurs when organisms escape from the anaerobic colon to aerobic sites such as the peritoneum where O(2) concentrations average 6%. Thus in addition to the classic virulence factors, resistance to oxidative stress is essential and may be involved in the initiation and persistence of infection. In fact, B. fragilis is highly O(2) tolerant, surviving extended periods (>24h) of O(2) exposure without a significant affect on viability. For protection against this oxidative stress B. fragilis mounts a complex physiological response that includes induction of >28 proteins involved in detoxification of oxygen radicals, protection of macromolecules, and adaptive physiology. One experimental strategy used to characterize this oxidative stress response is the direct detection of genes and proteins induced during exposure to O(2) or H(2)O(2). The methods employed have included RNA differential display to capture unique mRNA transcripts produced during oxidative stress, and native or 2D-gel electrophoresis to isolate and identify newly formed stress-induced proteins. Using these and other approaches a wide array of genes induced by oxidative stress have been discovered. These include genes for catalase, superoxide dismutase, thioredoxin-peroxidase, p20-peroxidase, cytochrome c peroxidase, Dps, alkyl hydroperoxidase, aerobic ribonucleotide reductase, ruberythrin, starch utilization, aspartate decarboxylase, and an RNA binding protein. The genes encoding these activities fall into three regulatory classes: (1) induced by O(2) only, (2) induced by H(2)O(2) only, and (3) induced by either O(2) or H(2)O(2). Such a complex regulatory response will likely involve multiple regulators. Thus far one regulator has been identified, OxyR, which controls a subset of the class 3 genes that are induced by either O(2) or H(2)O(2). OxyR responds rapidly to oxidative stress and transcriptional analyses have shown that OxyR-controlled genes are activated by as little as 0.5% O(2) or 10 microM H(2)O(2). Maximal expression of most OxyR regulon genes was reached at 50 microM H(2)O(2) and 2% O(2). These oxidant concentrations are similar to environmental levels that would be experienced by the organisms in tissues outside of the colon suggesting that the OxyR regulon would be induced during the course of an infection.