Michael H. Malamy

The strict anaerobe Bacteroides fragilis grows in and benefits from nanomolar concentrations of oxygen.

Strict anaerobes cannot grow in the presence of greater than 5 µM dissolved oxygen. Despite this growth inhibition, many strict anaerobes of the Bacteroides class of eubacteria can survive in oxygenated environments until the partial pressure of O2 (pO2) is sufficiently reduced. For example, the periodontal pathogens Porphyromonas gingivalis and Tannerella forsythensis colonize subgingival plaques of mammals, whereas several other Bacteroides species colonize the gastrointestinal tract of animals. It has been suggested that pre-colonization of these sites by facultative anaerobes is essential for reduction of the pO2 and subsequent colonization by strict anaerobes. However, this model is inconsistent with the observation that Bacteroides fragilis can colonize the colon in the absence of facultative anaerobes. Thus, this strict anaerobe may have a role in reduction of the environmental pO2. Although some strictly anaerobic bacteria can consume oxygen through an integral membrane electron transport system, the physiological role of this system has not been established in these organisms. Here we demonstrate that B. fragilis encodes a cytochrome bd oxidase that is essential for O2 consumption and is required, under some conditions, for the stimulation of growth in the presence of nanomolar concentrations of O2. Furthermore, our data suggest that this property is conserved in many other organisms that have been described as strict anaerobes.

Beta-lactamase-mediated imipenem resistance in Bacteroides fragilis.

Imipenem has excellent antimicrobial activity owing in part to beta-lactamase stability. We found that only 2 of over 350 Bacteroides fragilis group clinical isolates were resistant to imipenem, with an MIC of more than 16 micrograms/ml. These two isolates from the Tufts Anaerobe Laboratory (TAL) were resistant to all other beta-lactam agents tested. The organisms were able to inactivate imipenem in broth cultures and contained similar beta-lactamases that were able to hydrolyze carbapenems, cephamycins, cephalosporins, and penicillins. The molecular sizes of the beta-lactamases in TAL2480 and TAL3636 were estimated to be 44,000 daltons. The novel beta-lactamase contained Zn2+ as a cofactor. An additional factor contributing to resistance was determined. The outer membranes of these two organisms were found to limit free diffusion of the drugs into the periplasm. This novel beta-lactamase, associated with a barrier to drug permeation, resulted in high-grade beta-lactam drug resistance.

Electron Microscopy of Polar Insertions in the lac Operon of Escherichia coli

SummarySeveral strongly polar mutations in the omega region of the z gene of the lac operon result from insertions consisting of only two specific sequences of DNA, one about 870 and the other 1170 nucleotide pairs long (based on single-strand measurements). No sequence homology was detected between the shorter (IS1) and longer (IS3) insertions. The IS1 insertion was shown to possess a specific attachment site, but it can be inserted with either orientation at several sites in the z gene. Four insertion sites in the omega region of gene z were identified and the position of the lac5 substitution and the SR2 deletion in the λplac DNA were determined by heteroduplex mapping.

A mitochondrial-like aconitase in the bacterium Bacteroides fragilis: Implications for the evolution of the mitochondrial Krebs cycle

Aconitase and isocitrate dehydrogenase (IDH) enzyme activities were detected in anaerobically prepared cell extracts of the obligate anaerobe Bacteroides fragilis. The aconitase gene was located upstream of the genes encoding the other two components of the oxidative branch of the Krebs cycle, IDH and citrate synthase. Mutational analysis indicates that these genes are cotranscribed. A nonpolar in-frame deletion of the acnA gene that encodes the aconitase prevented growth in glucose minimal medium unless heme or succinate was added to the medium. These results imply that B. fragilis has two pathways for α-ketoglutarate biosynthesis—one from isocitrate and the other from succinate. Homology searches indicated that the B. fragilis aconitase is most closely related to aconitases of two other Cytophaga–Flavobacterium–Bacteroides (CFB) group bacteria, Cytophaga hutchinsonii and Fibrobacter succinogenes. Phylogenetic analysis indicates that the CFB group aconitases are most closely related to mitochondrial aconitases. In addition, the IDH of C. hutchinsonii was found to be most closely related to the mitochondrial/cytosolic IDH-2 group of eukaryotic organisms. These data suggest a common origin for these Krebs cycle enzymes in mitochondria and CFB group bacteria.

