Exochelin genes in Mycobacterium smegmatis: identification of an ABC transporter and two non-ribosomal peptide synthetase genes

Many strains of mycobacteria produce two ferric chelating substances that are termed exochelin (an excreted product) and mycobactin (a cell-associated product). These agents may function as iron acquisition siderophores. To examine the genetics of the iron acquisition system in mycobacteria, ultraviolet (UV) and transposon (Tn611) mutagenesis techniques were used to generate exochelin-deficient mutants of Mycobacterium smegmatis strains ATCC 607 and LR222 respectively. Mutants were identified on CAS siderophore detection agar plates. Comparisons of the amounts of CAS-reactive material excreted by the possible mutant strains with that of the wild-type strains verified that seven UV mutant strains and two confirmed transposition mutant strains were deficient in exochelin production. Cell-associated mycobactin production in the mutants appeared to be normal. From the two transposon mutants, the mutated gene regions were cloned and identified by colony hybridization with an IS6100 probe, and the DNA regions flanking the transposon insertion sites were then used as probes to clone the wild-type loci from M. smegmatis LR222 genomic DNA. Complementation assays showed that an 8 kb PstI fragment and a 4.8 kb PstI/SacI subclone of this fragment complemented one transposon mutant (LUN2) and one UV mutant (R92). A 10.1 kb SacI fragment restored exochelin production to the other transposon mutant (LUN1). The nucleotide sequence of the 15.3 kb DNA region that spanned the two transposon insertion sites overlapped the 5' region of the previously reported exochelin biosynthetic gene fxbA and contained three open reading frames that were transcribed in the opposite orientation to fxbA. The corresponding genes were designated exiT, fxbB and fxbC. The deduced amino acid sequence of ExiT suggested that it was a member of the ABC transporter superfamily, while FxbB and FxbC displayed significant homology with many enzymes (including pristinamycin I synthetase) that catalyse non-ribosomal peptide synthesis. We propose that the peptide backbone of the siderophore exochelin is synthesized in part by enzymes resembling non-ribosomal peptide synthetases and that the ABC transporter ExiT is responsible for exochelin excretion.     

Gene knockout reveals a novel gene cluster for the synthesis of a class of cell wall lipids unique to pathogenic mycobacteria

Surface-exposed unusual lipids containing phthiocerol and phenolphthiocerol are found only in the cell wall of slow-growing pathogenic mycobacteria and are thought to play important roles in host-pathogen interaction. The enzymology and molecular genetics of biosynthesis of phthiocerol and phenolphthiocerol are unknown. We postulate the domain organization of a set of multifunctional enzymes and a cluster of genes (pps) that would encode these enzymes for the biosynthesis of phthiocerol and phenolphthiocerol. A cosmid containing the postulated pps gene cluster was identified by screening a genomic library of Mycobacterium bovis BCG with the postulated homologous domains from mycocerosic acid synthase and fatty acid synthase genes as probes. Homologous cosmids were also identified in the genomic libraries of Mycobacterium tuberculosis and Mycobacterium leprae. M. bovis BCG was transformed with a pps disruption construct, made from the BCG cosmid by introducing the hygromycin resistance gene as the positive-selectable marker and the sacB gene as the counter-selectable marker. Gene disruption by homologous recombination with double crossover was confirmed by polymerase chain reaction and Southern hybridization. Chromatographic analysis showed that the phenolphthiocerol derivative, mycoside B, and phthiocerol dimycocerosates were not produced by the gene knockout mutants. This result confirms the identity of the pps genes. With the identification of the pps gene clusters in both M. tuberculosis and M. leprae, it should be possible to test the postulated roles of these unique lipids in tuberculosis and leprosy.     

