Biosynthesis of mycobacterial arabinogalactan: identification of a novel alpha(1-->3) arabinofuranosyltransferase

The cell wall mycolyl-arabinogalactan–peptidoglycan complex is essential in mycobacterial species, such as Mycobacterium tuberculosis and is the target of several antitubercular drugs. For instance, ethambutol targets arabinogalactan biosynthesis through inhibition of the arabinofuranosyltransferases Mt-EmbA and Mt-EmbB. A bioinformatics approach identified putative integral membrane proteins, MSMEG2785 in Mycobacterium smegmatis , Rv2673 in Mycobacterium tuberculosis and NCgl1822 in Corynebacterium glutamicum , with 10 predicted transmembrane domains and a glycosyltransferase motif (DDX), features that are common to the GT-C superfamily of glycosyltransferases. Deletion of M. smegmatis MSMEG2785 resulted in altered growth and glycosyl linkage analysis revealed the absence of AG α(1→3)-linked arabinofuranosyl (Ara f ) residues. Complementation of the M. smegmatis deletion mutant was fully restored to a wild-type phenotype by MSMEG2785 and Rv2673, and as a result, we have now termed this previously uncharacterized open reading frame, a rabino f uranosyl t ransferase C ( aftC ). Enzyme assays using the sugar donor β- d -arabinofuranosyl-1-monophosphoryl-decaprenol (DPA) and a newly synthesized linear α(1→5)-linked Ara₅ neoglycolipid acceptor together with chemical identification of products formed, clearly identified AftC as a branching α(1→3) arabinofuranosyltransferase. This newly discovered glycosyltransferase sheds further light on the complexities of Mycobacterium cell wall biosynthesis, such as in M. tuberculosis and related species and represents a potential new drug target.     

Investigating the function of the putative mycolic acid methyltransferase UmaA: divergence between the Mycobacterium smegmatis and Mycobacterium tuberculosis proteins

Mycolic acids are major and specific lipid components of the cell envelope of mycobacteria that include the causative agents of tuberculosis and leprosy, Mycobacterium tuberculosis and Mycobacterium leprae, respectively. Subtle structural variations that are known to be crucial for both their virulence and the permeability of their cell envelope occur in mycolic acids. Among these are the introduction of cyclopropyl groups and methyl branches by mycolic acid S-adenosylmethionine-dependent methyltransferases (MA-MTs). While the functions of seven of the M. tuberculosis MA-MTs have been either established or strongly presumed nothing is known of the roles of the remaining umaA gene product and those of M. smegmatis MA-MTs. Mutants of the M. tuberculosis umaA gene and its putative M. smegmatis orthologue, MSMEG0913, were created. The lipid extracts of the resulting mutants were analyzed in detail using a combination of analytical techniques such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and proton nuclear magnetic resonance spectroscopy, and chemical degradation methods. The M. smegmatis mutants no longer synthesized subtypes of mycolates containing a methyl branch adjacent to either trans cyclopropyl group or trans double bond at the "proximal" position of both alpha- and epoxy-mycolates. Complementation with MSMEG0913, but not with umaA, fully restored the wild-type phenotype in M. smegmatis. Consistently, no modification was observed in the structures of mycolic acids produced by the M. tuberculosis umaA mutant. These data proved that despite their synteny and high similarity umaA and MSMEG0913 are not functionally orthologous.     

Identification of a novel alpha(1-->6) mannopyranosyltransferase MptB from Corynebacterium glutamicum by deletion of a conserved gene, NCgl1505, affords a lipomannan- and lipoarabinomannan-deficient mutant

