Analysis of the Mycobacterium tuberculosis 85A antigen promoter region.

A mycobacterial expression-secretion vector was constructed in which the Escherichia coli alkaline phosphatase (phoA) reporter gene was placed under the control of the Mycobacterium tuberculosis 85A promoter and secretion signal sequences. In recombinant Mycobacterium smegmatis and Mycobacterium bovis BCG, PhoA activity could readily be detected on the mycobacterial cell surface and in the culture supernatant, indicating that the 85A signals can drive heterologous expression and secretion in both species. In contrast to the mycobacteria, the 85A promoter did not function in E. coli. We mapped the promoter region by progressive deletions using BAL 31 exonuclease and by primer extension analysis. Insertion and deletion mutations within the promoter region indicated that, unlike most E. coli promoters but similar to Streptomyces promoters, the position of the putative -35 region was not critical for efficient promoter activity. In addition, we investigated the ability of the identified signals to drive the production and secretion in BCG of recombinant Schistosoma mansoni glutathione S-transferase (Sm28GST), a protective antigen against schistosomiasis. BALB/c mice immunized with the recombinant BCG by a single dose exhibited a weak but specific T-cell response to Sm28GST.     

Properties of the Corynebacterium glutamicum metC gene encoding cystathionine beta-lyase

The metC gene encoding the cystathionine beta-lyase, the third enzyme in the methionine biosynthetic pathway, was isolated from Corynebacterium glutamicum by heterologous complementation of the Escherichia coli metC mutant. A DNA-sequence analysis of the cloned DNA identified two open-reading frames (ORFs) of ORF1 and ORF2 that consisted of 1,107 and 978 bp, respectively. A SDS-PAGE analysis identified a putative cystathionine beta-lyase band with approximate Mr of 41,000 that consisted of 368 amino acids encoded from ORF1. The translational product of the gene showed no significant homology with that of the metC gene from other organisms. Introduction of the plasmid containing the metC gene into C. glutamicum resulted in a 5-fold increase in the activity of the cystathionine beta-lyase. The putative protein product of ORF2, encoding a protein product of 35,574 Da, consisted of 325 amino acids and was identical to the previously reported aecD gene product, except for the existence of two different amino acids. Like the aecD gene, when present in multiple copies, the metC gene conferred resistance to S-(betaaminoethyl)-cysteine, which is a toxic lysine analog. However, genetic and biochemical evidence suggests that the natural activity of the metC gene product is to mediate methionine biosynthesis in C. glutamicum. Mutant strains of metC were constructed, and the strains showed methionine prototrophy. The mutant strains completely lost their ability to show resistance to the S-(beta-aminoethyl)-cysteine. These results suggest that, in addition to the transsulfuration, other biosynthetic pathway(s), such as a direct sulfhydrylation pathway, may be functional in C. glutamicum as a parallel biosynthetic route for methionine.     

Cloning of the argF gene encoding the ornithine carbamoyltransferase from Corynebacterium glutamicum.

The argF gene encoding ornithine carbamoyl-transferase (OTCase; EC2.1.3.3) has been cloned from Corynebacterium glutamicum by transforming the Escherichia coli arginine auxotroph with the genomic DNA library. The cloned DNA also complements the E. coli argG mutant, suggesting a clustered organization of the genes in the genome. We have determined the DNA sequence of the minimal fragment complementing the E. coli argF mutant. The coding region of the cloned gene is 957 nucleotides long with a deduced molecular mass of about 35 kDa polypeptide. The enzyme activity and size of the expressed protein in the E. coli auxotroph carrying the argF gene revealed that the cloned gene indeed codes for OTCase. Analysis of the amino acid sequence of the predicted protein revealed a strong similarity to the corresponding protein of other bacteria.     

The ptsI gene encoding enzyme I of the phosphotransferase system of Corynebacterium glutamicum.

The phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) is widespread among bacteria where it mediates carbohydrate uptake and often serves in carbon control. Here we present cloning and analysis of the monocistronic ptsI gene of Corynebacterium glutamicum R, which encodes PTS Enzyme I (EI). EI catalyzes the first reaction of PTS and the reported ptsI was shown to complement the corresponding defect in Escherichia coli. The deduced 59.2-kDa EI of 564 amino acids shares more than 50% homology with EIs from Bacillus stearothermophilus, Bacillus subtilis, and Lactobacillus sake. Chromosomal inactivation of ptsI demonstrated that EI plays an indispensable role in PTS of C. glutamicum R and this system represents a dominant sugar uptake system. Cellobiose was only transported and utilized in adaptive mutants of C. glutamicum R. Cellobiose transport was also found to be PTS-dependent and repressed by PTS sugar glucose.     

