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Summary Mycolic acids are vital components of the cell wall of the tubercle bacillus Mycobacterium tuberculosis and are required for viability and virulence. While mycolic acid biosynthesis is studied extensively, components involved in mycolate transport remain unidentified. We investigated the role of large membrane proteins encoded by mmpL genes in mycolic acid transport in mycobacteria and the related corynebacteria. MmpL3 was found to be essential in mycobacteria and conditional depletion of MmpL3 in Mycobacterium smegmatis resulted in loss of cell wall mycolylation, and of the cell wall-associated glycolipid, trehalose dimycolate. In parallel, an accumulation of trehalose monomycolate (TMM) was observed, suggesting that mycolic acids were transported as TMM. In contrast to mycobacteria, we found redundancy in the role of two mmpL genes, in Corynebacterium glutamicum; a complete loss of trehalose-associated and cell wall bound corynomycolates was observed in an NCgl0228-NCgl2769 double mutant, but not in individual single mutants. Our studies highlight the role of mmpL genes in mycolic acid metabolism and identify potential new targets for anti-TB drug development., Highlights ► MmpL3 is an essential gene in mycobacteria ► MmpL3 is involved in mycolic acid transport in mycobacteria ► Two mmpL genes are involved in corynomycolate metabolism in corynebacteria

Lipomannan (LM) and lipoarabinomannan (LAM) are key Corynebacterineae glycoconjugates that are integral components of the mycobacterial cell wall, and are potent immunomodulators during infection. LAM is a complex heteropolysaccharide synthesized by an array of essential glycosyltransferase family C (GT-C) members, which represent potential drug targets. Herein, we have identified and characterized two open reading frames from Corynebacterium glutamicum that encode for putative GT-Cs. Deletion of NCgl2100 and NCgl2097 in C. glutamicum demonstrated their role in the biosynthesis of the branching α(1→2)-Manp residues found in LM and LAM. In addition, utilizing a chemically defined nonasaccharide acceptor, azidoethyl 6-O-benzyl-α-D-mannopyranosyl-(1→6)-[α-D-mannopyranosyl-(1→6)](7) -D-mannopyranoside, and the glycosyl donor C(50) -polyprenol-phosphate-[(14) C]-mannose with membranes prepared from different C. glutamicum mutant strains, we have shown that both NCgl2100 and NCgl2097 encode for novel α(1→2)-mannopyranosyltransferases, which we have termed MptC and MptD respectively. Complementation studies and in vitro assays also identified Rv2181 as a homologue of Cg-MptC in Mycobacterium tuberculosis. Finally, we investigated the ability of LM and LAM from C. glutamicum, and C. glutamicumΔmptC and C. glutamicumΔmptD mutants, to activate Toll-like receptor 2. Overall, our study enhances our understanding of complex lipoglycan biosynthesis in Corynebacterineae and sheds further light on the structural and functional relationship of these classes of polysaccharides.

A bioinformatics approach identified a putative integral membrane protein, NCgl0543, in Corynebacterium glutamicum , with 13 predicted transmembrane domains and a glycosyltransferase motif (RXXDE), features that are common to the glycosyltransferase C superfamily of glycosyltransferases. The deletion of C. glutamicum NCgl0543 resulted in a viable mutant. Further glycosyl linkage analyses of the mycolyl-arabinogalactan-peptidoglycan complex revealed a reduction of terminal rhamnopyranosyl-linked residues and, as a result, a corresponding loss of branched 2,5-linked arabinofuranosyl residues, which was fully restored upon the complementation of the deletion mutant by NCgl0543. As a result, we have now termed this previously uncharacterized open reading frame, r hamno p yranosyl t ransferase A ( rptA ). Furthermore, an analysis of base-stable extractable lipids from C. glutamicum revealed the presence of decaprenyl-monophosphorylrhamnose, a putative substrate for the cognate cell wall transferase.

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.

Addition of l -valine (50 to 200 mM) to glucose minimal medium had no effect on the growth of wild-type Corynebacterium glutamicum ATCC 13032 but inhibited the growth of the derived valine production strain VAL1 [13032 Δ ilvA Δ panBC (pJC1 ilvBNCD )] in a concentration-dependent manner. In order to explore this strain-specific valine effect, genomewide expression profiling was performed using DNA microarrays, which showed that valine caused an increased ilvBN mRNA level in VAL1 but not in the wild type. This unexpected result was confirmed by an increased cellular level of the ilvB protein product, i.e., the large subunit of acetohydroxyacid synthase (AHAS), and by an increased AHAS activity of valine-treated VAL1 cells. The conclusion that valine caused the limitation of another branched-chain amino acid was confirmed by showing that high concentrations of l -isoleucine could relieve the valine effect on VAL1 whereas l -leucine had the same effect as valine. The valine-caused isoleucine limitation was supported by the finding that the inhibitory valine effect was linked to the ilvA deletion that results in isoleucine auxotrophy. Taken together, these results implied that the valine effect is caused by competition for uptake of isoleucine by the carrier BrnQ, which transports all branched-chained amino acids. Indeed, valine inhibition could also be relieved by supplementing VAL1 with the dipeptide isoleucyl-isoleucine, which is taken up by a dipeptide transport system rather than by BrnQ. Interestingly, addition of external valine stimulated valine production by VAL1. This effect is most probably due to a reduced carbon usage for biomass production and to the increased expression of ilvBN , indicating that AHAS activity may still be a limiting factor for valine production in the VAL1 strain.

