Overexpression, purification and characterization of Mycobacterium bovis BCG alcohol dehydrogenase.

A previous study of the effect of zinc deprivation on Mycobacterium bovis BCG pointed out the potential importance of an alcohol dehydrogenase for maintaining the hydrophobic character of the cell envelope. In this report, the effect of the overexpression of the M. bovis BCG alcohol dehydrogenase (ADH) in Mycobacterium smegmatis and M. bovis BCG is described. The purification of the enzyme was performed to apparent homogeneity from overexpressing M. bovis BCG cells and its kinetic parameters were determined. The enzyme showed a strong preference for both aromatic and aliphatic aldehydes while the corresponding alcohols were processed 100-1000-fold less efficiently. The best kcat/Km values were found with benzaldehyde > 3-methoxybenzaldehyde > octanal > coniferaldehyde. A phylogenetic analysis clearly revealed that the M. bovis BCG ADH together with the ADHs from Bacillus subtilis and Helicobacter pylori formed a sister group of the class C medium-chain alcohol dehydrogenases, the plant cinnamyl alcohol dehydrogenases (CADs). Comparison of the kinetic properties of our ADH with some related class C enzymes indicated that the mycobacterial enzyme substrate profile resembled that of the CADs involved in plant defence rather than those implicated in lignification. A possible role for the M. bovis BCG ADH in the biosynthesis of the lipids composing the mycobacterial cell envelope is proposed.     

Molecular and biochemical characterisation of Mycobacterium smegmatis alcohol dehydrogenase C

The gene encoding of an alcohol dehydrogenase C (ADHC) from Mycobacterium smegmatis was cloned and sequenced. The protein encoded by this gene has 78% identity with Mycobacterium tuberculosis and Mycobacterium bovis BCG ADHC. The M. smegmatis ADHC was purified from M. smegmatis and the kinetic parameters of this enzyme showed that using NADPH as electron donor it has a strong preference for aliphatic and aromatic aldehyde substrates. Like the M. bovis BCG ADHC, this enzyme is more likely to act as an aldehyde reductase than as an alcohol dehydrogenase. The discovery of such an ADHC in a fast-growing, and easily engineered mycobacterial species opens the way to the utilisation of this M. smegmatis enzyme as a convenient model for the study of the physiological role of this alcohol dehydrogenase in mycobacteria.     

Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent M. bovis.

The live attenuated bacillus Calmette-Guérin (BCG) vaccine for the prevention of disease associated with Mycobacterium tuberculosis was derived from the closely related virulent tubercle bacillus, Mycobacterium bovis. Although the BCG vaccine has been one of the most widely used vaccines in the world for over 40 years, the genetic basis of BCG's attenuation has never been elucidated. We employed subtractive genomic hybridization to identify genetic differences between virulent M. bovis and M. tuberculosis and avirulent BCG. Three distinct genomic regions of difference (designated RD1 to RD3) were found to be deleted from BCG, and the precise junctions and DNA sequence of each deletion were determined. RD3, a 9.3-kb genomic segment present in virulent laboratory strains of M. bovis and M. tuberculosis, was absent from BCG and 84% of virulent clinical isolates. RD2, a 10.7-kb DNA segment containing a novel repetitive element and the previously identified mpt-64 gene, was conserved in all virulent laboratory and clinical tubercle bacilli tested and was deleted only from substrains derived from the original BCG Pasteur strain after 1925. Thus, the RD2 deletion occurred after the original derivation of BCG. RD1, a 9.5-kb DNA segment found to be deleted from all BCG substrains, was conserved in all virulent laboratory and clinical isolates of M. bovis and M. tuberculosis tested. The reintroduction of RD1 into BCG repressed the expression of at least 10 proteins and resulted in a protein expression profile almost identical to that of virulent M. bovis and M. tuberculosis, as determined by two-dimensional gel electrophoresis. These data indicate a role for RD1 in the regulation of multiple genetic loci, suggesting that the loss of virulence by BCG is due to a regulatory mutation. These findings may be applicable to the rational design of a new attenuated tuberculosis vaccine and the development of new diagnostic tests to distinguish BCG vaccination from tuberculosis infection.     