Mycobacterium tuberculosis and Corynebacterium glutamicum share a similar cell wall structure and orthologous enzymes involved in cell wall assembly. Herein, we have studied C. glutamicum NCgl1505, the orthologue of putative glycosyltransferases Rv1459c from M. tuberculosis and MSMEG3120 from Mycobacterium smegmatis. Deletion of NCgl1505 resulted in the absence of lipomannan (Cg-LM-A), lipoarabinomannan (Cg-LAM) and a multi-mannosylated polymer (Cg-LM-B) based on a 1,2-di-O-C(16)/C(18:1)-(alpha-D-glucopyranosyluronic acid)-(1-->3)-glycerol (GlcAGroAc(2)) anchor, while syntheses of triacylated-phosphatidyl-myo-inositol dimannoside (Ac(1)PIM(2)) and Man(1)GlcAGroAc(2) were still abundant in whole cells. Cell-free incubation of C. glutamicum membranes with GDP-[(14)C]Man established that C. glutamicum synthesized a novel alpha(1-->6)-linked linear form of Cg-LM-A and Cg-LM-B from Ac(1)PIM(2) and Man(1)GlcAGroAc(2) respectively. Furthermore, deletion of NCgl1505 also led to the absence of in vitro synthesized linear Cg-LM-A and Cg-LM-B, demonstrating that NCgl1505 was involved in core alpha(1-->6) mannan biosynthesis of Cg-LM-A and Cg-LM-B, extending Ac(1)PI[(14)C]M(2) and [(14)C]Man(1)GlcAGroAc(2) primers respectively. Use of the acceptor alpha-D-Manp-(1-->6)-alpha-D-Manp-O-C(8) in an in vitro cell-free assay confirmed NCgl1505 as an alpha(1-->6) mannopyranosyltransferase, now termed MptB. While Rv1459c and MSMEG3120 demonstrated similar in vitroalpha(1-->6) mannopyranosyltransferase activity, deletion of the Rv1459c homologue in M. smegmatis did not result in loss of mycobacterial LM/LAM, indicating a functional redundancy for this enzyme in mycobacteria.     

The two chorismate mutases from both Mycobacterium tuberculosis and Mycobacterium smegmatis: biochemical analysis and limited regulation of promoter activity by aromatic amino acids

Chorismate mutase (CM) catalyzes the rearrangement of chorismate to prephenate in the biosynthetic pathway that forms phenylalanine and tyrosine in bacteria, fungi, plants, and apicomplexan parasites. Since this enzyme is absent from mammals, it represents a promising target for the development of new antimycobacterial drugs, which are needed to combat Mycobacterium tuberculosis, the causative agent of tuberculosis. Until recently, two putative open reading frames (ORFs), Rv0948c and Rv1885c, showing low sequence similarity to CMs have been described as "conserved hypothetical proteins" in the M. tuberculosis genome. However, we and others demonstrated that these ORFs are in fact monofunctional CMs of the AroQ structural class and that they are differentially localized in the mycobacterial cell. Since homologues to the M. tuberculosis enzymes are also present in Mycobacterium smegmatis, we cloned the coding sequences corresponding to ORFs MSMEG5513 and MSMEG2114 from the latter. The CM activities of both ORFs was determined, as well as their translational start sites. In addition, we analyzed the promoter activities of three M. tuberculosis loci related to phenylalanine and tyrosine biosynthesis under a variety of conditions using M. smegmatis as a surrogate host. Our results indicate that the aroQ (Rv0948c), *aroQ (Rv1885c), and fbpB (Rv1886c) genes from M. tuberculosis are constitutively expressed or subjected to minor regulation by aromatic amino acids levels, especially tryptophan.     

New insights into the biosynthesis of mycobacterial lipomannan arising from deletion of a conserved gene

Genetic construction of a mutant strain (designated MSMEG4245) of Mycobacterium smegmatis, defective in a broadly conserved gene for a putative glycosyltransferase of the glycosyltransferase-C superfamily, results in a phenotype marked by the virtual absence of the phosphatidylinositol-containing lipomannan and lipoarabinomannan, replaced instead by a novel truncated form of lipomannan. The normal spectrum of phosphatidylinositol mannosides, long presumed precursors of these lipoglycans, was retained. Matrix-assisted laser desorption/ionization-time of flight/mass spectrometry of the mutated form of lipomannan shows a family of phosphatidylinositol-anchored lipomannans with from only 5 to 20 Manp residues as compared with lipomannan from the wild type strain consisting of 21-34 Manp residues but with few changes in the branching pattern. Thus, MSMEG4245 is apparently a key mannosyltransferase, required for the proper elongation of lipomannan to its normal state and subsequent synthesis of lipoarabinomannan. The corresponding ortholog in Mycobacterium tuberculosis H37Rv has been identified as Rv2174. This previously unrecognized feature of the biosynthesis of lipomannan/lipoarabinomannan allows a significant revision of structural and biosynthetic schemata and provides a molecular basis of selectivity in biosynthesis, as conferred by the MSMEG4245 gene.     

