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Similarities between Mycobacterium tuberculosis phoP-phoR mutants and the attenuated laboratory strain M. tuberculosis H37Ra in terms of morphological and cytochemical properties, lipid content, gene expression and virulence attenuation prompted us to analyze the functionality of this two-component regulator in the latter strain. Sequence analysis revealed a base substitution resulting in a one-amino-acid change in the likely DNA-binding region of PhoP in H37Ra relative to H37Rv. Using gel-shift assays, we show that this mutation abrogates the ability of the H37Ra PhoP protein to bind to a 40-bp segment of its own promoter. Consistent with this result, the phoP gene from H37Rv but not that from H37Ra was able to restore the synthesis of sulfolipids, diacyltrehaloses and polyacyltrehaloses in an isogenic phoP-phoR knock-out mutant of M. tuberculosis Moreover, complementation of H37Ra with phoP from H37Rv fully restored sulfolipid, diacyltrehalose and polyacyltrehalose synthesis, clearly indicating that the lack of production of these lipids in H37Ra is solely due to the point mutation in phoP. Using a pks2-3/4 knock-out mutant of M. tuberculosis H37Rv, evidence is further provided that the above-mentioned polyketide-derived acyltrehaloses do not significantly contribute to the virulence of the tubercle bacillus in a mouse model of infection. Reasons for the attenuation of H37Ra thus most likely stand in other virulence factors, many of which are expected to belong to the PhoP regulon and another of which, unrelated to PhoP, appears to be the lack of production of phthiocerol dimycocerosates in this strain.

The Mycobacterium tuberculosis acyl-coenzyme A (CoA) carbnxylases provide the building blocks for de novo fatty acid biosynthesis by fatty acid synthase I (FAS I) and for the elongation of FAS I end products by the FAS II complex to produce meromycolic acids. The M. tuberculosis genome contains three biotin carboxylase subunits (AccA1 to -3) and six carboxyltransferase subunits (AccD1 to -6), with accD6 located in a genetic locus that contains members of the FAS II complex. We found by quantitative real-time PCR analysis that the transcripts of accA3, accD4, accD5, and accD6 are expressed at high levels during the exponential growth phases of M. tuberculosis in vitro. Microarray analysis of M. tuberculosis transcripts indicated that the transcripts for accA3, accD4, accD5, accD6, and accE were repressed during later growth stages. AccD4 and AccD5 have been previously studied, but there are no reports on the function of AccD6. We expressed AccA3 ([[alpha].sub.3]) and AccD6 ([[beta.sub.6]) in E. coli and purified them by affinity chromatography. We report here that reconstitution of the [[alpha.sub.3]-[[beta.sub.6] complex yielded an active acyl-CoA carboxylase. Kinetic characterization of this carboxylase showed that it preferentially carboxylated acetyl-CoA (1.1 nmol/mg/min) over propionyl-CoA (0.36 nmol/mg/min). The activity of the [[alpha].sub.3]-[[beta.sub.6] complex was inhibited by the e, subunit. The [[alpha].sub.3]-[[beta.sub.6] carboxylase was inhibited significantly by dimethyl itaconate, C75, haloxyfop, cerulenin, and 1,2-cyclohexanedione. Our results suggest that the [[beta.sub.6] subunit could play an important role in mycolic acid biosynthesis by providing malonyl-CoA to the FAS II complex.

Mycobacterium tuberculosis under stress stores triacylglycerol (TG). There are 15 genes in M. tuberculosis that belong to a novel family of TG synthase genes (tgs), but it is not known which of them is responsible for this accumulation of TG. In this paper, it is reported that M. tuberculosis H37Rv accumulated TG under acidic, static or hypoxic growth conditions, or upon treatment with NO, whereas TG accumulation was drastically reduced in the tgs1 (Rv3130c) disrupted mutant. Complementation with tgs1 restored this TG accumulation. [C.sub.26] was a major fatty acid in this TG, indicating that the TGS1 gene product uses [C.sub.26] fatty acid, which is known to be produced by the mycobacterial fatty acid synthase. TGS1 expressed in Escherichia coli preferred [C.sub.26:0]-CoA for TG synthesis. If TG storage is needed for the long-term survival of M. tuberculosis under dormant conditions, the tgs1 product could be a suitable target for antilatency drugs.

Endoplasmic reticulum (ER) stress has been found to be associated with neurodegenerative diseases and diabetes mellitus. Whether ER stress is involved in the development of heart disease is not known. Cardiac-specific expression of monocyte chemoattractant protein-1 (MCP-1) in mice causes the development of ischemic heart disease. Here we report that microarray analysis of gene expression changes in the heart of these transgenic mice revealed that a cluster of ER stress-related genes was transcriptionally activated in the heart during the development of ischemic heart disease. The gene array results were verified by quantitative real-time PCR that showed highly elevated transcript levels of genes involved in unfolded protein response such as ER and cytoplasmic chaperones, oxidoreductases, protein disulfide isomerase (PDI) family, and ER-associated degradation system such as ubiquitin. Immunoblot analysis confirmed the expression of chaperones, PDI, and ubiquitin. Immunohistochemical analyses showed that ER stress proteins were associated mainly with the degenerating cardiomyocytes. A novel ubiquitin fold modifier (Ufm1) that has not been previously associated with ER stress and not found to be induced under any condition was also found to be upregulated in the hearts of MCP mice (transgenic mice that express MCP-1 specifically in the heart). The present results strongly suggest that activation of ER stress response is involved in the development of ischemic heart disease in this murine model. monocyte chemoattractant protein-1; transgenic; myocardial inflammation doi: 10.1152/ajpheart.01378.2005