Cloning and characterization of arylamine N-acetyltransferase genes from Mycobacterium smegmatis and Mycobacterium tuberculosis: increased expression results in isoniazid resistance

Arylamine N-acetyltransferases (NATs) are found in many eukaryotic organisms, including humans, and have previously been identified in the prokaryote Salmonella typhimurium. NATs from many sources acetylate the antitubercular drug isoniazid and so inactivate it. nat genes were cloned from Mycobacterium smegmatis and Mycobacterium tuberculosis, and expressed in Escherichia coli and M. smegmatis. The induced M. smegmatis NAT catalyzes the acetylation of isoniazid. A monospecific antiserum raised against pure NAT from S. typhimurium recognizes NAT from M. smegmatis and cross-reacts with recombinant NAT from M. tuberculosis. Overexpression of mycobacterial nat genes in E. coli results in predominantly insoluble recombinant protein; however, with M. smegmatis as the host using the vector pACE-1, NAT proteins from M. tuberculosis and M. smegmatis are soluble. M. smegmatis transformants induced to express the M. tuberculosis nat gene in culture demonstrated a threefold higher resistance to isoniazid. We propose that NAT in mycobacteria could have a role in acetylating, and hence inactivating, isoniazid.     

A human homolog of bacterial acetolactate synthase genes maps within the CADASIL critical region

CADASIL, a recently identified autosomal dominant condition characterized by the recurrence of subcortical infarcts leading to dementia, was previously mapped to chromosome 19p13.1 within a 2-cM interval, D19S226-D19S199. No recombination event was observed with D19S841, a highly polymorphic microsatellite marker isolated from a cosmid mapped to this region. We recently identified within this cosmid a conserved sequence that we used to screen a fetal brain cDNA library and isolated an ubiquitous and abundantly transcribed gene. We did not detect any mutation of this gene in CADASIL patients, suggesting that it is not implicated in this disorder. Interestingly, this gene encodes a putative protein homologous to several thiamine pyrophosphate-binding proteins previously identified in bacteria, yeast, and plants. The proteins with the highest degree of similarity were the acetolactate synthase enzymes which, in prokaryotes, are involved in the branched chain amino acid biosynthetic pathway, raising fascinating questions on the yet unknown function of this gene in mammals.     

Cloning of a Vibrio cholerae vibriobactin gene cluster: identification of genes required for early steps in siderophore biosynthesis

Vibrio cholerae secretes the catechol siderophore vibriobactin in response to iron limitation. Vibriobactin is structurally similar to enterobactin, the siderophore produced by Escherichia coli, and both organisms produce 2,3-dihydroxybenzoic acid (DHBA) as an intermediate in siderophore biosynthesis. To isolate and characterize V. cholerae genes involved in vibriobactin biosynthesis, we constructed a genomic cosmid bank of V. cholerae DNA and isolated clones that complemented mutations in E. coli enterobactin biosynthesis genes. V. cholerae homologs of entA, entB, entC, entD, and entE were identified on overlapping cosmid clones. Our data indicate that the vibriobactin genes are clustered, like the E. coli enterobactin genes, but the organization of the genes within these clusters is different. In this paper, we present the organization and sequences of genes involved in the synthesis and activation of DHBA. In addition, a V. cholerae strain with a chromosomal mutation in vibA was constructed by marker exchange. This strain was unable to produce vibriobactin or DHBA, confirming that in V. cholerae VibA catalyzes an early step in vibriobactin biosynthesis.     

Inhibition of breast cancer in nude mouse model by anti-angiogenesis

The type II secretion system (main terminal branch of the general secretion pathway) is used by diverse gram-negative bacteria to secrete extracellular proteins. Proteins secreted by this pathway are synthesized with an N-terminal signal peptide which is removed upon translocation across the inner membrane, but the signals which target the mature proteins for secretion across the outer membrane are unknown. The plant pathogens Erwinia chrysanthemi and Erwinia carotovora secrete several isozymes of pectate lyase (Pel) by the out-encoded type II pathway. However, these two bacteria cannot secrete Pels encoded by heterologously expressed pel genes from the other species, suggesting the existence of species-specific secretion signals within these proteins. The functional cluster of E. chrysanthemi out genes carried on cosmid pCPP2006 enables Escherichia coli to secrete E. chrysanthemi, but not E. carotovora, Pels. We exploited the high sequence similarity between E. chrysanthemi PelC and E. carotovora Pel1 to construct 15 hybrid proteins in which different regions of PelC were replaced with homologous sequences from Pell. The differential secretion of these hybrid proteins by E. coli(pCPP2006) revealed M118 to D175 and V215 to C329 as regions required for species-specific secretion of PelC. We propose that the primary targeting signal is contained within the external loops formed by G274 to C329 but is dependent on residues in M118 to D170 and V215 to G274 for proper positioning.     