Identification of the mycobacterial glucosyl-3-phosphoglycerate synthase.

Mycobacteria synthesize unique polysaccharides that regulate fatty acid synthesis, namely the methylglucose lipopolysaccharide (MGLP) and the methylmannose polysaccharide. Glucosyl-(1-->2)-glycerate is found at the reducing end of MGLP. The mycobacterial gene encoding a glucosyl-3-phosphoglycerate synthase (GpgS), primarily found in actinobacteria and sharing very low amino acid identity with known homo-functional GpgSs, has been identified. This gene has been annotated as an inverting family 2 glycosyltransferase of unknown function. The gpgS genes from the fast-growing Mycobacterium smegmatis strain 1102 and from the slow-growing Mycobacterium bovis BCG in Escherichia coli were expressed, and the recombinant enzymes were purified and characterized. The substrates for optimal activity were UDP-glucose and d-3-phosphoglycerate but ADP-glucose was also an efficient donor. The enzymes had maximal activity around 45 degrees C, pH 8.0, and were strictly dependent on Mg(2+). In Mycobacterium tuberculosis H37Rv, the gene encoding GpgS (Rv1208) is identical to the homologue in Mycobacterium bovis BCG and was considered to be essential for growth. It is shown that these genes encode retaining family 81 glycosyltransferases regardless of the low amino acid identity with other known enzymes of this family.     

Capsular glucan and intracellular glycogen of Mycobacterium tuberculosis: biosynthesis and impact on the persistence in mice

Mycobacterium tuberculosis and other pathogenic mycobacterial species produce large amounts of a glycogen-like alpha-glucan that represents the major polysaccharide of their outermost capsular layer. To determine the role of the surface-exposed glucan in the physiology and virulence of these bacteria, orthologues of the glg genes involved in the biosynthesis of glycogen in Escherichia coli were identified in M. tuberculosis H37Rv and inactivated by allelic replacement. Biochemical analyses of the mutants and complemented strains indicated that the synthesis of glucan and glycogen involves the alpha-1,4-glucosyltransferases Rv3032 and GlgA (Rv1212c), the ADP-glucose pyrophosphorylase GlgC (Rv1213) and the branching enzyme GlgB (Rv1326c). Disruption of glgC reduced by half the glucan and glycogen contents of M. tuberculosis, whereas the inactivation of glgA and Rv3032 affected the production of capsular glucan and glycogen, respectively. Attempts to disrupt Rv3032 in the glgA mutant were unsuccessful, suggesting that a functional copy of at least one of the two alpha-1,4-glucosyltransferases is required for growth. Importantly, the glgA mutant was impaired in its ability to persist in mice, suggesting a role for the capsular glucan in the persistence phase of infection. Unexpectedly, GlgB was found to be an essential enzyme.     

Inactivation of the mycobacterial rhamnosyltransferase, which is needed for the formation of the arabinogalactan-peptidoglycan linker, leads to irreversible loss of viability

Temperature-sensitive mutant 2-20/32 of Mycobacterium smegmatis mc(2)155 was isolated and genetically complemented with a Mycobacterium tuberculosis H37Rv DNA fragment that contained a single open reading frame. This open reading frame is designated Rv3265c in the M. tuberculosis H37Rv genome. Rv3265c shows homology to the Escherichia coli gene wbbL, which encodes a dTDP-Rha:alpha-D-GlcNAc-pyrophosphate polyprenol, alpha-3-L-rhamnosyltransferase. In E. coli this enzyme is involved in O-antigen synthesis, but in mycobacteria it is required for the rhamnosyl-containing linker unit responsible for the attachment of the cell wall polymer mycolyl-arabinogalactan to the peptidoglycan. The M. tuberculosis wbbL homologue, encoded by Rv3265c, was shown to be capable of restoring an E. coli K12 strain containing an insertionally inactivated wbbL to O-antigen positive. Likewise, the E. coli wbbL gene allowed 2-20/32 to grow at higher non-permissive temperatures. The rhamnosyltransferase activity of M. tuberculosis WbbL was demonstrated in 2-20/32 as was the loss of this transferase activity in 2-20/32 at elevated temperatures. The wbbL of the temperature-sensitive mutant contained a single-base change that converted what was a proline in mc(2)155 to a serine residue. Exposure of 2-20/32 to higher non-permissive temperatures resulted in bacteria that could not be recovered at the lower permissive temperatures.     