谷氨酸棒杆菌高丝氨酸脱氢酶编码基因hom的敲除

本研究以谷氨酸棒杆菌（Corynebacterium glutamicum）标准菌株ATCC 13032染色体为模板,设计引物PCR扩增高丝氨酸脱氢酶编码基因（hom）,在hom基因内部插入一段来源于质粒pET28a的卡那霉素抗性基因（Km）,得到基因元件hom::Km;通过电击转化法将hom::Km转入出发菌株替换原菌株的hom,在含卡那霉素的平板上挑取阳性转化子,通过PCR验证得到高丝氨酸脱氢酶缺陷的重组菌。发酵结果表明重组菌C.g- hom::Km -8发酵60小时赖氨酸产量达到4.7 g／L,是出发菌株谷氨酸棒杆菌ATCC 13032（0.7 g／L）的6.7倍。     

A coupled assay measuring Mycobacterium tuberculosis antigen 85C enzymatic activity

The prevalence of drug-resistant strains of Mycobacterium tuberculosis (M. tb) emphasizes the need for new antitubercular drugs. An essential component of the drug discovery process is the development of tools to rapidly screen potential drug libraries against important biological targets. Similarly to well-documented M. tb targets, the antigen 85 (Ag85) enzymes are involved in the maintenance of the mycobacterial cell wall. The products synthesized by these mycolyltransferases are the cell wall components most responsible for the reduced permeability of drugs into the bacterial cell, thereby linking Ag85 activity directly with drug resistance. This article presents the development of a high-throughput colorimetric assay suitable for direct monitoring of the enzymatic activity. The assay uses a synthetic substrate containing three chemical moieties: an octanoyl fatty acid, β-d-glucose, and p-nitrophenyl. In the context of the assay, Ag85 catalyzes the removal of the fatty acid and releases p-nitrophenyl-β-d-glucoside. The glucoside is hydrolyzed by β-glucosidase to release the p-nitrophenolate chromophore. With this assay, the K_M and k_cat values of Ag85C were determined to be 0.047 ± 0.008 mM and 0.062 s⁻¹, respectively. In addition, the assay exhibits a Z′ value of 0.81 ± 0.06, indicating its suitability for high-throughput screening applications and drug development.     

A tetracycline inducible expression vector for Corynebacterium glutamicum allowing tightly regulable gene expression

Here we report on the construction of a tetracycline inducible expression vector that allows a tightly regulable gene expression in Corynebacterium glutamicum which is used in industry for production of small molecules such as amino acids. Using the green fluorescent protein (GFP) as a reporter protein we show that this vector, named pCLTON1, is characterized by tight repression under non-induced conditions as compared to a conventional IPTG inducible expression vector, and that it allows gradual GFP synthesis upon gradual increase of anhydrotetracycline addition.     

Cloning and analysis of the aroB gene encoding dehydroquinate synthase from Corynebacterium glutamicum

The aroB gene encoding dehydroquinate synthase of Corynebacterium glutamicum has been cloned by complementation of an aro auxotrophic mutant of Escherichia coli with the genomic DNA library. The recombinant plasmid contained a 1.4-kb fragment that complemented the Escherichia coli dehydroquinate-synthase-deficient mutant. The nucleotide sequences of the subcloned DNA has been determined. The sequences contain an open reading frame of 360 codons, from which a protein with a molecular mass of about 38 kDa could be predicted. This is consistent with the size of the AroB protein expressed in E. coli. Alignment of different prokaryotic and eukaryotic aroB gene products reveals an overall identity ranging from 29 to 57% and the presence of several highly conserved regions.     

谷氨酸棒杆菌海藻糖合成酶基因的克隆及功能鉴定

利用生物信息学手段,在GenBank数据库进行氨基酸的同源性检索分析,发现来自谷氨酸棒杆茵(Corynebacterium glutamicum)一功能未确定的ORF序列被注释为假设的海藻糖酶(putative trehalose sesynthase),它与已报道的海藻糖合成酶的氨基酸序列有60％以上的同源性。本研究把这段ORF克隆到大肠杆茵进行表达及进行功能鉴定。实验表明这段ORF序列为一新的海藻糖合成酶基因,其表达产物能将麦芽糖分子转化成海藻糖分子。重组酶性质的初步研究表明重组酶在pH7．0～7．5,30℃转化麦芽糖效率最高。     

