Lawrence G. Wayne

Role of narK2X and narGHJI in Hypoxic Upregulation of Nitrate Reduction by Mycobacterium tuberculosis

Mycobacterium tuberculosis is one of the strongest reducers of nitrate in the genus Mycobacterium: Under microaerobic conditions, whole cells exhibit upregulation of activity, producing approximately eightfold more nitrite than those of aerobic cultures of the same age. Assays of cell extracts from aerobic cultures and hypoxic cultures yielded comparable nitrate reductase activities. Mycobacterium bovis produced only low levels of nitrite, and this activity was not induced by hypoxia. M. tuberculosis has two sets of genes, narGHJI and narX of the narK2X operon, that exhibit some degree of homology to prokaryotic dissimilatory nitrate reductases. Each of these were knocked out by insertional inactivation. The narG mutant showed no nitrate reductase activity in whole culture or in cell-free assays, while the narX mutant showed wild-type levels in both assays. A knockout of the putative nitrite transporter narK2 gene produced a strain that had aerobic levels of nitrate reductase activity but failed to show hypoxic upregulation. Insertion of the M. tuberculosis narGHJI into a nitrate reductase Escherichia coli mutant allowed anaerobic growth in the presence of nitrate. Under aerobic and hypoxic conditions, transcription of narGHJI was constitutive, while the narK2X operon was induced under hypoxia, as measured with a lacZ reporter system and by quantitative real-time reverse PCR. This indicates that nitrate reductase activity in M. tuberculosis is due to the narGHJI locus with no detectable contribution from narX and that the hypoxic upregulation of activity is associated with the induction of the nitrate and nitrite transport gene narK2.

Microaerophilic Induction of the Alpha-Crystallin Chaperone Protein Homologue (hspX) mRNA of Mycobacterium tuberculosis

Among the products that are expressed when Mycobacterium tuberculosis undergoes hypoxic shiftdown to nonreplicating persistence (NRP) is the alpha-crystallin chaperone protein homologue (Acr). This expression coincides with the previously reported appearance of a respiratory type of nitrate reductase activity, the increase in glycine dehydrogenase activity, and the production of a unique antigen, URB-1. In a timed sampling study, using a slowly stirred oxygen depletion culture model, we have demonstrated that the hspX mRNA that codes for Acr protein as well as the protein itself is induced just as the bacilli enter the microaerophilic NRP stage 1 (NRP-1). In contrast to the induction observed for hspX mRNA, levels of 16S rRNA, fbpB mRNA (encoding the 85B alpha antigen), and aroB mRNA (encoding dehydroquinate synthase) demonstrate relatively small to no change upon entering NRP-1. Acr protein was shown to be identical to URB-1 by Western analysis with anti-URB-1 antibody. The fact that antibody to Acr is found in a high percentage of tuberculosis patients suggests that the hypoxic shiftdown of tubercle bacilli to the NRP state that occurs in vitro, resulting in production of the alpha-crystallin protein, occurs in vivo as well. Simultaneous abrupt increases in hspX mRNA and Acr protein suggest that Acr protein expression is controlled at the level of transcription.

Actions of the Judicial Commission of the International Committee on Systematic Bacteriology on Requests for Opinions Published Between January 1985 and July 1993

Eleven requests were published between January 1985 and July 1993. Four of these requests were granted and have been published as Opinion 64, replacing and recognizing type strains of Methanobacterium formicicum and Methanobacterium bryantii, respectively; Opinion 65, replacing the type strain of Selenomonas sputigena; Opinion 66, replacing the type strain of Streptococcus mitis; and Opinion 67, rejecting Citrobacter diversum. Six requests were denied, including requests for conservation or recognition of “Rhodococcus lentifragmentus,” “Pediococcus acidilactici,” and “Salmonella enterica,” rejection of Erwinia carnegieana, Pectobacterium carnegieana, and Lactobacillus paracasei, and transfer of the type strain of Methanosaeta concilii to Methanothrix soehngenii. A request based on a proposal for reinterpretation of the position of Xanthomonas maltophilia was not considered by the Judicial Commission because it represented a substantive taxonomic issue rather than a nomenclatural question governed by the Bacteriological Code.

Serological, Taxonomic, and Kinetic Studies of the T and M Classes of Mycobacterial Catalase

Two types of catalase may be found in extracts of mycobacteria, the heat-labile T class and the heat-stable M class. The T-catalase is resistant to 3-amino-1,2,4-triazole and has a Michaelis constant in the range of 3.1 to 6.8 mM H2O2, whereas the M-catalase is inhibited by 3-amino-1,2,4-triazole and has a Michaelis constant in the range of 143 to 156 mM. Some species of mycobacteria produce only one class of catalase, and others produce both. Of the species studied, only Mycobacterium terrae, M. triviale, and M. nonchromogenicum failed to exhibit T-catalase, although all three of these species had M-catalase. Conversely, M. tuberculosis, M. bovis, M. intracellulare, M. avium, M. gastri, M. marinum, and M. xenopi yielded T-catalase but not M-catalase. Six species, M. szulgai, M. simiae, M. kansasii, M. gordonae, M. scrofulaceum, and M. asiaticum, produced both classes. The differences in resistance to heat and 3-amino-1,2,4-triazole were exploited in the development of methods for quantitative serological characterization of one class of catalase in the presence of the other. These techniques were used with three reference sera to produce a branching diagram of divergence of the T-catalases from 13 species of mycobacteria based on measurements of immunological distance. No T-catalase could be demonstrated in another three species. A first-stage study was also carried out with a single reference antiserum to M-catalase from M. kansasii. Representatives of nine mycobacterial species, including the three that produced no T-catalase, were characterized with this reference system, which tends to yield larger immunological distance values than the T-catalase system.