Characterization of genes involved in molybdenum transport in Azotobacter vinelandii

Expression of alternative nitrogenases in Azotobacter vinelandii is repressed by molybdenum. Two strains with Tn5 insertion mutations showed alternative nitrogenase-dependent diazotrophic growth in the presence of Mo. The mutations were in a region which contained four open reading frames (ORFs 1-4). The genetic structure and predicted products of ORFs 2, 3 and 4 are typical of the membrane-associated elements of the ATP-binding cassette (ABC) superfamily of transport systems. The products of ORF3 and ORF4 are homologous with the products of the Escherichia coli genes chlD and the partially sequenced chlJ, respectively, both of which are implicated in molybdenum transport. ORF1, which is in the relative position of bacterial permease genes commonly specifying periplasmic binding proteins, encodes a 29 kDa protein with a novel primary structure. It lacks a potential signal sequence, and its C-terminal half consists of a tandem repeat of a segment which is homologous with the M(r) 7 kDa molybdenum-pterin binding protein Mop from Clostridium pasteurianum. This suggests that a substituted pterin may be involved in the initial capture or early metabolism of molybdenum.     

Identification of a gene cluster of biosynthetic genes of rubradirin substructures in S. achromogenes var. rubradiris NRRL3061

Rubradirin, an ansamycin antibiotic has been purified from Streptomyces achromogenes var. rubradiris NRRL3061. It consists of four distinct structural moieties, rubransarol, 3-amino-4-hydroxy-coumarin, dihydroxydipicolinic acid, and 2,6-dideoxynitrosugar (DNS). Polymerase chain reaction (PCR) primers were designed based on consensus sequences of dTDP-D-glucose 4,6-dehydratase, one of enzymes involved in the biosynthesis of 2,6-dideoxysugar. A PCR product was obtained from S. achromogenes var. rubradiris. Hybridization of the PCR product to a cosmid library constructed from S. achromogenes genomic DNA has led to the identification of three unlinked regions of DNA. One of three kinds of cosmid clones contains homologues of dTDP-D-glucose 4,6-dehydratase, 3-amino-5-hydroxybenzoic acid (AHBA) synthase, and eryA genes. The size of the gene homologous to eryA is 30 kb, and the AHBA synthase gene homologue resides between the eryA homologous genes. A gene cluster of rubransarol and 2,6-dideoxynitrosugar is around 50 kb. Sequencing of the PCR product from the AHBA synthase gene homologue isolated from S. achromogenes revealed 85% amino acid sequence homology (73/86) with the AHBA synthase from a rifamycin-producer. dTDP-D-glucose 4,6-dehydratase gene homologue was subcloned from one of the isolated cosmid clones and sequenced. It showed 65% homology (43/66) with dTDP-D-glucose 4,6-dehydratase from a streptomycin-producer.     

A cluster of genes involved in polysaccharide biosynthesis from Enterococcus faecalis OG1RF

Our previous work identified a cosmid clone containing a 43-kb insert from Enterococcus faecalis OG1RF that produced a nonprotein antigen in Escherichia coli. In the present work, we studied this clone in detail. Periodate treatment of lysates of the clone confirmed that the antigen was carbohydrate in nature. Analysis of DNA sequences and transposon insertion mutants suggested that the insert contained a multicistronic gene cluster. Database comparison showed that the cluster contained genes similar to genes involved in the biosynthesis of dTDP-rhamnose, glycosyltransferases, and ABC transporters involved in the export of sugar polymers from both gram-positive and gram-negative organisms. Insertions in several genes within the cluster abolished the immunoreactivity of the clone. This is the first report on a gene cluster of E. faecalis involved in the biosynthesis of an antigenic polysaccharide.     

