Inhibitor-associated transposition events in Corynebacterium glutamicum.

In up to 100% of all bacteria grown in the presence of initially inhibitory concentrations of five diverse inhibitors, an extra copy of the resident insertion element IS 31831 was found in specific chromosomal regions, the sites of which apparently depended on the inhibitor used. Thus, in nine out of nine independently isolated cyanide-associated transpositions, the acquired copy was located within an ORF encoding a protein related to the hypothetical but conserved protein YeiH of Escherichia coli. A putative Sox box upstream of the yeiH gene implicates superoxide as a potential regulator of the gene, a possibility further supported by the finding that superoxide dismutase (SodA) is overexpressed in cells cultured in cyanide-containing medium. Neither the cyanide-associated nor any of the other transposition mutations appeared to confer any discernible phenotypic advantage upon cells grown in the presence or absence of the inhibitors, as revealed most stringently by mixed-cell experiments. An alternative, albeit heterodox, explanation for the emergence of the mutants postulates a very high rate of transpositional activity in the presence of inhibitors. The initial emergence of the mutants was found to depend crucially upon the cell density. Thus, when growth medium was supplemented with 50 mM fluoropyruvate and inoculated to a density of 2 x 10(7) cfu/ml, single colonies with heterogeneous restriction fragment length polymorphisms (RFLPs) were routinely isolated at a frequency of 6 to 16% after 1-2 days of incubation. After 3 days, 10-36% of the colonies showed RFLPs, but the type was now dominated by the fluoropyruvate-specific RFLP, which, at higher resolution, invariably proved to be heterogeneous. This heterogeneity proved that these specific mutants were of multiple origin, indicating that clonal enrichment was irrelevant to their emergence. It is suggested that the presence of the inhibitor induces the development of hyper-transpositional activity, which is regulated by a soluble bacterial product.

Common solvent toxicity: autoxidation of respiratory redox-cyclers enforced by membrane derangement.

Unspecific biological effects of chemically diverse solvents strikingly reveal the unifying motif of oxidant toxicity both in higher organisms and in aerobic bacteria. In a few spectacular cases, solvent metabolites with oxidant properties were demonstrated, which however cannot explain extrahepatic toxicity, e.g. in muscle and nerve cells. A common source of solvent-inducible oxidants, by contrast, is suggested to be located in mitochondria or, more general, in membranes where the respiratory chain operates. Orderly respiration depends on membrane integrity, which is invariably compromised by exposure to most solvents and many other lipophils. In rat mitochondria, toluene-induced membrane derangement has been directly implicated with superoxide production, resulting from autoxidation of the membrane-located respiratory redox-cycler ubisemiquinone. A related mechanism may occur in bacteria: Exposure of Escherichia coli to lipophils such as ethanol, tetralin, indole, chlorpromazine and procaine, or to heat shock, induces anti-oxidant proteins, which are reliable indicators of increased oxidant levels. Although many molecular details remain to be elucidated, this review documents that oxidant toxicity of lipophilic compounds is a common physiological phenomenon correlated with derangement of membranes where respiratory processes take place. Subjective consequences of acute oxidant injury are probably the hangover from alcohol and nicotine consumption, and the sudden death from recreational solvent abuse. Suggestions concerning oxidants as major contributors to ageing remain unchallenged.

Indolyl carboxylic acids by condensation of indoles with alpha-keto acids.

The novel indole derivatives 2,2-bis(3,3'-indolyl)propionic acid (1); 1,1,1,-tris(3,3',3"-indolyl)ethane (2); and 2,2-bis(3, 3'-indolyl)isocaproic acid (3) were isolated from solvent extracts of indole-supplemented supernatants of Escherichia coli and corynebacteria. The compounds were also obtained by chemical synthesis: compounds 1 and 2 from indole and pyruvic acid and compound 3 from indole and alpha-ketoisocaproic acid, following incubation at 37 degrees C in aqueous medium. Tryptophan and pyruvic acid gave the novel 2-(2-tryptophanyl)lactic acid (4). The condensation reaction between indoles and alpha-keto acids was of general nature, and the mild reaction conditions suggested it may proceed in vivo. Examples for endogenous occurrence may be the neuro-degenerative diseases phenylketonuria and maple syrup urine disease, both characterized by elevated blood levels of alpha-keto acids.

