Heterologous gene expression in Bacteroides fragilis.

Bacteroides fragilis and other gastrointestinal tract Bacteroides are unusual gram-negative eubacteria in that genes from other gram-negative eubacteria are not expressed when introduced into these organisms. To analyze gene expression in Bacteroides, expression vector and promoter probe (detection) vector systems were developed. The essential feature of the expression vector was the incorporation of a Bacteroides insertion sequence element, IS4351, which possesses promoter activity directed outward from its ends. Genes inserted into the multiple cloning site downstream from an IS4351 DNA fragment were readily expressed in B. fragilis. The chloramphenicol acetyltransferase (cat) structural gene from Tn9 was tested and conferred chloramphenicol resistance on B. fragilis. Both chloramphenicol resistance and CAT activity were shown to be dependent on the IS4351 promoters. Similar results were obtained with the Escherichia coli beta-glucuronidase gene (uidA) but activity was just 30% of the levels seen with cat. Two tetracycline resistance determinants, tetM from Streptococcus agalactiae and tetC from E. coli, also were examined. tetC did not result in detectable tetracycline resistance but the gram-positive tetM gene conferred high-level resistance to tetracycline and minocycline in Bacteroides hosts. Based on the cat results, promoter probe vectors containing the promoterless cat gene were constructed. These vectors were used to clone random B. fragilis promoters from partial genomic libraries and the recombinants displayed a range of CAT activities and chloramphenicol MICs in B. fragilis hosts. In addition, known E. coli promoters (Ptet, Ptac, Ptrc, Psyn, and P1P2rrnB) were tested for activity in B. fragilis. No chloramphenicol resistance or CAT activity was observed in B. fragilis with these promoters.     

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 xylanase 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 xylanase 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.     

Heat shock stress in Bacteroides fragilis

The response to heat shock was investigated in the obligate anaerobe Bacteroides fragilis. The cells responded quickly to stress and synthesised seven heat shock proteins immediately upon exposure to heat. The apparent molecular weights of the seven proteins differed from the apparent molecular weights of the proteins induced by UV irradiation, O₂ and H₂O₂. Heat shock did not induce phage reactivation whereas UV irradiation, O₂ and H₂O₂ did induce phage reactivation systems. Ethanol did not elicit the heat shock response. Two heat resistant B. fragilis mutants were isolated. Both mutants lost the ability to synthesise the same two heat shock proteins. It is concluded that the heat shock response and the responses to UV irradiation, O₂ and H₂O₂ represent two independent groups of stress responses in B. fragilis.     

Cellular control of abscess formation: role of T cells in the regulation of abscesses formed in response to Bacteroides fragilis

Although abscesses are a major sequela of infection, little is known about which cellular events initiate and which prevent this pathologic response. These studies are the first to indicate a role for T cells in the important pathogenic process of abscess development and also in immunity to abscesses induced by Bacteroides fragilis. We have shown that T cells initiate the formation of abscesses in mice after i.p. challenge with B. fragilis. These T cells bear both Ly-1 and Ly-2 surface markers. Nude mice (which have been shown by others to have T cell or T cell precursors) are also able to form abscesses. Cyclophosphamide-treated mice (with depressed T cell function) were not capable of developing abscesses. Reconstitution with normal or nude mouse spleen cells restored this ability. However, reconstitution with anti-Thy-1.2-treated, anti-Ly-1, or anti-Ly-2-treated spleen cells (or a mixture of the two cell populations) failed to allow abscess formation after bacterial challenge. Immunity to abscesses caused by B. fragilis requires two T cells. The first Ly-1-2+ T cell has an IJ surface marker and has been shown to release a small m.w. soluble factor (ITF) that is antigen specific. Immunity to abscesses, however, depends on the interaction of ITF with a second Ly-1-2+ T cell, demonstrated in reconstitution experiments with nude mice. The data presented document a critical role for T cells in abscess induction and suggest the existence of a suppressor-like T cell circuit in immunity to abscesses.     

Cloning and expression of the Bacteroides fragilis YCH46 neuraminidase gene in Eschirichia coli and Bacteroides uniformis

Abstract A neuraminidase-encoding gene nanH of Bacteroides fragilis strain YCH46 was cloned into the cosmid vector pHC79. The nanH gene was subcloned from the cosmid and was located within a 2.2-kb Xho I- Kpn I fragment. Southern hybridization experiments demonstrated that the gene was present as a single copy on the bacterial chromosome. Neuraminidase activity expressed in the initial Escherichia coli clone was approximately 3600-fold lower than that expressed in B. fragilis YCH46. However, when nanH was transferred from E. coli to B. uniformis by mobilization of a shuttle plasmid, the transconjugant expressed 1100-fold higher activity than the E. coli donor did. These results suggest that modes of nanH expression in E. coli and Bacteroides are heterologous.     