Molecular analysis and nucleotide sequence of the adh1 gene encoding an NADPH-dependent butanol dehydrogenase in the Gram-positive anaerobe Clostridium acetobutylicum

The nucleotide sequence of a 2081-bp fragment of Clostridium acetobutylicum DNA containing the adh1 gene was determined. The butanol dehydrogenase gene is referred to as the adh1 gene since it was shown to have activity using butanol and ethanol as substrates. The adh1 gene consisted of 1164 bp and encoded an alcohol dehydrogenase (ADH) enzyme of 388 aa residues with an Mr of 43,274. The adh1 gene was separated from an upstream open reading frame by an intergenic region of 354 bp. No promoter consensus sequences were identified in the intergenic upstream region and the adh1 gene did not appear to be expressed off its own promoter in Escherichia coli. Three separate types of ADH have been recognized. The ADH1 from C. acetobutylicum exhibited 39% homology with the Fe-containing ADH2 from Zymomonas mobilis and 37% homology with the ADH4 from Saccharomyces cerevisiae, but showed little or no homology with the other characterised types of ADH.     

Cloning and analysis of a Candida maltosa gene which confers resistance to formaldehyde in Saccharomyces cerevisiae.

A gene (FDH1) of Candida maltosa which confers resistance to formaldehyde in Saccharomyces cerevisiae was cloned and its nucleotide sequence determined. The gene has a single intron which possesses the highly conserved splicing signals found in S. cerevisiae introns. We demonstrated that processing of the pre-mRNA of the cloned gene occurred identically in both S. cerevisiae and C. maltosa. The predicted amino acid sequence from the cloned gene showed 65.5% identity to human alcohol dehydrogenase (ADH) class III and 23.9% identity to S. cerevisiae ADH1. The most probable mechanism of resistance to formaldehyde is thought to be the glutathione-dependent oxidation of formaldehyde which is characteristic for ADH class III. The cloned FDH1 gene was successfully employed as a dominant selectable marker in the transformation of S. cerevisiae.     

Complete nucleotide sequence of the 16S rRNA gene of Mycobacterium bovis BCG.

The complete nucleotide sequence of the 16S rRNA gene of Mycobacterium bovis BCG was determined. Its coding region was estimated to be 1,536 base pairs long. The nucleotide sequence of the gene in M. bovis BCG has homologies of 75 and 89% with those of Escherichia coli and Streptomyces lividans, respectively.     

Complete amino acid sequence of rat liver alcohol dehydrogenase deduced from the cDNA sequence

Alcohol dehydrogenase (ADH) catalyzes the rate-determining reaction in the metabolism of ethanol. We report here the complete nucleotide sequence of a cDNA encoding rat liver ADH, and the deduced amino acid (aa) sequence of the protein. The rat enzyme contains a cluster of aa substitutions and an aa insertion in the region between aa residues 111 and 118, which is near the intron-exon junction reported for the human ADH gene. It also contains an additional cysteine in the highly variable region from aa residues 108-125 which may account for the unusual lability of rat ADH compared with ADH from other species.     

Homology of Saccharomyces cerevisiae ADH4 to an iron-activated alcohol dehydrogenase from Zymomonas mobilis

Summary Insertion of the transposable element Ty at the ADH4 locus results in increased levels of a new alcohol dehydrogenase (ADH) activity in Saccharomyces cerevisiae. The DNA sequence of this locus has been determined. It contains a long open reading frame which is not homologous to the other ADH isozymes that have been characterized in S. cerevisiae nor does it show obvious homology to Drosophila ADH. The hypothetical ADH does, however, show strong homology to the sequence of an iron-activated ADH from the bacterium Zymomonas mobilis. Thus ADH4 appears to encode an ADH structural gene which, along with the Zymomonas enzyme, may define a new family of alcohol dehydrogenases.Now The Plant Cell Research Institute, Inc., 6560 Trinity Court, Dublin, CA 94568, USA     