Indole-inducible proteins in bacteria suggest membrane and oxidant toxicity.

Oxidant toxicity of indole was demonstrated by the induction of alkylhydroperoxide reductase subunit C (AhpC) in Escherichia coli K12 and by the constitutive overproduction of AhpC in a variant of E. coli JM109 with enhanced resistance to indole. Oxidant toxicity was also indicated in an indole-adapted variant of Brevibacterium flavum by the indole-inducible overproduction of a novel 36-kDa protein with N-terminal sequence similarity to proteins involved in superoxide and singlet oxygen resistance. It is proposed that indole dissolved in membrane lipids, which caused membrane derangement and enabled direct interaction of redox-cycling isoprenoid quinones and dioxygen, resulting in the generation of superoxide. A direct indication of membrane derangement in E. coli may be the indole-inducible overproduction of spheroplast protein y (Spy).

Response to reactive nitrogen intermediates in Mycobacterium tuberculosis: induction of the 16-kilodalton alpha-crystallin homolog by exposure to nitric oxide donors.

In contrast to the apparent paucity of Mycobacterium tuberculosis response to reactive oxygen intermediates, this organism has evolved a specific response to nitric oxide challenge. Exposure of M. tuberculosis to NO donors induces the synthesis of a set of polypeptides that have been collectively termed Nox. In this work, the most prominent Nox polypeptide, Nox16, was identified by immunoblotting and by N-terminal sequencing as the alpha-crystallin-related, 16-kDa small heat shock protein, sHsp16. A panel of chemically diverse donors of nitric oxide, with the exception of nitroprusside, induced sHsp16 (Nox16). Nitroprusside, a coordination complex of Fe2+ with a nitrosonium (NO+) ion, induced a 19-kDa polypeptide (Nox19) homologous to the nonheme bacterial ferritins. We conclude that the NO response in M. tuberculosis is dominated by increased synthesis of the alpha-crystallin homolog sHsp16, previously implicated in stationary-phase processes and found in this study to be a major M. tuberculosis protein induced upon exposure to reactive nitrogen intermediates.

Major surface glycoprotein genes from Pneumocystis carinii f. sp. ratti.

Pneumocystis carinii occurs in a variety of mammals, each of which harbors one or more genetically distinct "special forms" of the microbe. Laboratory rats can be infected by two special forms, P. carinii f. sp. ratti and P. carinii f. sp. carinii. P. carinii f. sp. carinii has a variable antigen, the major surface glycoprotein (MSG), the expression of which is controlled by genetic recombination. Recombination may involve the CRJE, a 23-bp DNA sequence element invariant among P. carinii f. sp. carinii MSG genes. To better understand the role of the CRJE in MSG gene expression and to explore the possible role of MSG in P. carinii infection in rats, P. carinii f. sp. ratti MSG genes were studied. These genes were found to be related to MSG genes of P. carinii f. sp. carinii, but less so than MSG genes from P. carinii f. sp. carinii are to each other. P. carinii f. sp. ratti MSG genes were present throughout the genome and were expressed as an abundant mRNA species slightly smaller than that found in P. carinii f. sp. carinii. P. carinii f. sp. ratti MSG transcripts included a CRJE-like sequence only 78% identical to the CRJE of P. carinii f. sp. carinii. Comparison of MSG proteins from the two rat special forms of P. carinii to those from human, ferret, and mouse P. carinii did not support the hypothesis that growth in the rat lung requires certain primary MSG peptide sequences.

Isoniazid induces expression of the antigen 85 complex in Mycobacterium tuberculosis.

Exposure to isoniazid induced the expression of several secreted proteins in Mycobacterium tuberculosis H37Rv. Two-dimensional gel electrophoresis and immunoblot analyses indicated that two of the prominent isonicotinic acid hydrazide-inducible polypeptides were members of the antigen 85 complex, recently demonstrated to have mycolyltransferase activity. We postulate the existence of an intermediate, whose production is inhibited by isonicotinic acid hydrazide, which plays a negative feedback regulatory role in the metabolism of mycolic acids are revealed by the overexpression of the antigen 85 complex. The approach described here relies on analyses of differential gene expression following exposure to inhibitors and may become a more general tool in dissecting the effects of antimicrobial agents.

