Identification of the INO1 gene of Mycobacterium tuberculosis H37Rv reveals a novel class of inositol-1-phosphate synthase enzyme.

1L-myo-inositol (inositol) is vital for the biogenesis of mycothiol, phosphatidylinositol and glycosylphosphatidylinositol anchors linked to complex carbohydrates in Mycobacterium tuberculosis. All these cellular components are thought to play important roles in host-pathogen interactions and in the survival of the pathogen within the host. However, the inositol biosynthetic pathway in M. tuberculosis is not known. To delineate the pathways for inositol formation, we employed a unique combination of tertiary structure prediction and yeast-based functional assays. Here, we describe the identification of the gene for mycobacterial INO1 that encodes inositol-1-phosphate synthase distinct in many respects from the eukaryotic analogues.

Functional characterization of the repeated UASINO element in the promoters of the INO1 and CHO2 genes of yeast.

In yeast, INO1 and CHO2 gene expression is subject to repression in response to inositol and choline supplementation. The response by both genes to inositol is controlled by a single set of regulatory factors and the highly conserved and repeated UASINO element (consensus: 5' CATGTGAAAT 3') that is found in multiple copies in both promoters. However, none of the native elements found in the INO1 and CHO2 promoters constitutes an exact match to the consensus element and the functionality of individual elements from these two promoters has not been tested. In this study, the function of individual putative UASINO elements from both promoters was tested by placing promoter fragments into a reporter construct which lacked a UAS element but contained the TATA element and start of transcription from the yeast CYC1 gene fused to the Escherichia coli lacZ gene. In addition, a set of oligonucleotides containing the consensus UASINO element with the first position systematically modified was also tested for UASINO function. These studies indicated that elements that contain a C or an A as the first base at the 5' end are functional to varying degrees. The majority of potential UASINO elements from the INO1 promoter were found to be inactive, whereas all of the elements from the CHO2 promoter tested were active. These results are discussed in light of the differential regulation of the two promoters.

Functional characterization of an inositol-sensitive upstream activation sequence in yeast. A cis-regulatory element responsible for inositol-choline mediated regulation of phospholipid biosynthesis.

A repeated element, the inositol-sensitive upstream activation sequence (UASINO), having the consensus sequence, 5'-CATGTGAAAT-3', is present in the promoters of genes encoding enzymes of phospholipid biosynthesis that are regulated in response to the phospholipid precursors, inositol and choline. None of the naturally occurring variants of the UASINO element exactly recapitulates the consensus (for review, see Carman, G. M., and Henry, S. A. (1989) Annu. Rev. Biochem. 58, 635-669 and Paltauf, F., Kolwhein, S., and Henry, S. A. (1992) in Molecular Biology of the Yeast Saccharomyces cerevisiae (Broach, J., Jones, E., and Pringle, J., eds) Vol. 2, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). The first six bases of the UASINO element are homologous with canonical binding motif for proteins of the basic helix-loop-helix (bHLH) family. Two bHLH regulatory proteins, Ino2p and Ino4p from yeast, were previously shown to bind to promoter fragments containing this element. In the present study, an extensive analysis of UASINO function has been conducted. We report that any base substitution within the putative bHLH binding site resulted either in a dramatic reduction or in a complete obliteration of UASINO function as tested in an expression assay in vivo. Base substitutions in the 5' region that flanks the 10-base pair repeat, as well as sequences within the repeat itself at its 3' end outside the bHLH core, were also assessed. The two bases immediately flanking the 5' end of the element proved to be very important to its function as a UAS element as did the two bases immediately 3' of the bHLH core motif. Substitutions of the final two bases of the original ten base pair consensus (i.e. 5'-CATGTGAAAT-3') had less dramatic effects. We also tested a subset of the altered elements for their ability to serve as competitors in an assay of Ino2p x Ino4p binding. The strength of any given sequence as a UASINO element, as assayed in vivo, was strongly correlated with its strength as a competitor for Ino2p x Ino4p binding. We also tested a subset of the modified UASINO elements for their effects on expression in vivo in a strain carrying an opi1 mutation. The opi1 mutation renders the coregulated enzymes of phospholipid synthesis constitutive in the presence of phospholipid precursors. All elements that retained some residual UASINO activity when tested in the wild-type strain were constitutively expressed at a level comparable with the wild-type derepressed level when tested in the opi1 mutant.(ABSTRACT TRUNCATED AT 400 WORDS)

Repressor mutations in the marRAB operon that activate oxidative stress genes and multiple antibiotic resistance in Escherichia coli.

Resistance to multiple antibiotics and certain oxidative stress compounds was conferred by three independently selected mutations (marR1, soxQ1, and cfxB1) that mapped to 34 min on the Escherichia coli chromosome. Mutations at this locus can activate the marRAB operon, in which marR encodes a putative repressor of mar transcription and marA encodes a putative transcriptional activator of defense genes against antibiotics and oxidants. Overexpression of the wild-type MarR protein reversed the phenotypes (antibiotic resistance and increased antioxidant enzyme synthesis) of all three mutants. DNA sequence analysis showed that, like marR1, the other two mutations were alterations of marR: a 285-bp deletion in cfxB1 and a GC-->AT transition at codon 70 (Ala-->Thr) in soxQ1. All three mutations cause increased amounts of mar-specific RNA, which supports the hypothesis that MarR has a repressor function in the expression of the marRAB operon. The level of mar RNA was further induced by tetracycline in both the marR1 and soxQ1 strains but not in the cfxB1 deletion mutant. In the cfxB1 strain, the level of expression of a truncated RNA, with or without tetracycline exposure, was the same as the fully induced level in the other two mutants. Overproduction of MarR in the cfxB1 strain repressed the transcription of the truncated RNA and restored transcriptional inducibility by tetracycline. Thus, induction of the marRAB operon results from the relief of the repression exerted by MarR. The marRAB operon evidently activates both antibiotic resistance and oxidative stress genes.