Cloning, sequencing and expression of the L-2-hydroxyisocaproate dehydrogenase-encoding gene of Lactobacillus confusus in Escherichia coli

The gene (L-HicDH) encoding L-2-hydroxyisocaproate dehydrogenase (L-HicDH) from Lactobacillus confusus was cloned in Escherichia coli. A 69-mer oligodeoxyribonucleotide probe, derived to be complementary to the N-terminal amino acid (aa) coding sequence, was used for screening. The complete nucleotide (nt) sequence of the L-HicDH gene was determined. The 5'-end of the mRNA was mapped by primer extension and the promoter identified. Downstream from the L-HicDH gene is a typical Rho-independent terminator. The aa sequence of L-HicDH, deduced from the nt sequence, has an overall similarity of 30% to the aa sequence of L-lactate dehydrogenase (L-LDH) from Lactobacillus casei. The aa residues involved in binding of coenzyme and substrate are highly conserved in L-HicDH with respect to prokaryotic and eukaryotic L-LDHs. The L-HicDH gene could be expressed under control of phage lambda 'Leftward' and 'rightward' promoters in E. coli up to 35% of total cell protein. The enzyme produced under these conditions exhibits full specific activity and is found exclusively in soluble form.     

Cloning, sequencing, and characterization of the gene encoding the smallest subunit of the three-component membrane-bound alcohol dehydrogenase from Acetobacter pasteurianus.

The membrane-bound alcohol dehydrogenase (ADH) of Acetobacter pasteurianus NCI1452 consists of three different subunits, a 78-kDa dehydrogenase subunit, a 48-kDa cytochrome c subunit, and a 20-kDa subunit of unknown function. For elucidation of the function of the smallest subunit, this gene was cloned from this strain by the oligonucleotide-probing method, and its nucleotide sequence was determined. Comparison of the deduced amino acid sequence and the NH2-terminal sequence determined for the purified protein indicated that the smallest subunit contained a typical signal peptide of 28 amino acids, as did the larger two subunits. This gene complemented the ADH activity of a mutant strain which had lost the smallest subunit. Disruption of this gene on the chromosome resulted in loss of ADH activity in Acetobacter aceti, indicating that the smallest subunit was essential for ADH activity. Immunoblot analyses of cell lysates prepared from various ADH mutants suggested that the smallest subunit was concerned with the stability of the 78-kDa subunit and functioned as a molecular coupler of the 78-kDa subunit to the 48-kDa subunit on the cytoplasmic membrane.     

Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA.

Using a set of synthetic oligonucleotides homologous to broadly conserved sequences in-vitro amplification via the polymerase chain reaction followed by direct sequencing results in almost complete nucleotide determination of a gene coding for 16S ribosomal RNA. As a model system the nucleotide sequence of the 16S rRNA gene of M.kansasii was determined and found to be 98.7% homologous to that of M.bovis BCG. This is the first report on a contiguous sequence information of an entire amplified gene spanning 1.5 kb without any subcloning procedures.     

The Mycobacterium bovis homologous protein of the Mycobacterium tuberculosis serine/threonine protein kinase Mbk (PknD) is truncated

We previously identified a 70-kDa serine/threonine protein kinase (MbK or PknD) from Mycobacterium tuberculosis Erdman containing a transmembrane domain and bearing a 270-amino acid N-terminal kinase domain. With the use of a polyclonal serum, Mbk has now been identified by Western blotting in protein extracts from M. tuberculosis and confirmed to be localised in the envelope. An identical mbk gene has been found by sequencing different M. tuberculosis and M. africanum strains. Surprisingly, in two virulent M. bovis strains and four different strains of M. bovis BCG, an additional adenine after position 829 of the open reading frame was found that produces a frame shift resulting in a predicted truncated, presumably free cytoplasmic protein, encoding only the N-terminal 30-kDa Mbk kinase domain. This sequence polymorphism has been confirmed by Western blot analysis of M. bovis BCG protein extracts.     