Determination of the structure of exochelin MN, the extracellular siderophore from Mycobacterium neoaurum

BACKGROUND: Siderophores are compounds produced by bacteria to acquire iron. Exochelin MN, the extracellular siderophore from Mycobacterium neoaurum, is of particular interest because it has been shown to transport iron into M. leprae, which is responsible for the disease leprosy. Exochelins from other species cannot mediate iron transport in M. leprae, suggesting a specific uptake mechanism involving exochelin MN. We set out to determine the structure of exochelin MN and identify the features of the molecule that may account for this specificity. RESULTS: The structure of exochelin MN was elucidated by a combination of techniques including nuclear magnetic resonance, mass spectrometry, derivatization and gas chromatography. Exochelin MN is a peptide, containing the unusual amino acid beta-hydroxyhistidine and an unusual N-methyl group. The peptide coordinates iron(III) octahedrally using its two cis-hydroxamate groups plus the hydroxyl and imidazole nitrogen of the beta-hydroxyhistidine. The three-dimensional structure of the hexadentate exochelin/gallium complex was deduced from NMR data. CONCLUSIONS: Exochelin MN has some structural features in common with other siderophores, but has a unique three-dimensional structure, which is presumably important for its specific activity in M. leprae. Exochelin MN may be a target for drug design in the fight against infection with this pathogen.     

Identification, genomic organization, and analysis of the group III capsular polysaccharide genes kpsD, kpsM, kpsT, and kpsE from an extraintestinal isolate of Escherichia coli (CP9, O4/K54/H5)

Group III capsular polysaccharides (e.g., K54) of extraintestinal isolates of Escherichia coli, similar to group II capsules (e.g., K1), are important virulence traits that confer resistance to selected host defense components in vitro and potentiate systemic infection in vivo. The genomic organization of group II capsule gene clusters has been established as a serotype-specific region 2 flanked by regions 1 and 3, which contain transport genes that are highly homologous between serotypes. In contrast, the organization of group III capsule gene clusters is not well understood. However, they are defined in part by an absence of genes with significant nucleotide homology to group II capsule transport genes in regions 1 and 3. Evaluation of isogenic, TnphoA-generated, group III capsule-minus derivatives of a clinical blood isolate (CP9, O4/K54/H5) has led to the identification of homologs of the group II capsule transport genes kpsDMTE. These genes and their surrounding regions were sequenced and analyzed. The genomic organization of these genes is distinctly different from that of their group II counterparts. Although kps(K54)DMTE are significantly divergent from their group II homologs at both the DNA and protein levels phoA fusions and computer-assisted analyses suggest that their structures and functions are similar. The putative proteins Kps(K54)M and Kps(K54)T appear to be the integral membrane component and the peripheral ATP-binding component of the ABC-2 transporter family, respectively. The putative Kps(K54)E possesses features similar to those of the membrane fusion protein family that facilitates the passage of large molecules across the periplasm. At one boundary of the capsule gene cluster, a truncated kpsM (kpsM(truncated) and its 5' noncoding regulatory sequence were identified. In contrast to the complete kps(K54)M, this region was highly homologous to the group II kpsM. Fifty-three base pairs 3' from the end of kpsM(truncated) was a sequence 75% homologous to the 39-bp inverted repeat in the IS110 insertion element from Streptomyces coelicolor. Southern analysis established that two copies of this element are present in CP9. These findings are consistent with the hypothesis that CP9 previously possessed group II capsule genes and acquired group III capsule genes via IS110-mediated horizontal transfer.     