Arsenate resistant mutants of Escherichia coli and phosphate transport

Abstract Arsenate is a substrate for an inorganic phosphate transport system in E . coli U7. Mutants selected for growth in the presence of high concentrations of arsenate lose the capacity to transport arsenate at all concentrations. Most arsenate resistant mutants retain the ability to grow on orthophosphate. Phosphate transport in these mutants is completely abolished in the presence of cyanide, but in the wild type partial activity remains under the same conditions. Strains completely dependent on organic phosphate have also been found. Phosphate transport in the wild type U7 must be mediated by at least two separable systems, one specific for phosphate and the other active for both phosphate and arsenate.

Identification, cloning and expression of the mouse N-acetylglutamate synthase gene.

In ureotelic animals, N-acetylglutamate (NAG) is an essential allosteric activator of carbamylphosphate synthetase I (CPSI), the first enzyme in the urea cycle. NAG synthase (NAGS; EC 2.3.1.1) catalyses the formation of NAG from glutamate and acetyl-CoA in liver and intestinal mitochondria. This enzyme is supposed to regulate ureagenesis by producing variable amounts of NAG, thus modulating CPSI activity. Moreover, inherited deficiencies in NAGS have been associated with hyperammonaemia, probably due to the loss of CPSI activity. Although the existence of the NAGS protein in mammals has been known for decades, the gene has remained elusive. We identified the mouse (Mus musculus) and human NAGS genes using their similarity to the respective Neurospora crassa gene. NAGS was cloned from a mouse liver cDNA library and was found to encode a 2.3 kb message, highly expressed in liver and small intestine with lower expression levels in kidney, spleen and testis. The deduced amino acid sequence contains a putative mitochondrial targeting signal at the N-terminus. The cDNA sequence complements an argA (NAGS)-deficient Escherichia coli strain, reversing its arginine auxotrophy. His-tagged versions of the pre-protein and two putative mature proteins were each overexpressed in E. coli, and purified to apparent homogeneity by using a nickel-affinity column. The pre-protein and the two putative mature proteins catalysed the NAGS reaction but one of the putative mature enzymes had significantly higher activity than the pre-protein. The addition of l-arginine increased the catalytic activity of the purified recombinant NAGS enzymes by approx. 2-6-fold.

Sialic Acid (N-Acetyl Neuraminic Acid) Utilization by Bacteroides fragilis Requires a Novel N-Acetyl Mannosamine Epimerase

We characterized the nanLET operon in Bacteroides fragilis, whose products are required for the utilization of the sialic acid N-acetyl neuraminic acid (NANA) as a carbon and energy source. The first gene of the operon is nanL, which codes for an aldolase that cleaves NANA into N-acetyl mannosamine (manNAc) and pyruvate. The next gene, nanE, codes for a manNAc/N-acetylglucosamine (NAG) epimerase, which, intriguingly, possesses more similarity to eukaryotic renin binding proteins than to other bacterial NanE epimerase proteins. Unphosphorylated manNAc is the substrate of NanE, while ATP is a cofactor in the epimerase reaction. The third gene of the operon is nanT, which shows similarity to the major transporter facilitator superfamily and is most likely to be a NANA transporter. Deletion of any of these genes eliminates the ability of B. fragilis to grow on NANA. Although B. fragilis does not normally grow with manNAc as the sole carbon source, we isolated a B. fragilis mutant strain that can grow on this substrate, likely due to a mutation in a NAG transporter; both manNAc transport and NAG transport are affected in this strain. Deletion of the nanE epimerase gene or the rokA hexokinase gene, whose product phosphorylates NAG, in the manNAc-enabled strain abolishes growth on manNAc. Thus, B. fragilis possesses a new pathway of NANA utilization, which we show is also found in other Bacteroides species.