Taxonomic Probability Matrix for Use With Slowly Growing Mycobacteria

A probability matrix for identification was developed from data derived from a series of cooperative studies of slowly growing mycobacteria. The matrix includes feature frequencies exhibited by 14 numerical taxonomy clusters in 34 tests. The clusters correspond to 13 defined species. The matrix is designed primarily to screen strains either for membership in 1 of the 14 taxa or for exclusion from these taxa and, thus, to determine whether the strains are in need of further characterization. The matrix was used in the analysis of 298 strains. Two related parameters were used as decision thresholds. First, the probability of the most likely taxon must be 99 times greater than that of the second most likely taxon. Second, the absolute likelihood of the strain being in the most likely taxon must be at least 0.01 times that of a “perfect” strain of the taxon. By using these thresholds and additional judgments, 83 strains were found to be appropriate for further study, with a likelihood that 53% of these strains belong to unrepresented taxa.

ald of Mycobacterium tuberculosis Encodes both the Alanine Dehydrogenase and the Putative Glycine Dehydrogenase

The putative glycine dehydrogenase of Mycobacterium tuberculosis catalyzes the reductive amination of glyoxylate to glycine but not the reverse reaction. The enzyme was purified and identified as the previously characterized alanine dehydrogenase. The Ald enzyme was expressed in Escherichia coli and had both pyruvate and glyoxylate aminating activities. The gene, ald, was inactivated in M. tuberculosis, which resulted in the loss of all activities. Both enzyme activities were found associated with the cell and were not detected in the extracellular filtrate. By using an anti-Ald antibody, the protein was localized to the cell membrane, with a smaller fraction in the cytosol. None was detected in the extracellular medium. The ald knockout strain grew without alanine or glycine and was able to utilize glycine but not alanine as a nitrogen source. Transcription of ald was induced when alanine was the sole nitrogen source, and higher levels of Ald enzyme were measured. Ald is proposed to have several functions, including ammonium incorporation and alanine breakdown.

Actions of the judicial commission of the international committee on systematic bacteriology on requests for opinions published in 1982

Three requests for Opinions were published in 1982. Two of these, one requesting rejection of the name Nocardia farcinica and one requesting substitution of the family name Escherichiaceae for Enterobacteriaceae, have been denied. The third, requesting confirmation of types designated in the Approved Lists as nomenclatural types, has been awarded.

Reciprocal Immunological Distances of Catalase Derived from Strains of Mycobacterium avium, Mycobacterium tuberculosis, and Closely Related Species

The structural divergence of catalase derived from different mycobacteria has previously been studied in terms of immunological distances as determined by titration with antiserum against catalase extracted from Mycobacterium tuberculosis. This study has been extended by addition of a second reference antiserum: One raised against catalase from M. avium. The two systems show a high reciprocality of immunological distance against one another. The catalase from M. intracellulare is serologically very similar to that of M. avium, and the enzyme from M. bovis is very similar to that of M. tuberculosis. Catalase extracted from BCG, a pathovar of M. bovis, appears to exhibit structural modification which diverges from the parent M. bovis and converges on M. avium. Two classes of catalase have been separated from M. scrofulaceum: One is nonreactive with either reference antiserum, and the other exhibits immunological distances that suggest that M. avium is in a position between M. scrofulaceum and M. tuberculosis.

Immunoprecipitation Studies of Mycobacterial Catalase

Serological techniques have been used to detect small changes in amino acid sequences of specific proteins in closely related biological systems. These changes are believed to reflect evolutionary divergence. In the present study, the differences in binding capacities of catalases from different mycobacteria for a reference antiserum were measured very specifically by assaying the unbound functional enzyme after exposure to antibody. Catalase derived from Mycobacterium tuberculosis has been used to immunize rabbits. The antibody so produced precipitated the enzyme but did not inactivate it. This antibody also precipitated catalase from sonic lysates of other mycobacterial species. The binding capacity of catalase derived from a number of heterologous species for the M. tuberculosis antibody was always lower than that of homologous enzyme. Some species produced catalase that failed to react at all with the reference serum. In other cases, there was evidence of at least two serologically unrelated catalases in a single strain. There was also a limited correlation between rate of inactivation of catalase by heat and the relative antibody-binding capacity of the enzyme. The serological study of catalases offers promise of providing a useful tool for clarifying evolutionary relationships among mycobacterial species.

Actions of the Judicial Commission of the International Committee on Systematic Bacteriology on Requests for Opinions Published in 1983 and 1984

Seven requests for opinions were published in 1983 and 1984. Five of these, requesting replacement of the type strain of Acetobacter aceti subsp. xylinum, requesting rejection of Yersinia pseudotuberculosis subsp. pestis and conservation of Yersinia pestis, requesting a change in the type of Pasteuria ramosa, and requesting conservation of the genera Methanococcus and Methanosarcina with new type species, have been awarded. Two others, one requesting a change in the type strain of Listeria monocytogenes and the other requesting conservation of the genus Micropolyspora over Faenia, have been denied.