Response of Mycobacterium tuberculosis to reactive oxygen and nitrogen intermediates.

BACKGROUND: Mycobacterium tuberculosis is a significant human pathogen capable of replicating in mononuclear phagocytic cells. Exposure to reactive oxygen and nitrogen intermediates is likely to represent an important aspect of the life cycle of this organism. The response of M. tuberculosis to these agents may be of significance for its survival in the host. MATERIALS AND METHODS: Patterns of de novo proteins synthesized in M. tuberculosis H37Rv exposed to compounds that generate reactive oxygen and nitrogen intermediates were studied by metabolic labeling and two-dimensional electrophoresis. RESULTS: Menadione, a redox cycling compound which increases intracellular superoxide levels, caused enhanced synthesis of seven polypeptides, six of which appeared to be heat shock proteins. Chemical release of nitric oxide induced eight polypeptides of which only one could be identified as a heat shock protein. Nitric oxide also exhibited a mild inhibitory action on general protein synthesis in the concentration range tested. Hydrogen peroxide did not cause differential gene expression and exerted a generalized inhibition in a dose-dependent manner. Cumene hydroperoxide caused mostly inhibition but induction of two heat shock proteins was detectable. CONCLUSIONS: The presented findings indicate major differences between M. tuberculosis and the paradigms of oxidative stress response in enteric bacteria, and are consistent with the multiple lesions found in oxyR of this organism. The effect of hydrogen peroxide, which in Escherichia coli induces eight polypeptides known to be controlled by the central regulator oxyR, appears to be absent in M. tuberculosis. Superoxide and nitric oxide responses, which in E. coli overlap and are controlled by the same regulatory system soxRS, represent discrete and independent phenomena in M. tuberculosis.

X-ray structure analysis of the iron-dependent superoxide dismutase from Mycobacterium tuberculosis at 2.0 Angstroms resolution reveals novel dimer-dimer interactions.

The X-ray structure of the tetrameric iron-dependent superoxide dismutase from Mycobacterium tuberculosis has been refined to an R-factor of 0.167 and a correlation coefficient of 0.954 at 2.0 A resolution. The crystals are monoclinic P2(1) and have four subunits related by strong non-crystallographic 222 (or D2) symmetry in the asymmetric unit. 198 of the 207 amino acids of each subunit are defined by the electron density which shows that they adopt the conserved fold of other iron- or manganese-dependent SODs. The structure can be divided into two domains, the N-terminal domain involving an extended region followed by two projecting antiparallel alpha-helices, and the C-terminal domain containing four more helical segments with a three-stranded antiparallel beta-sheet inserted sequentially between the fourth and fifth helices. The catalytic iron is co-ordinated by five ligands: three histidines (residues 28, 76 and 164), one aspartate (160) and a solvent molecule. The inferred positions of protons at the active site are consistent with the solvent ligand being a hydroxide ion. This ligand interacts with His145 in the Mycobacterium tuberculosis SOD. In the highly homologous Mycobacterium leprae Mn-SOD, the histidine is replaced by glutamine, this being the only significant residue difference within 10 A of the Fe3+. The nature of the amino acid at this position may influence the metal ion specificity of these enzymes. The subunits of the Mycobacterium tuberculosis SOD associate by polar contacts to form dimers, which closely resemble those of other dimeric or tetrameric Fe- or Mn-SODs. However, the dimer-dimer interactions within the tetramer are novel, being dominated by dimerisation of the 144 to 152 loop regions which connect the outer two beta-strands of the three-membered beta-sheet. This contrasts strongly with the other tetrameric Fe- or Mn-SODs where the dimer-dimer association is dominated by the projecting alpha alpha-turn in the N-terminal domain.

Molecular characterization of clustered variants of genes encoding major surface antigens of human Pneumocystis carinii.