Mycobacterium tuberculosis enters the host by inhalation of an infectious aerosol and replicates in the alveolar macrophages until the host's immune defense causes bacteriostasis, which leads the pathogen to go into nonreplicative drug-resistant dormancy. The dormant pathogen can survive for decades till the host's immune system is weakened and active tuberculosis develops. Even though fatty acids are thought to be the major energy source required for the persistence phase, the source of fatty acids used is not known. We postulate that the pathogen uses triacylglycerol (TG) as a storage form of fatty acids. Little is known about the biosynthesis of TG in M. tuberculosis. We show that 15 mycobacterial genes that we identified as putative triacylglycerol synthase (tgs) when expressed in Escherichia coli showed TGS activity, and we report some basic catalytic characteristics of the most active enzymes. We show that several tgs genes are induced when the pathogen goes into the nonreplicative drug-resistant state caused by slow withdrawal of 02 and also by NO treatment, which is known to induce dormancy-associated genes. The gene (Rv3130c) that shows the highest TGS activity when expressed in E. coli shows the highest induction by hypoxia and NO treatment. Biochemical evidence shows that TG synthesis and accumulation occur under both conditions. We conclude that TG may be a form of energy storage for use during long-term dormancy. Therefore, TG synthesis may be an appropriate target for novel anti-latency drugs that can prevent the organism from surviving dormancy and thus assist in the control of tuberculosis.

We show that the disruption of one of the mycocerosic acid synthase (mas)-like genes, msl5 (pks8 plus pks17) in Mycobacterium tuberculosis H37Rv generates a mutant incapable of producing monomethyl branched unsaturated [C.sub.16] to [C.sub.20] fatty acids that are minor constituents of acyltrehaloses and sulfolipids. The msl5 mutation did not cause any significant change in the acyl lipid composition and also did not affect growth in culture, in mouse alveolar macrophage cell line MH-S, or in the murine lung.

The cell envelope of pathogenic mycobacteria is highly distinctive in that it contains a large number of structurally related very long multiple methyl-branched fatty acids. These complex molecules are thought to play important roles in cell envelope organization and virulence. The genetic and enzymic characterization of the polyketide synthase Mas, which is responsible for the synthesis of one such family of fatty acids (the mycocerosic acids), paved the way towards the identification of other enzymes involved in the synthesis of methyl-branched fatty acids in M. tuberculosis. In an effort to elucidate the origin of these complex fatty acids and their possible involvement in pathogenesis, the two mas-like polyketide genes pks5 and pks7 were disrupted in M. tuberculosis and the effects of their inactivation on fatty acid composition and virulence were analysed. While the disruption of pks7 resulted in a mutant deficient in the production of phthiocerol dimycocerosates, the cell envelope composition of the pks5 mutant was found to be identical to that of the wild-type parental strain M. tuberculosis H37Rv. Interestingly, both the pks5 and pks7 mutants displayed severe growth defects in mice.

Research reveals that germinating conidia of Colletotrichum gloeosporiodes on contact with hard-surface produce appressorium specific proteins. However, the bacterium maintains the ability to form appressorium even in the absence of the genes coding the proteins suggesting that the genes are not involved in the pathogenesis.

The hard-surface contact primes the conidia of Colletrotrichum gloeosporioides to react to plant surface waxes and ethylene to germinate and produce the appressoria needed for infection of the host. This hard-surface contact was observed to utilize the calmodulin and calmodulin kinase signaling pathway. Also, a selective inhibitor, the putative calmodulin kinase, inhibits the melanization of appressorium but scytalone can partially reverse this inhibition.

A study was conducted to examine the formation of an infection structure called appresorium which allows the phytopathogenic fungi and avocado pathogen Colletrichum gloeosporioides gene expressed in Saccharomyces cerevisiae to penetrate its host. An analysis of the differential display of RNA from C. gloeosporioides during hard-surface contact revealed that the contact was essential to the initiation of appressorium formation by the chemical signals ethylene and avocado wax. The contact was activated by ubiquitin-conjugating enzyme.

Research was conducted to test whether acyltransferase (AT) and beta-ketoacyl synthase (KS) domains from Mycobacterium bovis influence selectivity for methylmalonyl-CoA. Extracts from the cultures expressing AT and KS were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis while a routine assay was carried out to determine the nature of the products. Results showed that the production of mycocerosic acids is catalyzed by a multifunctional FAS-like enzyme called mycocerosic acid synthase.

The adjacent 900-bp open reading frame (ORF3) of the mycocerosic acid synthase mas gene was expressed in Escherichia coli and Mycobacterium bovis to characterize its protein product. ORF3 protein that was characterized as a 61-kDa C-terminal fusion protein in Escherichia coli encoded an acyl coenzyme A synthase when expressed in Mycobacterium bovis BCG. Furthermore, the purified protein product from ORF3 catalyzed coenzyme A ester synthesis without affecting mycocerosic acid synthesis.

Aspergillus flavus cells, containing a mutant serine proteinase (sep) gene, have higher levels of mep20 mRNA and protein than wild-type cells. The mutants have no serine proteinase activity but their elastinolytic activity is similar to the wild-type cells. This is due to the increased expression of the mep20 metalloproteinase gene. The increase in mep20 expression is probably due to the lack of sep transcription in the mutants. A study indicates that serine and metalloproteinase encoding genes have a common regulatory system.