Evolution of ATP-binding cassette transporter genes

The transport of molecules across lipid membranes is an essential function of all living organisms. One of the families of genes that have evolved to carry out this function is that which encodes the ATP-binding cassette proteins. These molecules use active transport to pump specific molecules across membranes, and the genes that encode them are found in abundance in the genomes of both prokaryotes and eukaryotes. By using gene disruption techniques and by studying homologous genes in model organisms, significant progress has been made during the last few years in evaluating the physiological functions of ABC proteins in higher eukaryotes.     

Molecular and immunological analyses of the Mycobacterium avium homolog of the immunodominant Mycobacterium leprae 35-kilodalton protein

The analysis of host immunity to mycobacteria and the development of discriminatory diagnostic reagents relies on the characterization of conserved and species-specific mycobacterial antigens. In this report, we have characterized the Mycobacterium avium homolog of the highly immunogenic M. leprae 35-kDa protein. The genes encoding these two proteins were well conserved, having 82% DNA identity and 90% identity at the amino acid level. Moreover both proteins, purified from the fast-growing host M. smegmatis, formed multimeric complexes of around 1000 kDa in size and were antigenically related as assessed through their recognition by antibodies and T cells from M. leprae-infected individuals. The 35-kDa protein exhibited significant sequence identity with proteins from Streptomyces griseus and the cyanobacterium Synechoccocus sp. strain PCC 7942 that are up-regulated under conditions of nutrient deprivation. The 67% amino acid identity between the M. avium 35-kDa protein and SrpI of Synechoccocus was spread across the sequences of both proteins, while the homologous regions of the 35-kDa protein and the P3 sporulation protein of S. griseus were interrupted in the P3 protein by a divergent central region. Assessment by PCR demonstrated that the gene encoding the M. avium 35-kDa protein was present in all 30 M. avium clinical isolates tested but absent from M. intracellulare, M. tuberculosis, or M. bovis BCG. Mice infected with M. avium, but not M. bovis BCG, developed specific immunoglobulin G antibodies to the 35-kDa protein, consistent with the observation that tuberculosis patients do not recognize the antigen. Strong delayed-type hypersensitivity was elicited by the protein in guinea pigs sensitized with M. avium.     

Structure of a hydroxymycolic acid potentially involved in the synthesis of oxygenated mycolic acids of the Mycobacterium tuberculosis complex

Mycolic acids are believed to play a crucial role in the architecture of the mycobacterial envelope. However, very few steps of their biosynthetic pathway have yet been elucidated. We previously isolated [Dubnau, E., Lanéelle, M. A., Soares, S., Bénichou, A., Vaz, T., Promé, D., Promé, J. C., Daffé, M. & Quémard, A. (1997) Mycobacterium bovis BCG genes involved in the biosynthesis of cyclopropyl keto- and hydroxy-mycolic acids, Mol. Microbiol. 23, 313-322] a gene cluster from Mycobacterium bovis BCG, cmaA-D, which confers upon M. smegmatis the ability to synthesize cyclopropanated ketomycolic acid, and a new type of mycolic acid which is hydroxylated. A meticulous analysis of all the mycolic-like fatty acids of M. bovis BCG and M. tuberculosis showed that these organisms produce small amounts of the hydroxymycolic acid. The structure of this molecule, determined by NMR spectroscopy, mass spectrometry and stereochemical studies, strongly suggests that there is a direct biosynthetic relationship between the keto- and the hydroxy-mycolic acids.     

Purification, gene cloning, targeted knockout, overexpression, and biochemical characterization of the major pyrazinamidase from Mycobacterium smegmatis

The pyrazinamidase from Mycobacterium smegmatis was purified to homogeneity to yield a product of approximately 50 kDa. The deduced amino-terminal amino acid sequence of this polypeptide was used to design an oligonucleotide probe for screening a DNA library of M. smegmatis. An open reading frame, designated pzaA, which encodes a polypeptide of 49.3 kDa containing motifs conserved in several amidases was identified. Targeted knockout of the pzaA gene by homologous recombination yielded a mutant, pzaA::aph, with a more-than-threefold-reduced level of pyrazinamidase activity, suggesting that this gene encodes the major pyrazinamidase of M. smegmatis. Recombinant forms of the M. smegmatis PzaA and the Mycobacterium tuberculosis pyrazinamidase/nicotinamidase (PncA) were produced in Escherichia coli and were partially purified and compared in terms of their kinetics of nicotinamidase and pyrazinamidase activity. The comparable Km values obtained from this study suggested that the unique specificity of pyrazinamide (PZA) for M. tuberculosis was not based on an unusually high PZA-specific activity of the PncA protein. Overexpression of pzaA conferred PZA susceptibility on M. smegmatis by reducing the MIC of this drug to 150 micrograms/ml.     

