The actinobacterial WhiB-like (Wbl) family of transcription factors.

The WhiB-like (Wbl) family of proteins are exclusively found in Actinobacteria. Wbls have been shown to play key roles in virulence and antibiotic resistance in Mycobacteria and Corynebacteria, reflecting their importance during infection by the human pathogens Mycobacterium tuberculosis, Mycobacterium leprae and Corynebacterium diphtheriae. In the antibiotic-producing Streptomyces, several Wbls have important roles in the regulation of morphological differentiation, including WhiB, a protein that controls the initiation of sporulation septation and the founding member of the Wbl family. In recent years, genome sequencing has revealed the prevalence of Wbl paralogues in species throughout the Actinobacteria. Wbl proteins are small (generally ~80-140 residues) and each contains four invariant cysteine residues that bind an O2 - and NO-sensitive [4Fe-4S] cluster, raising the question as to how they can maintain distinct cellular functions within a given species. Despite their discovery over 25 years ago, the Wbl protein family has largely remained enigmatic. Here I summarise recent research in Mycobacteria, Corynebacteria and Streptomyces that sheds light on the biochemical function of Wbls as transcription factors and as potential sensors of O2 and NO. I suggest that Wbl evolution has created diversity in protein-protein interactions, [4Fe-4S] cluster-sensitivity and the ability to bind DNA.

Characterization of some bacterial strains isolated from animal clinical materials and identified as Corynebacterium xerosis by molecular biological techniques.

Eighteen Corynebacterium xerosis strains isolated from different animal clinical specimens were subjected to phenotypic and molecular genetic studies. On the basis of the results of the biochemical characterization, the strains were tentatively identified as C. xerosis. Phylogenetic analysis based on comparative analysis of the sequences of 16S rRNA and rpoB genes revealed that the 18 strains were highly related to C. xerosis, C. amycolatum, C. freneyi, and C. hansenii. There was a good concordance between 16S rRNA and partial rpoB gene sequencing results, although partial rpoB gene sequencing allowed better differentiation of C. xerosis. Alternatively, C. xerosis was also differentiated from C. freneyi and C. amycolatum by restriction fragment length polymorphism analysis of the 16S-23S rRNA gene intergenic spacer region. Phenotypic characterization indicated that besides acid production from D-turanose and 5-ketogluconate, 90% of the strains were able to reduce nitrate. The absence of the fatty acids C(14:0), C(15:0), C(16:1)omega 7c, and C(17:1)omega 8c can also facilitate the differentiation of C. xerosis from closely related species. The results of the present investigation demonstrated that for reliable identification of C. xerosis strains from clinical samples, a combination of phenotypic and molecular-biology-based identification techniques is necessary.

The reductase that catalyzes mycolic motif synthesis is required for efficient attachment of mycolic acids to arabinogalactan.

Mycolic acids are essential components of the cell walls of bacteria belonging to the suborder Corynebacterineae, including the important human pathogens Mycobacterium tuberculosis and Mycobacterium leprae. Mycolic acid biosynthesis is complex and the target of several frontline antimycobacterial drugs. The condensation of two fatty acids to form a 2-alkyl-3-keto mycolate precursor and the subsequent reduction of this precursor represent two key and highly conserved steps in this pathway. Although the enzyme catalyzing the condensation step has recently been identified, little is known about the putative reductase. Using an extensive bioinformatic comparison of the genomes of M. tuberculosis and Corynebacterium glutamicum, we identified NCgl2385, the orthologue of Rv2509 in M. tuberculosis, as a potential reductase candidate. Deletion of the gene in C. glutamicum resulted in a slow growing strain that was deficient in arabinogalactan-linked mycolates and synthesized abnormal forms of the mycolate-containing glycolipids trehalose dicorynomycolate and trehalose monocorynomycolate. Analysis of the native and acetylated trehalose glycolipids by MALDI-TOF mass spectrometry indicated that these novel glycolipids contained an unreduced beta-keto ester. This was confirmed by analysis of sodium borodeuteride-reduced mycolic acids by gas chromatography mass spectrometry. Reintroduction of the NCgl2385 gene into the mutant restored the transfer of mature mycolic acids to both the trehalose glycolipids and cell wall arabinogalactan. These data indicate that NCgl2385, which we have designated CmrA, is essential for the production of mature trehalose mycolates and subsequent covalent attachment of mycolic acids onto the cell wall, thus representing a focus for future structural and pathogenicity studies.