Heterologous expression and localization of gentisate transporter Ncg12922 from Corynebacterium glutamicum ATCC 13032

Ralstonia sp. strain U2 metabolizes naphthalene via gentisate (2,5-dihydroxybenzoate) to central metabolites, but it was found unable to utilize gentisate as growth substrate. A putative gentisate transporter encoded by ncg12922 from Corynebacterium glutamicum ATCC 13032 was functionally expressed in Ralstonia sp. strain U2, converting strain U2 to a gentisate utilizer. After ncg12922 was inserted into plasmid pGFPe with green fluorescence protein gene gfp, the expressed fusion protein Ncg12922-GFP could be visualized in the periphery of Escherichia coli cells under confocal microscope, consistent with a cytoplasmic membrane location. In contrast, GFP was ubiquitous in the cytoplasm of E. coli cells carrying pGFPe only. Gentisate 1,2-dioxygenase activity was present in the cell extract from strain U2 induced with gentisate but at a much lower level (one-fifth) than that obtained with salicylate. However, it exhibited a similar level in strain U2 containing Ncg12922 induced either by salicylate or gentisate.     

T cell expression cloning of a Mycobacterium tuberculosis gene encoding a protective antigen associated with the early control of infection

Infection of C57BL/6 mice with Mycobacterium tuberculosis results in the development of a progressive disease during the first 2 wk after challenge. Thereafter, the disease is controlled by the emergence of protective T cells. We have used this infection model in conjunction with direct T cell expression cloning to identify Ags involved with the early control of the disease. A protective M. tuberculosis-specific CD4 T cell line derived from mice at 3 wk postchallenge was used to directly screen an M. tuberculosis genomic expression library. This screen resulted in the identification of a genomic clone comprising two putative adjacent genes with predicted open reading frames of 10 and 41 kDa, MTB10 and MTB41, respectively (the products of Rv0916c and Rv0915c, respectively, in the TubercuList H37Rv database). MTB10 and MTB41 belong to the PE and PPE family of proteins recently identified to comprise 10% of the M. tuberculosis genome. Evaluation of the recombinant proteins revealed that MTB41, but not MTB10, is the Ag recognized by the cell line and by M. tuberculosis-sensitized human PBMC. Moreover, C57BL/6 mice immunized with MTB41 DNA developed both CD4- (predominantly Th1) and CD8-specific T cell responses to rMTB41 protein. More importantly, immunization of C57BL/6 mice with MTB41 DNA induced protection against infection with M. tuberculosis comparable to that induced by bacillus Calmette-Guérin. Thus, the use of a proven protective T cell line in conjunction with the T cell expression cloning approach resulted in the identification of a candidate Ag for a subunit vaccine against tuberculosis.     

Significance of the Cgl1427 gene encoding cytidylate kinase in microaerobic growth of Corynebacterium glutamicum

The Cgl1427 gene was previously found to be relevant to the microaerobic growth of Corynebacterium glutamicum (Ikeda et al. Biosci Biotechnol Biochem 73:2806–2808, 2009). In the present work, Cgl1427 was identified as a cytidylate kinase gene (cmk) by homology analysis of its deduced amino acid sequence with that of other bacterial cytidylate kinases (CMP kinases) and on the basis of findings that deletion of Cgl1427 results in loss of CMP kinase activity. Deletion of the cmk gene significantly impaired the growth of C. glutamicum in oxygen-limiting static culture, and the impaired growth was restored by introducing a plasmid containing the cmk gene, suggesting that this gene plays an important role in the microaerobic growth of C. glutamicum. On the other hand, in the main culture with aerobic shaking, a prolonged lag phase was observed in the cmk disruptant, despite an unchanged growth rate, compared to the behavior of the wild-type strain. The prolongation was observed when using seed culture grown to later growth stages in which oxygen limitation occurred, but it was not observed when using seed culture grown to an earlier growth stage in which oxygen remained relatively plentiful. Since nucleotide biosynthesis in C. glutamicum requires oxygen, we hypothesized that the ability of the cmk disruptant to synthesize nucleotides was influenced by oxygen limitation in the later growth stages of the seed culture, which caused the prolongation of the lag phase in the following shaken culture. To verify this hypothesis, a plasmid containing genes encoding all components of a homologous ribonucleotide reductase, a key enzyme for nucleotide synthesis that requires oxygen for its reaction, was introduced into the cmk disruptant, which significantly ameliorated the lag phase prolongation. Furthermore, this experimental setup almost completely restored the growth of the cmk disruptant in the oxygen-limiting static culture. These results indicate that CMP kinase plays an important role in normal nucleotide biosynthesis under an oxygen-limiting environment.     