Transfer of the first arabinofuranose residue to galactan is essential for Mycobacterium smegmatis viability

The mycobacterial arabinan is an elaborate component of the cell wall with multiple glycosyl linkages and no repeating units. In Mycobacterium spp., the Emb proteins (EmbA, EmbB, and EmbC) have been identified as putative mycobacterial arabinosyltransferases implicated in the biogenesis of the cell wall arabinan. Furthermore, it is now evident that the EmbA and EmbB proteins are involved in the assembly of the nonreducing terminal motif of arabinogalactan and EmbC is involved in transferring arabinose, perhaps in the early stage of arabinan synthesis in lipoarabinomannan. It has also been shown that the Emb proteins are a target of the antimycobacterial drug ethambutol (EMB). In the search for additional mycobacterial arabinosyltransferases in addition to the Emb proteins, we disrupted MSMEG_6386 (an orthologue of Rv3792 and a gene upstream of embC) in Mycobacterium smegmatis. Allelic exchange at the chromosomal MSMEG_6386 locus of M. smegmatis could only be achieved in the presence of a rescue plasmid carrying a functional copy of MSMEG_6386 or Rv3792, strongly suggesting that MSMEG_6386 is essential. An in vitro arabinosyltransferase assay using a membrane preparation from M. smegmatis expressing Rv3792 and synthetic beta-d-Galf-(1-->5)-beta-D-Galf-(1-->6)-beta-D-Galf-octyl and beta-D-Galf-(1-->6)-beta-D-Galf-(1-->5)-beta-D-Galf-octyl showed that Rv3792 gene product can transfer an arabinose residue to the C-5 position of the internal 6-linked galactose. The reactions were insensitive to EMB, and when alpha-d-Manp-(1-->6)-alpha-D-Manp-(1-->6)-alpha-D-Manp-octylthiomethyl was used as an acceptor, no product was formed. These observations indicate that transfer of the first arabinofuranose residue to galactan is essential for M. smegmatis viability.     

Identification of the required acyltransferase step in the biosynthesis of the phosphatidylinositol mannosides of mycobacterium species

Fatty acyl functions of the glycosylated phosphatidylinositol (GPI) anchors of the phosphatidylinositol mannosides (PIM), lipomannan (LM), and lipoarabinomannan (LAM) of mycobacteria play a critical role in both the physical properties and biological activities of these molecules. In a search for the acyltransferases that acylate the GPI anchors of PIM, LM, and LAM, we examined the function of the mycobacterial Rv2611c gene that encodes a putative acyltransferase involved in the early steps of phosphatidylinositol mannoside synthesis. A Rv2611c mutant of Mycobacterium smegmatis was constructed which exhibited severe growth defects and contained an increased amount of phosphatidylinositol mono- and di-mannosides and a decreased amount of acylated phosphatidylinositol di-mannosides compared with the wild-type parental strain. In cell-free assays, extracts from M. smegmatis overexpressing the M. tuberculosis Rv2611c gene incorporated [14C]palmitate into acylated phosphatidylinositol mono- and di-mannosides, and transferred cold endogenous fatty acids onto 14C-labeled phosphatidylinositol mono- and di-mannosides more efficiently than extracts from the wild-type strain. Cell-free extracts from the Rv2611c mutant of M. smegmatis were greatly impaired in these respects. This work provides evidence that Rv2611c is the acyltransferase that catalyzes the acylation of the 6-position of the mannose residue linked to position 2 of myo-inositol in phosphatidylinositol mono- and di-mannosides, with the mono-mannosylated lipid acceptor being the primary substrate of the enzyme. We also provide the first evidence that two distinct pathways lead to the formation of acylated PIM2 from PIM1 in mycobacteria.     