A 13-kb genomic fragment from human Pneumocystis carinii was cloned as repetitive DNA. The fragment contains a cluster of three related genes, each 3 kb in size, and the 5' end of a fourth gene. The predicted polypeptide of the first gene in the cluster comprises 1,030 amino acid residues with a total molecular mass of 116 kDa. The gene's predicted amino acid sequence bears 32% identity to predicted sequences of recently described gene fragments of ferret P. carinii, which encode an immunodominant surface glycoprotein (gpA) (P. J. Haidaris, T. W. Wright, F. Gigliotti, and C. G. Haidaris, J. Infect. Dis. 166:1113-1123, 1992), and 36% identity to the predicted sequence of a rat P. carinii major surface glycoprotein gene (msg) (J. A. Kovacs, F. Powell, J. C. Edman, B. Lundgren, A. Martinez, B. Drew, and C. W. Angus, J. Biol. Chem. 268:6034-6040). DNA hybridization showed that sequences related to the cloned msg genes reside on at least 12 chromosomes of human P. carinii at various degrees of multiplicity and/or homology. Affinity-purified antibodies with specificity to a fusion protein made from the human P. carinii msgI gene recognized two bands on a Western immunoblot containing total human P. carinii protein; they also recognized fusion proteins derived from the other two genes of the cluster. Monoclonal antibodies with reactivity to Msg of human P. carinii recognized fusion proteins produced from two msg genes. Fusion proteins were also recognized by sera from healthy humans and from patients. The msg genes are candidates for the development of immunotherapy and subunit vaccines for the treatment and prevention of P. carinii pneumonia.

Transformation of mycobacterial species using hygromycin resistance as selectable marker.

Electroporation with shuttle plasmids carrying a kanamycin resistance gene as a selectable marker failed to generate transformants in two mycobacterial species currently being used in human vaccine trials (Mycobacterium w and Mycobacterium vaccae). In contrast, efficient transformation [10(3)-10(5) transformants (micrograms DNA)-1] was obtained using novel vectors with selection based on expression of resistance to hygromycin. The hygromycin resistance vector was also found to be more efficient than kanamycin resistance vectors for transformation of Mycobacterium smegmatis and Mycobacterium bovis BCG. The hygromycin resistance vector was used to overexpress superoxide dismutase of Mycobacterium tuberculosis in M. vaccae in a form suitable for detailed structural analysis. The potential use of this approach for generation of novel recombinant mycobacterial vaccines is discussed.

Heat shock proteins and infection: interactions of pathogen and host.

Invasive microorganisms encounter defensive attempts of the host to starve, destroy and eliminate the infection. In experimental model systems aiming to imitate defensive actions of the host, microorganisms respond by the rapid acceleration in the rate of expression of heat shock and other stress proteins. Heat shock proteins (hsp) of most if not all pathogens are major immune targets for both B- and T-cells. Host cells involved in the defensive action cannot avoid exposure to their own reactive compounds, such as oxygen radicals, resulting in premature cell death and tissue damage. Long-term consequences to the host may include cancer. In cells in tissue culture, induction of host-specific hsps occurs upon exposure to oxidants and in viral infections. Drugs that bind to members of the hsp70 family induce peroxisome proliferation and hepatocarcinoma, but may open the way for the development of novel drugs in support of antimetabolite treatment of infections and cancer.

Heat shock proteins and antigens of Mycobacterium tuberculosis.

The heat shock response of Mycobacterium tuberculosis has been characterized in detail by one- and two-dimensional polyacrylamide gel electrophoresis after metabolic labeling with [35S]methionine and 14C-amino acids. A temperature increase from 37 to 42 degrees C induced elevated synthesis of three major proteins corresponding to the DnaK, GroEL, and GroES proteins of M. tuberculosis previously identified as prominent antigens. At higher temperatures (45 to 48 degrees C), synthesis of GroEL decreased and novel heat shock proteins with molecular masses of 90, 28, 20, and 15 kDa were observed. These new proteins did not comigrate with known antigens during two-dimensional gel electrophoresis. The heat shock response is discussed with regard to the possible importance of transcriptional regulation of mycobacterial genes in vivo.

Lipoprotein antigens of Mycobacterium tuberculosis.

Detergent phase separation and metabolic labelling have been used to screen for the presence of lipoproteins amongst the antigens of Mycobacterium tuberculosis. Both techniques indicated that four antigens, with subunit molecular weights of 19, 26, 27 and 38 kilodaltons (kDa), are lipoproteins. This finding is consistent with the presence of conserved cysteine residues characteristic of other bacterial lipoproteins within the amino terminal sequences of the 38 kDa and 19 kDa proteins. It is proposed that lipoproteins are involved in the induction of humoral and cellular immune responses to mycobacteria and have a functional role in the transport of nutrients through the mycobacterial cell wall.