Interactions between subunits of the human epithelial sodium channel

The human epithelial sodium channel (hENaC) mediates Na+ transport across the apical membrane of epithelia, and mutations in hENaC result in hypertensive and salt-wasting diseases. In heterologous expression systems, maximal hENaC function requires co-expression of three homologous proteins, the alpha, beta, and gammahENaC subunits, suggesting that hENaC subunits interact to form a multimeric channel complex. Using a co-immunoprecipitation assay, we found that hENaC subunits associated tightly to form homo- and heteromeric complexes and that the association between subunits occurred early in channel biosynthesis. Deletion analysis of gammahENaC revealed that the N terminus was sufficient but not necessary for co-precipitation of alphahENaC, and that both the N terminus and the second transmembrane segment (M2) were required for gamma subunit function. The biochemical studies were supported by functional studies. Co-expression of gamma subunits lacking M2 with full-length hENaC subunits revealed an inhibitory effect on hENaC channel function that appeared to be mediated by the cytoplasmic N terminus of gamma, and was consistent with the assembly of nonfunctional subunits into the channel complex. We conclude that the N terminus of gammahENaC is involved in channel assembly.     

Export of recombinant Mycobacterium tuberculosis superoxide dismutase is dependent upon both information in the protein and mycobacterial export machinery. A model for studying export of leaderless proteins by pathogenic mycobacteria

We have investigated the expression and extracellular release of enzymatically active superoxide dismutase, one of the 10 major extracellular proteins of Mycobacterium tuberculosis, both in its native host and in the heterologous host Mycobacterium smegmatis. We found that the M. tuberculosis superoxide dismutase gene, encoding a leaderless polypeptide of Mr approximately 23,000 representing one of the four identical subunits of the enzyme, is expressed constitutively under normal growth conditions and at a 5-fold increased level under conditions of hydrogen peroxide stress. The highly pathogenic mycobacterium M. tuberculosis expresses 93-fold more superoxide dismutase than the nonpathogenic mycobacterium M. smegmatis, and it exports a much higher proportion of expressed enzyme (76 versus 21%); taking both expression and export into consideration, M. tuberculosis exports approximately 350-fold more enzyme than M. smegmatis. In M. smegmatis, recombinant M. tuberculosis superoxide dismutase is expressed at 8.4 times the level of the endogenous enzyme and the proportion exported (66%) approaches that in the homologous host; hence M. smegmatis exports up to 26-fold more of the recombinant than endogenous enzyme. Interestingly, subunits of the M. tuberculosis and M. smegmatis enzymes readily and stoichiometrically exchange with each other, forming five different complexes of four subunits, both when the enzymes are expressed in the recombinant host and when the purified enzymes are incubated together; however, each subunit retains its characteristic metal ion, iron for M. tuberculosis and manganese for M. smegmatis. Compared with the cell-associated enzyme, the supernatant enzyme of recombinant M. smegmatis is enriched for M. tuberculosis enzyme subunits, consistent with preferential export of the M. tuberculosis enzyme. Recombinant M. tuberculosis superoxide dismutase transcomplements a superoxide dismutase-deficient Escherichia coli, resulting in a reduction of sensitivity of the strain to oxidative stress, but the enzyme is not exported from this nonmycobacterial host. Our findings indicate that the information for export of the M. tuberculosis superoxide dismutase is contained within the protein but that export additionally requires export machinery specific to mycobacteria.