The RD1 proteins of Mycobacterium tuberculosis: expression in Mycobacterium smegmatis and biochemical characterization

A 9.5-kb section of DNA called region of deletion 1 (RD1) is present in virulent Mycobacterium tuberculosis strains but is deleted in all attenuated Mycobacterium bovis BCG vaccine strains. This region codes for at least nine genes. Some or all RD1 gene products may be involved in virulence and pathogenesis, and at least two, ESAT-6 and CFP-10, represent potent T- and B-cell antigens. In order to produce the entire set of RD1 proteins with their natural posttranslational modifications, a robust expression system for M. tuberculosis proteins in the fast-growing saprophytic strain Mycobacterium smegmatis was developed. Our system employs the inducible acetamidase promoter and allows translational fusion of recombinant M. tuberculosis proteins with polyhistidine or influenza hemagglutinin epitope tags for affinity purification. Using eGFP as reporter gene, we showed that the acetamidase promoter is tightly regulated in M. smegmatis and that this promoter is much stronger than the widely used constitutive groEL2 promoter. We then cloned 11 open reading frames (ORFs) found within RD1 and successfully expressed and purified the respective proteins. Sera from tuberculosis patients and M. tuberculosis-infected mice reacted with 10 purified RD1 proteins, thus demonstrating that Rv3871, Rv3872, Rv3873, CFP-10, ESAT-6, Rv3876, Rv3878, Rv3879c and ORF-14 are expressed in vivo. Finally, glycosylation of the RD1 proteins was analyzed. We present preliminary evidence that the PPE protein Rv3873 is glycosylated at its C terminus, thus highlighting the ability of M. smegmatis to produce M. tuberculosis proteins bearing posttranslational modifications.     

Definition of the first mannosylation step in phosphatidylinositol mannoside synthesis. PimA is essential for growth of mycobacteria

We examined the function of the pimA (Rv2610c) gene, located in the vicinity of the phosphatidylinositol synthase gene in the genomes of Mycobacterium tuberculosis and Mycobacterium smegmatis, which encodes a putative mannosyltransferase involved in the early steps of phosphatidylinositol mannoside synthesis. A cell-free assay was developed in which membranes from M. smegmatis overexpressing the pimA gene incorporate mannose from GDP-[(14)C]Man into di- and tri-acylated phosphatidylinositol mono-mannosides. Moreover, crude extracts from Escherichia coli producing a recombinant PimA protein synthesized diacylated phosphatidylinositol mono-mannoside from GDP-[(14)C]Man and bovine phosphatidylinositol. To determine whether PimA is an essential enzyme of mycobacteria, we constructed a pimA conditional mutant of M. smegmatis. The ability of this mutant to synthesize the PimA mannosyltransferase was dependent on the presence of a functional copy of the pimA gene carried on a temperature-sensitive rescue plasmid. We demonstrate here that the pimA mutant is unable to grow at the higher temperature at which the rescue plasmid is lost. Thus, the synthesis of phosphatidylinositol mono-mannosides and derived higher phosphatidylinositol mannosides in M. smegmatis appears to be dependent on PimA and essential for growth. This work provides the first direct evidence of the essentiality of phosphatidylinositol mannosides for the growth of mycobacteria.     

p-Hydroxybenzoic acid synthesis in Mycobacterium tuberculosis

Glycosylated p-hydroxybenzoic acid methyl esters and structurally related phenolphthiocerol glycolipids are important virulence factors of Mycobacterium tuberculosis. Although both types of molecules are thought to be derived from p-hydroxybenzoic acid, the origin of this putative biosynthetic precursor in mycobacteria remained to be established. We describe the characterization of a transposon mutant of M. tuberculosis deficient in the production of all forms of p-hydroxybenzoic acid derivatives. The transposon was found to be inserted in Rv2949c, a gene located in the vicinity of the polyketide synthase gene pks15/1, involved in the elongation of p-hydroxybenzoate to phenolphthiocerol in phenolic glycolipid-producing strains. A recombinant form of the Rv2949c enzyme was produced in the fast-growing non-pathogenic Mycobacterium smegmatis and purified to near homogeneity. The recombinant enzyme catalyzed the removal of the pyruvyl moiety of chorismate to form p-hydroxybenzoate with an apparent K(m) value for chorismate of 19.7 microm and a k(cat) value of 0.102 s(-1). Strong inhibition of the reaction by p-hydroxybenzoate but not by pyruvate was observed. These results establish Rv2949c as a chorismate pyruvate-lyase responsible for the direct conversion of chorismate to p-hydroxybenzoate and identify Rv2949c as the sole enzymatic source of p-hydroxybenzoic acid in M. tuberculosis.     