A polyketide synthase catalyzes the last condensation step of mycolic acid biosynthesis in mycobacteria and related organisms.

Mycolic acids are major and specific constituents of the cell envelope of Corynebacterineae, a suborder of bacterial species including several important human pathogens such as Mycobacterium tuberculosis, Mycobacterium leprae, or Corynebacterium diphtheriae. These long-chain fatty acids are involved in the unusual architecture and impermeability of the cell envelope of these bacteria. The condensase, the enzyme responsible for the final condensation step in mycolic acid biosynthesis, has remained an enigma for decades. By in silico analysis of various mycobacterial genomes, we identified a candidate enzyme, Pks13, that contains the four catalytic domains required for the condensation reaction. Orthologs of this enzyme were found in other Corynebacterineae species. A Corynebacterium glutamicum strain with a deletion in the pks13 gene was shown to be deficient in mycolic acid production whereas it was able to produce the fatty acids precursors. This mutant strain displayed an altered cell envelope structure. We showed that the pks13 gene was essential for the survival of Mycobacterium smegmatis. A conditional M. smegmatis mutant carrying its only copy of pks13 on a thermosensitive plasmid exhibited mycolic acid biosynthesis defect if grown at nonpermissive temperature. These results indicate that Pks13 is the condensase, a promising target for the development of new antimicrobial drugs against Corynebacterineae.

Production of volatile compounds by cheese-ripening yeasts: requirement for a methanethiol donor for S-methyl thioacetate synthesis by Kluyveromyces lactis.

Five cheese-ripening yeasts (Geotrichum candidum, Saccharomyces cerevisiae, Kluyveromyces lactis, Yarrowia lipolytica and Debaryomyces hansenii) were compared with respect to their ability to generate volatile aroma compounds. K. lactis produced a variety of esters - ethylacetate (EA) being the major one - and relatively limited amounts of volatile sulphur compounds (VSCs). Conversely, G. candidum produced significant amounts of VSCs [with the thioester S-methyl thioacetate (MTA) being the most prevalent] and lower quantities of non-sulphur volatile compounds than K. lactis. We suspect that K. lactis is able to produce and/or accumulate acetyl CoA - a common precursor of MTA and EA - but that it produces limited amounts of methanethiol (MTL); both acetyl CoA and MTL are precursors for MTA synthesis. When supplemented with exogenous MTL, MTA production greatly increased in K. lactis cultures whereas it was unchanged in G. candidum cultures, suggesting that MTL is a limiting factor for MTA synthesis in K. lactis but not in G. candidum. Our results are discussed with respect to L-methionine catabolism.

L-methionine degradation potentialities of cheese-ripening microorganisms.

Volatile sulphur compounds are major flavouring compounds in many traditional fermented foods including cheeses. These compounds are products of the catabolism of L-methionine by cheese-ripening microorganisms. The diversity of L-methionine degradation by such microorganisms, however, remains to be characterized. The objective of this work was to compare the capacities to produce volatile sulphur compounds by five yeasts, Geotrichum candidum, Yarrowia lipolytica, Kluyveromyces lactis, Debaryomyces hansenii, Saccharomyces cerevisiae and five bacteria, Brevibacterium linens, Corynebacterium glutamicum, Arthrobacter sp., Micrococcus lutens and Staphylococcus equorum of technological interest for cheese-ripening. The ability of whole cells of these microorganisms to generate volatile sulphur compounds from L-methionine was compared. The microorganisms produced a wide spectrum of sulphur compounds including methanethiol, dimethylsulfide, dimethyldisulfide, dimethyltrisulfide and also S-methylthioesters, which varied in amount and type according to strain. Most of the yeasts produced methanethiol, dimethylsulfide, dimethyldisulfide and dimethyltrisulfide but did not produce S-methylthioesters, apart from G. candidum that produced S-methyl thioacetate. Bacteria, especially Arth. sp. and Brevi. linens, produced the highest amounts and the greatest variety of volatile sulphur compounds includling methanethiol, sulfides and S-methylthioesters, e.g. S-methyl thioacetate, S-methyl thiobutyrate, S-methyl thiopropionate and S-methyl thioisovalerate. Cell-free extracts of all the yeasts and bacteria were examined for the activity of enzymes possibly involved in L-methionine catabolism, i.e. L-methionine demethiolase, L-methionine aminotransferase and L-methionine deaminase. They all possessed L-methionine demethiolase activity, while some (K. lactis, Deb. hansenii, Arth. sp., Staph. equorum) were deficient in L-methionine aminotransferase, and none produced L-methionine deaminase. The catabolism of L-methionine in these microorganisms is discussed.