Characterization of the BatI (Bacteroides aerotolerance) operon in Bacteroides fragilis: isolation of a B. fragilis mutant with reduced aerotolerance and impaired growth in in vivo model systems

YT135.2.8, a Tn4400′ insertion mutant of Bacteroides fragilis strain TM4000, grows poorly when used to infect Monika or Chinese hamster ovary (CHO) cell monolayers and is outcompeted by wild‐type strains in mixed infections. YT135.2.8 also shows defects in the rat granuloma pouch model system in monoculture and is completely outcompeted by the wild‐type strain in a mixed infection. In addition, this mutant shows defects in a new model system consisting of CHO suspension cell columns. All of these defects may be explained by the finding that YT135.2.8 shows decreased tolerance to exposure to atmospheric oxygen (less aerotolerant). The monolayer growth defect (MGD) of YT135.2.8 can be influenced significantly by the presence of sulphur‐containing reducing agents (cysteine, dithiothreitol, thiodiglycol) or the non‐sulphur reducing agent Tris‐(2‐carboxylethyl)phosphine (TCEP). The defects in YT135.2.8 can be complemented by a 6.6 kb fragment of the B. fragilis chromosome. DNA sequencing of this fragment and of the regions flanking the Tn4400′ insertion in the B. fragilis chromosome revealed the presence of five open reading frames, corresponding to genes bat (Bacteroides aerotolerance) A, B, C, D, E, which form the BatI operon; Tn4400′ inserted within batD. All of the hypothetical proteins possess one or more membrane‐spanning domains. BatA and BatB show high similarity to each other but, like BatD, they show no match to sequences of known function in the databases. BatC and BatE contain 2–4 repeated sequences similar to the tetratricopeptide repeats (TPRs) seen in many eukaryotic proteins. The function of TPR sequences in protein interactions in other systems leads to the suggestion that the Bat proteins form a complex. The BatI complex may be involved in the generation or export of reducing power equivalents to the periplasm of the B. fragilis cell.

Role of the F factor oriV1 region in recA-independent illegitimate recombination: Stable replicon fusions of the F derivative pOX38 and pBR322-related plasmids☆

We have used a mating protocol to isolate recA-independent recombinants of pOX38 , an F factor derivative, and the non-conjugative plasmid pMBO311 . Plasmid pMBO311 is a derivative of pBR322 carrying a DNA insertion that contains IS121 and shows no extensive sequence homology to pOX38 . Twenty-seven cointegrate molecules formed during independent mobilizations of pMBO311 by pOX38 were examined by restriction and Southern hybridization analysis. In general, there were two classes of recombinants. A minority class appears to have been mediated by IS121 , resulting in the formation of cointegrate molecules containing IS121 at the junctions between the two plasmids. The majority class (23/27) apparently involved reciprocal recombination between sites on pOX38 and pMBO311 . IS121 does not seem to be responsible for the formation of this type of cointegrate molecule, since similar structures were generated at approximately the same frequency during mobilization of control plasmids that do not contain IS121 . We have localized the regions involved in this second class of recombination events and find that most (17/23) occur at or near oriV1 , the primary replication initiation site of pOX38 . Twelve of the cointegrate molecules showed identical restriction and Southern hybridization patterns demonstrating a preferred region on pMBO311 as well. This site was localized just distal to the tet genes, within a 640-base AvaI-PvuII segment in the pBR322 portion of the molecule.

Acetylornithine Transcarbamylase: a Novel Enzyme in Arginine Biosynthesis

Ornithine transcarbamylase is a highly conserved enzyme in arginine biosynthesis and the urea cycle. In Xanthomonas campestris, the protein annotated as ornithine transcarbamylase, and encoded by the argF gene, is unable to synthesize citrulline directly from ornithine. We cloned and overexpressed this X. campestris gene in Escherichia coli and show that it catalyzes the formation of N-acetyl-L-citrulline from N-acetyl-L-ornithine and carbamyl phosphate. We now designate this enzyme as an acetylornithine transcarbamylase. The K(m) values for N-acetylornithine and carbamyl phosphate were 1.05 mM and 0.01 mM, respectively. Additional putative transcarbamylases that might also be misannotated were found in the genomes of members of other xanthomonads, Cytophaga, and Bacteroidetes as well as in DNA sequences of bacteria from environmental isolates. It appears that these different paths for arginine biosynthesis arose very early in evolution and that the canonical ornithine transcarbamylase-dependent pathway became the prevalent form. A potent inhibitor, N(alpha)-acetyl-N(delta)-phosphonoacetyl-L-ornithine, was synthesized and showed a midpoint of inhibition at approximately 22 nM; this compound may prove to be a useful starting point for designing inhibitors specific to this novel family of transcarbamylases.