Initiation of Methylglucose Lipopolysaccharide Biosynthesis in Mycobacteria

Background

Mycobacteria produce two unique families of cytoplasmic polymethylated polysaccharides - the methylglucose lipopolysaccharides (MGLPs) and the methylmannose polysaccharides (MMPs) - the physiological functions of which are still poorly defined. Towards defining the roles of these polysaccharides in mycobacterial physiology, we generated knock-out mutations of genes in their putative biosynthetic pathways.

Methodology/Principal Findings

We report here on the characterization of the Rv1208 protein of Mycobacterium tuberculosis and its ortholog in Mycobacterium smegmatis (MSMEG_5084) as the enzymes responsible for the transfer of the first glucose residue of MGLPs. Disruption of MSMEG_5084 in M. smegmatis resulted in a dramatic decrease in MGLP synthesis directly attributable to the almost complete abolition of glucosyl-3-phosphoglycerate synthase activity in this strain. Synthesis of MGLPs in the mutant was restored upon complementation with wild-type copies of the Rv1208 gene from M. tuberculosis or MSMEG_5084 from M. smegmatis.

Conclusions/Significance

This is the first evidence linking Rv1208 to MGLP biosynthesis. Thus, the first step in the initiation of MGLP biosynthesis in mycobacteria has been defined, and subsequent steps can be inferred.     

结核分枝杆菌蛋白Rv3425对细菌表型及毒力影响的研究

为探索蛋白Rv3425在结核分枝杆菌(Mycobacterium tuberculosis,M. tuberculosis)中的功能,本研究以耻垢分枝杆菌(Mycobacterium smegmatis,M. smegmatis)为模式菌株,构建重组了耻垢分枝杆菌Ms-Rv3425。分别将构建菌株(Ms-Rv3425)、野生株(Ms)及空载对照(Ms-Pact)接种于7H9-OADC培养基中37 ℃培养,观察Ms-Rv3425与Ms及Ms-Pact之间在生长速率、菌落形态、生物膜以及聚集度方面的差异。分别用低pH值以及含有十二烷基磺酸钠(sodium dodecyl sulfate,SDS)、氨苄西林、异烟肼及利福平的培养基进行培养,计算存活率以分析抗逆和抗药能力;用上述压力条件培养结核分枝杆菌标准株H37Ra,分析Rv3425内源表达量的变化;进行THP-1细胞感染和BALB/c小鼠攻毒实验分析菌株的毒性变化。结果显示,与Ms及Ms-Pact相比,Ms-Rv3425的菌落形态更为粗糙且隆起,成膜及聚集能力增强;在压力条件下,Ms-Rv3425表现出更高的抗逆和抗药能力,H37Ra中Rv3425的表达量也显著上调;胞内存活率及小鼠致死率更高,各脏器病理损伤更为严重。综上所述,过表达Rv3425能够改变耻垢分枝杆菌的表型,提高抗逆性、抗药性和毒力。深入探讨PPE家族蛋白Rv3425的功能,将为结核病的防治带来新的视角。     

结核杆菌活动期高表达基因Rv1776c的克隆及其在耻垢分枝杆菌中的表达

目的构建表达结核分枝杆菌Rv1776c基因的重组耻垢分支杆菌,并鉴定该基因在重组耻垢分支杆菌中的活性。方法采用PCR技术克隆结核分枝杆菌Rv1776c基因,构建大肠埃希菌-分支杆菌穿梭表达质粒pMV-Rv1776c,通过酶切和测序鉴定其正确性,用电穿孔法将重组质粒转染到耻垢分支杆菌mc^2155中。以SDS-PAGE及Western blot检测证实Rv1776c蛋白在重组耻垢分支杆菌内的表达。结果重组耻垢分支杆菌构建成功,生长曲线说明重组质粒不会影响耻垢分支杆菌的体外生长;SDSPAGE及Western blot检测证实Rv1776c在耻垢分枝杆菌内表达出相对分子量约56kD的Rv1776c蛋白。结论成功构建了Rv1776c基因的穿梭质粒pMV-Rv1776c,且该质粒在耻垢分枝杆菌内具有生物活性,为进一步研究其表达产物的功能提供基础。     