Biological, chemical, immunological and staining properties of bacteria isolated from tissues of leprosy patients.

Two kinds of microorganisms are found in tissue of leprosy patients: Mycobacterium leprae (ML) and leprosy derived corynebacteria (LDC). ML from untreated patients has an alcohol-acid-fastness, which is lost upon treatment with antibiotics and immune response (tuberculoid leprosy). Vulnerable ML thus produced can be reversibly de-stained by organic solvent: in tissue sections from tuberculoid and treated patients, more bacteria are, thus, revealed by the Wade-Fite than by the Ziehl-Neelsen procedure. Organisms of genera Corynebacterium, Mycobacterium and Nocardia (CMN group), have DNA with %GC contents of 50-70, 69-72, and 68-70 respectively. GC values of DNA from ML and LDC are close to 56%. DNA from different LDC strains display high homology among them and low homology with reference corynebacteria. CMN cell wall consists of interconnected peptidoglycan and polysaccharide-mycolate complex. Peptidoglycan of LDC (and known CMN) has the polysaccharide backbone linked to a tetrapeptide of L-Ala, D-Glu, m-DAP (meso-diaminopimelate), D-Ala. In ML, L-Ala is replaced by glycine. Mycobacterial wall polysaccharides (that of ML is unknown) are branched arabinogalactans with end arabinoses linked to C70 to C90 mycolates. LDC peripheral polysaccharides are arabinogalactomannans with arabinose and mannose lateral strands. Mycolic acids of LDC are of corynomycolic type (C32, C34 and C36 with 1-4 double bonds) and those of ML are of mycobacterial type. Components of CMN wall and cytoplasm are immunologically active as antigens (polysaccharides, proteins), haptens (lipids) and adjuvants (peptidoglycans). Strong intrageneric and weak intergenera crossreactions are observed among CMN bacteria: LDC preparations, however, crossreact strongly with ML and mycobacteria, and weakly with reference corynebacteria. LDC in leprosy tissues can, thus, be revealed as well by fluorescent anti-LDC antisera as by anti-ML antisera. The main crossreacting component is antigen M1 of LDC, which corresponds to antigens Ag 7 of ML and Ag60 of BCG, the active components of lepromin and tuberculin (known reagents for cutaneous tests). Antigen M1 has a polysaccharide moiety crossreacting with the wall polysaccharide of LDC.(ABSTRACT TRUNCATED AT 400 WORDS)

Description of Corynebacterium tuberculostearicum sp. nov., a leprosy-derived Corynebacterium.

Leprosy-derived corynebacteria (LDC) have been extensively studied over the past decade. A composite of their biological properties (cell morphology, staining reactions, cellular inclusions and guanine-plus-cytosine content of their deoxyribonucleic acid; 16 strains studied) and their chemical structures (peptidoglycan type, major cell wall polysaccharide, major glycolipid as well as characteristic mycolic acids) appears to define them as members of the genus Corynebacterium. In relation to other corynebacteria found in humans, including "JK corynebacteria", they seem to be distinct. They are here named Corynebacterium tuberculostearicum sp. nov. because they produce a 10-methyloctadecanoic (tuberculostearic) acid (8 strains studied). This and some of their other attributes are considered in relation to properties of leprosy bacilli and Mycobacterium leprae.