Mycobacterium tuberculosis antigen 85A and 85C structures confirm binding orientation and conserved substrate specificity

The maintenance of the highly hydrophobic cell wall is central to the survival of Mycobacterium tuberculosis within its host environment. The antigen 85 proteins (85A, 85B, and 85C) of M. tuberculosis help maintain the integrity of the cell wall 1) by catalyzing the transfer of mycolic acids to the cell wall arabinogalactan and 2) through the synthesis of trehalose dimycolate (cord factor). Additionally, these secreted proteins allow for rapid invasion of alveolar macrophages via direct interactions between the host immune system and the invading bacillus. Here we describe two crystal structures: the structure of antigen 85C co-crystallized with octylthioglucoside as substrate, resolved to 2.0 A, and the crystal structure of antigen 85A, which was solved at a resolution of 2.7 A. The structure of 85C with the substrate analog identifies residues directly involved in substrate binding. Elucidation of the antigen 85A structure, the last of the three antigen 85 homologs to be solved, shows that the active sites of the three antigen 85 proteins are virtually identical, indicating that these share the same substrate. However, in contrast to the high level of conservation within the substrate-binding site and the active site, surface residues disparate from the active site are quite variable, indicating that three antigen 85 enzymes are needed to evade the host immune system.     

A Corynebacterium glutamicum gene encoding a two-domain protein similar to biotin carboxylases and biotin-carboxyl-carrier proteins

Following the analysis of transposon Tn5432-induced mutants of Corynebacterium glutamicum ATCC 13032, a gene encoding a protein with a biotin-binding motif was cloned. The DNA sequence of this gene revealed an open reading frame encoding 591 amino acids with a calculated mol. mass of 63.4 kDa. The protein is composed of two domains, an N-terminal biotin carboxylase and a C-terminal biotin-carboxyl-carrier protein, that are highly similar to corresponding subunits from prokaryotic and eukaryotic biotin enzymes. Over 70% identity was found to a protein from Mycobacterium leprae proposed to be part of an acyl-CoA carboxylase. Since it was not possible to inactivate the C. glutamicum gene, the gene most likely encodes a subunit of the essential acetyl-CoA carboxylase, which catalyzes the committed step in fatty acid synthesis. Received: 2 December 1995 / Accepted: 20 May 1996     

The antigen 85 complex: a major secretion product of Mycobacterium tuberculosis.

The large number of different proteins synthesized by the mycobacterial cell are currently classified and studied in terms of groups of proteins with certain common properties such as physical and chemical characteristics, function, and localization in the mycobacterial cell. Proteins that are actively secreted during culture on synthetic media represent a particular group of great current interest. At least eight proteins secreted by Mycobacterium tuberculosis have been isolated and characterized to various extents. The genes coding for five proteins secreted from M. tuberculosis and/or Mycobacterium bovis BCG have been cloned and sequenced. All of them contain typical signal sequences. The proteins of the antigen 85 complex, which form the main subject of this review, are often the most common proteins in M. tuberculosis culture fluid. The constituents denoted 85A, 85B, and 85C are encoded by three genes located at different sites in the mycobacterial genome and show extensive cross-reactivity as well as homology at amino acid and gene levels. The proteins differ slightly in molecular mass in the 30- to 31-kDa region, and all of them are fibronectin-binding proteins, but the significance of the latter observation and the role of these proteins in mycobacterial physiology and interaction with the infected host remain to be elucidated. The antigen 85 complex proteins are strongly immunogenic in natural and experimental mycobacterial infections in terms of both induction of antibody synthesis and T-cell-mediated reactions. The well-recognized difference in the efficacy of live and dead mycobacterial vaccines should be considered in relation to the group of secreted antigens. After inoculation, live bacteria in vaccines such as BCG multiply in the host, probably releasing several constituents belonging to the class of secreted proteins and hence resulting in more efficient stimulation of the immune system. Secreted mycobacterial antigens are expected to be of particular significance in induction of various immune responses that are responsible for development of protective immunity in some individuals and for clinical symptoms and complications of the ensuing disease in others.     

Mycobacterium tuberculosis gene expression in macrophages

This review provides a discussion on the current information about the response of Mycobacterium tuberculosis to the environment encountered in the macrophage. We focus on the types of genes shown to be upregulated when the pathogen grows in macrophages and discuss the possible roles of these genes in adaptation to the conditions in the eukaryotic cell, in the context of enhancing the survival of the pathogen during infection.