Comparative analysis of mycobacterial NADH pyrophosphatase isoforms reveals a novel mechanism for isoniazid and ethionamide inactivation

NADH pyrophosphatase (NudC) catalyses the hydrolysis of NAD(H) to AMP and NMN(H) [nicotinamide mononucleotide (reduced form)]. NudC multiple sequence alignment reveals that homologues from most Mycobacterium tuberculosis isolates, but not other mycobacterial species, have a polymorphism at the highly conserved residue 237. To elucidate the functional significance of this polymorphism, comparative analyses were performed using representative NudC isoforms from M. tuberculosis H37Rv (NudC(Rv)) and M. bovis BCG (NudC(BCG)). Biochemical analysis showed that the P237Q polymorphism prevents dimer formation, and results in a loss of enzymatic activity. Importantly, NudC(BCG) was found to degrade the active forms of isoniazid (INH), INH-NAD and ethionamide (ETH), ETH-NAD. Consequently, overexpression of NudC(BCG) in Mycobacterium smegmatis mc(2)155 and M. bovis BCG resulted in a high level of resistance to both INH and ETH. Further genetic studies showed that deletion of the nudC gene in M. smegmatis mc(2)155 and M. bovis BCG resulted in increased susceptibility to INH and ETH. Moreover, inactivation of NudC in both strains caused a defect in drug tolerance phenotype for both drugs in exposure assays. Taken together, these data suggest that mycobacterial NudC plays an important role in the inactivation of INH and ETH.     

Genetic determination of the meso-diaminopimelate biosynthetic pathway of mycobacteria.

The increasing incidence of multiple-drug-resistant mycobacterial infections indicates that the development of new methods for treatment of mycobacterial diseases should be a high priority. meso-Diaminopimelic acid (DAP), a key component of a highly immunogenic subunit of the mycobacterial peptidoglycan layer, has been implicated as a potential virulence factor. The mycobacterial DAP biosynthetic pathway could serve as a target for design of new antimycobacterial agents as well as the construction of in vivo selection systems. We have isolated the asd, dapA, dapB, dapD, and dapE genes involved in the DAP biosynthetic pathway of Mycobacterium bovis BCG. These genes were isolated by complementation of Escherichia coli mutations with an expression library of BCG DNA. Our analysis of these genes suggests that BCG may use more than one pathway for biosynthesis of DAP. The nucleotide sequence of the BCG dapB gene was determined. The activity of the product of this gene in Escherichia coli provided evidence that the gene may encode a novel bifunctional dihydrodipicolinate reductase and DAP dehydrogenase.     

Cloning and expression of superoxide dismutase from Mycobacterium bovis BCG

We have previously purified the superoxide dismutase (SOD) of Mycobacterium bovis bacillus Calmette-Guerin (BCG), and there is no signal peptide necessary for protein exportation [S.K. Kang, Y.J. Jung, C.H. Kim, C.Y. Song, Extracellular and cytosolic iron superoxide dismutase from Mycobacterium bovis BCG, Clin. Diagn. Lab. Immunol. 5 (1998) 784-789]. In the present study, SOD gene of M. bovis BCG was cloned and expressed in Escherichia coli, and its complete nucleotide sequence and deduced amino acid composition were determined. The open reading frame from the GTG initiation codon was 621 base pair (bp) in length for the SOD structural gene. The ribosomal-binding sequences (GGAAGG) were 6-12 bp upstream from the initiation codon. The amino acid sequence, deduced from the nucleotide sequence, revealed that the SOD consists of 207 amino acids residues with a molecular weight of 22.8 kDa. The N-terminal amino acid sequence predicted from the nucleotide sequence showed that the structural gene of the SOD is not preceded by leader sequences. There were no cysteine residues in the deduced amino acid composition, indicating that the SOD does not consist of disulfide bonds. Analyses of both nucleotide and amino acid sequences of the SOD showed significant similarity to other pathogenic mycobacterial SODs. Furthermore, the results of fractionation and two-dimensional electrophoresis showed that SOD is also associated with cell membrane, suggesting that there might be a specific mechanism for exportation of SOD in M. bovis BCG as well as other pathogenic mycobacteria. Overexpressed SOD in E. coli was purified from the inclusion bodies, and the histidine tag was removed from the protein using enterokinase. Enzyme activity was then determined by gel staining analysis.     