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 role of carbon dioxide in glucose metabolism of Bacteroides fragilis

The effect of CO₂ concentration on growth and glucose fermentation of Bacteroides fragilis was studied in a defined mineral medium. Batch culture experiments were done in closed tubes containing CO₂ concentrations ranging from 10% to 100% (with appropriate amounts of bicarbonate added to maintain the pH at 6.7). These experiments revealed that CO₂ had no influence on growth rate or cell yield when the CO₂ concentration was above 30% CO₂ (minimum available CO₂–HCO ₃ ^- , 25.5 mM), whereas a slight decrease in these parameters was observed at 20% and 10% CO₂ (available CO₂–HCO ₃ ^- , 17 and 8.5 mM, respectively). If CO₂–HCO ₃ ^- concentrations were below 10 mM, the lag phase lengthened and a decrease in maximal growth rate and cell yield were observed. The amount of acetate made decreased, while d-lactate concentration increased. A net production of CO₂ allowed growth under conditions of extremely low concentrations of added CO₂.When B. fragilis was grown in continuous culture with 100% CO₂ or 100% N₂, the dilution rate influenced the concentrations of acetate, succinate, propionate, d-lactate, l-malate and formate formed. Decreasing the dilution rate favored propionate and acetate production under both conditions. When the organism was grown with 100% N₂, the amount of propionate formed was greater than the amount of succinate formed at all dilution rates. Except at slow dilution rates the reverse was true when 100% CO₂ was used. B. fragilis was unable to grow at dilution rates faster than 0.154 h^-1 when grown with 100% N₂; the Y _glc ^max was 67.9 g DW cells/mol glucose and m _s was 0.064 mmol glucose/g DW·h. If the gas atmosphere was 100% CO₂ the organism was washed out of the culture when the dilution rate exceeded 0.38 h^-1; the Y _glc ^max was 59.4 g DW cells/mol glucose and m _s was 0.094 mmol glucose/g DW·h.Measurement of the phosphoenolpyruvate (PEP) carboxykinase (E.C. 4.1.1.49) with whole, permeabilized cells of B. fragilis showed an increase of specific enzyme activity with decreasing CO₂ concentrations. The mechanisms used by B. fragilis to adjust to low levels of CO₂ are discussed.     

Cloning and expression in Escherichia coli of a recA-like gene from Bacteroides fragilis

The recombinant plasmid pHG100, containing a 5.2-kb DNA fragment from Bacteroides fragilis, complemented defects in homologous recombination, DNA repair and prophage induction to various levels in an Escherichia coli recA mutant strain. There was no DNA homology between the cloned B. fragilis recA-like gene and E. coli chromosomal DNA. pHG100 produced two proteins with Mr of approx. 39,000 and 37,000 which cross-reacted with antibodies raised against E. coli RecA protein. The production of these proteins was not increased after UV induction. The cloned B. fragilis recA-like gene product did not enhance the production of native but defective E. coli RecA protein after UV irradiation.     

The whcA gene plays a negative role in oxidative stress response of Corynebacterium glutamicum

In this study, we analyzed the whcA gene from Corynebacterium glutamicum , which codes for a homologue of the WhiB-family of proteins. Deletion of the gene did not affect the growth of the mutant cells, indicating that the whcA gene was not essential under ordinary growth conditions. However, cells overexpressing the protein not only showed retarded growth as compared with the wild-type or the Δ whcA mutant cells but also showed increased sensitivity to a variety of oxidants, such as diamide, menadione, and hydrogen peroxide. Thioredoxin reductase activity was repressed in the whcA -overexpressing cells, whereas its activity in the Δ whcA mutant strain was derepressed regardless of the presence of oxidative stress. The whcA gene was constitutively expressed throughout the growth phase and its expression level was not affected by oxidative stress. A set of proteins under the control of whcA were identified by two-dimensional polyacrylamide gel electrophoresis and they were annotated as NADH oxidase, alcohol dehydrogenase, quinone reductase, and cysteine desulfurase. The corresponding genes encoding the identified proteins were not transcribed in Δ sigH mutant cells. Collectively, these data suggest that the whcA gene of C. glutamicum plays a negative role in the sigH -mediated stress response pathway.     

Prevalence of the Bacteroides fragilis Group and Enterotoxigenic Bacteroides fragilis in Immunodeficient Children

Members of the Bacteroides fragilis group are indigenous to the human and animal intestinal microbiota and they are responsible for several endogenous infections. Enterotoxigenic B. fragilis (ETBF) has been associated with acute diarrhea in children and farm animals. Immunodeficient patients are more predisposed to different opportunistic infections, including anaerobic infections. In this study, 130 stool samples were analysed from 56 immunodeficient and 74 healthy children. Enterotoxin production was detected by cytotoxicity assay on HT-29 cells and by PCR. B. fragilis sensu strictu was prevalent in both groups and ETBF species was detected from a single stool sample belonged to an immunodeficient child with AIDS.