Identification of a Mycobacterium tuberculosis gene that enhances mycobacterial survival in macrophages

Intracellular survival plays a central role in the pathogenesis of Mycobacterium tuberculosis. To identify M. tuberculosis genes required for intracellular survival within macrophages, an M. tuberculosis H37Rv plasmid library was constructed by using the shuttle vector pOLYG. This plasmid library was electroporated into Mycobacterium smegmatis 1-2c, and the transformants were used to infect the human macrophage-like cell line U-937. Because M. smegmatis does not readily survive within macrophages, any increased intracellular survival is likely due to cloned M. tuberculosis H37Rv DNA. After six sequential passages of M. smegmatis transformants through U-937 cells, one clone (p69) was enriched more than 70% as determined by both restriction enzyme and PCR analyses. p69 demonstrated significantly enhanced survival compared to that of the vector control, ranging from 2.4- to 5.3-fold at both 24 and 48 h after infection. DNA sequence analysis revealed three open reading frames (ORFs) in the insert of p69. ORF2 (1.2 kb) was the only one which contained a putative promoter region and a ribosome-binding site. Deletion analysis of the p69 insert DNA showed that disruption of ORF2 resulted in complete loss of the enhanced intracellular survival phenotype. This gene was named the enhanced intracellular survival (eis) gene. By using an internal region of eis as a probe for Southern analysis, eis was found in the genomic DNA of various M. tuberculosis strains and of Mycobacterium bovis BCG but not in that of M. smegmatis or 10 other nonpathogenic mycobacterial species. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis showed that all M. smegmatis eis-containing constructs expressed a unique protein of 42 kDa, the predicted size of Eis. The expression of this 42-kDa protein directly correlated to the enhanced survival of M. smegmatis p69 in U-937 cells. These results suggest a possible role for eis and its protein product in the intracellular survival of M. tuberculosis.     

Large Mg(2+)-dependent currents are associated with the increased expression of ALR1 in Saccharomyces cerevisiae

Mycobacteria adapt to a decrease in oxygen tension by entry into a non-replicative persistent phase. It was shown earlier that the two-component system, DevR-DevS, was induced in Mycobacterium tuberculosis and Mycobacterium bovis BCG cultures during hypoxia, suggesting that it may play a regulatory role in their adaptation to oxygen limitation. The presence of a homologous genetic system in Mycobacterium smegmatis was predicted by scanning its unfinished genome sequence with devR and devS genes of M. tuberculosis. Rv3134c, which is cotranscribed with devR-devS in M. tuberculosis, was also present in M. smegmatis at a similar location upstream from devR. The expression of all three genes was induced at the RNA and protein levels in M. smegmatis cultures grown under microaerobic and anaerobic conditions. The M. smegmatis genome also contained the hspX gene, encoding chaperone alpha-crystallin, Acr, that was induced during hypoxia. The similarity in sequences and hypoxia-responsive behaviour of devR-devS, Rv3134c and hspX genes in M. smegmatis and M. tuberculosis suggests that the molecular mechanisms involved in the dormancy response are likely conserved in these two species. M. smegmatis could therefore serve as a useful model for the delineation of the hypoxia response in general and DevR-DevS regulated pathways in particular.     

Salicylate uniquely induces a 27-kDa protein in tubercle bacillus

Salicylate was found to uniquely induce a 27-kDa protein in Mycobacterium tuberculosis complex organisms but not in Mycobacterium smegmatis or Escherichia coli. The structural analogue antitubercular para-amino-salicylate also induced the 27-kDa protein but to a somewhat lower level than salicylate. Other structural analogues such as benzoic acid and acetyl salicylic acid (aspirin) did not induce the 27-kDa protein. Western blot analysis indicated that the 27-kDa protein was localized mainly in the cytoplasm. The 27-kDa protein was not expressed at different growth phases in the absence of salicylate. The 27-kDa protein was identified as a putative benzoquinone methyltransferase (Rv0560c), which has several homologues in the M. tuberculosis genome. The cloned 27-kDa gene was found to express constitutively in E. coli, M. smegmatis and BCG with or without salicylate.