Improved protective efficacy of a species-specific DNA vaccine encoding mycolyl-transferase Ag85A from Mycobacterium ulcerans by homologous protein boosting

Vaccination with plasmid DNA encoding Ag85A from M. bovis BCG can partially protect C57BL/6 mice against a subsequent footpad challenge with M. ulcerans. Unfortunately, this cross-reactive protection is insufficient to completely control the infection. Although genes encoding Ag85A from M. bovis BCG (identical to genes from M. tuberculosis) and from M. ulcerans are highly conserved, minor sequence differences exist, and use of the specific gene of M. ulcerans could possibly result in a more potent vaccine. Here we report on a comparison of immunogenicity and protective efficacy in C57BL/6 mice of Ag85A from M. tuberculosis and M. ulcerans, administered as a plasmid DNA vaccine, as a recombinant protein vaccine in adjuvant or as a combined DNA prime-protein boost vaccine. All three vaccination formulations induced cross-reactive humoral and cell-mediated immune responses, although species-specific Th1 type T cell epitopes could be identified in both the NH2-terminal region and the COOH-terminal region of the antigens. This partial species-specificity was reflected in a higher--albeit not sustained--protective efficacy of the M. ulcerans than of the M. tuberculosis vaccine, particularly when administered using the DNA prime-protein boost protocol.     

Alcohol dehydrogenase: multiplicity and relatedness in the solvent-producing clostridia

Abstract: Alcohol dehydrogenase (ADH) is a key enzyme for the production of butanol, ethanol, and isopropanol by the solvent-producing clostridia. Initial studies of ADH in extracts of several strains of Clostridium acetobutylicum and C. beijerinckii gave conflicting molecular properties. A more coherent picture has emerged because of the following results: (i) identification of ADHs with different coenzyme specificities in these species; (ii) discovery of structurally conserved ADHs (type 3) in three solvent-producing species; (iii) isolation of mutants with deficiencies in butanol production and restoration of butanol production with a cloned alcohol/aldehyde dehydrogenase gene; and (iv) resolution of various ' C. acetobutylicum ' cultures into four species. The three ADH isozymes of C. beijerinckii NRRL B592 have high sequence similarities to ADH-1 of Clostridium sp. NCP 262 (formerly C. acetobutylicum P262) and to the ADH domain of the alcohol/aldehyde dehydrogenase of C. acetobutylicum ATCC 824/DSM 792. The NADH-dependent activity of the ADHs from C. beijerinckii NRRL B592 and the BDHs from C. acetobutylicum ATCC 824 is profoundly affected by the pH of the assay, and the relative importance of NADH and NADPH to butanol production may be misappraised when NAD(P)H-dependent activities were measured at different pH values. The primary/secondary ADH of isopropanol-producing C. beijerinckii is a type-1 enzyme and is highly conserved in Thermoanaerobacter brockii (formerly Thermoanaerobium brockii ) and Entamoeba histolytica . Several solvent-forming enzymes (primary ADH, aldehyde dehydrogenase, and 3-hydroxybutyryl-CoA dehydrogenase) are very similar between C. beijerinckii and the species represented by Clostridium sp. NCP 262 and NRRL B643. The realization of such relationships will facilitate the elucidation of the roles of different ADHs because each type of ADH can now be studied in an organism most amenable to experimental manipulations.