Mannose is an important constituent of the immunomodulatory glycoconjugates of the mycobacterial cell wall: lipoarabinomannan (LAM), lipomannan (LM) and the related phospho-myo-inositol mannosides (PIMs). In Mycobacterium tuberculosis and the related bacillus Corynebacterium glutamicum, mannose is either imported from the medium or derived from glycolysis, and is subsequently converted into the nucleotide-based sugar donor guanosine diphosphomannose (GDP-mannose). This can be utilized by the glycosyltranferases of the GT-A/B superfamily or converted to the lipid-based donor polyprenyl monophosphomannose, and used as a substrate by the transmembrane glycosyltransferases of the GT-C superfamily. To investigate GDP-mannose biosynthesis in detail, the gene encoding a putative ManC in C. glutamicum was deleted. Deletion of manC resulted in a slow-growing mutant, with reduced but not totally abrogated guanosine diphosphomannose pyrophosphorylase activity. However, a comprehensive cell wall analysis revealed that C. glutamicumΔmanC is deficient in PIMs and LM/LAM. Closer inspection suggests that promiscuous ManC activity is contributed by additional putative nucleotidyltransferases, PmmB, WbbL1, GalU and GlmU, and a hypothetical protein, NCgl0715. Furthermore, complementation analyses of C. glutamicumΔmanC with Rv3264c suggested that it is a true homologue of ManC in M. tuberculosis, and the essentiality of PIMs in M. tuberculosis makes it an attractive drug target.

Lipomannan (LM) and lipoarabinomannan (LAM) are key Corynebacterineae glycoconjugates that are integral components of the mycobacterial cell wall, and are potent immunomodulators during infection. LAM is a complex heteropolysaccharide synthesized by an array of essential glycosyltransferase family C (GT-C) members, which represent potential drug targets. Herein, we have identified and characterized two open reading frames from Corynebacterium glutamicum that encode for putative GT-Cs. Deletion of NCgl2100 and NCgl2097 in C. glutamicum demonstrated their role in the biosynthesis of the branching α(1→2)-Manp residues found in LM and LAM. In addition, utilizing a chemically defined nonasaccharide acceptor, azidoethyl 6-O-benzyl-α-D-mannopyranosyl-(1→6)-[α-D-mannopyranosyl-(1→6)](7) -D-mannopyranoside, and the glycosyl donor C(50) -polyprenol-phosphate-[(14) C]-mannose with membranes prepared from different C. glutamicum mutant strains, we have shown that both NCgl2100 and NCgl2097 encode for novel α(1→2)-mannopyranosyltransferases, which we have termed MptC and MptD respectively. Complementation studies and in vitro assays also identified Rv2181 as a homologue of Cg-MptC in Mycobacterium tuberculosis. Finally, we investigated the ability of LM and LAM from C. glutamicum, and C. glutamicumΔmptC and C. glutamicumΔmptD mutants, to activate Toll-like receptor 2. Overall, our study enhances our understanding of complex lipoglycan biosynthesis in Corynebacterineae and sheds further light on the structural and functional relationship of these classes of polysaccharides.

The amino acid-producing organism Corynebacterium glutamicum cannot utilize glycerol, a stoichiometric by-product of biodiesel production. By heterologous expression of Escherichia coli glycerol utilization genes, C. glutamicum was engineered to grow on glycerol. While expression of the E. coli genes for glycerol kinase ( glpK ) and glycerol 3-phosphate dehydrogenase ( glpD ) was sufficient for growth on glycerol as the sole carbon and energy source, additional expression of the aquaglyceroporin gene glpF from E. coli increased growth rate and biomass formation. Glutamate production from glycerol was enabled by plasmid-borne expression of E. coli glpF, glpK , and glpD in C. glutamicum wild type. In addition, a lysine-producing C. glutamicum strain expressing E. coli glpF, glpK , and glpD was able to produce lysine from glycerol as the sole carbon substrate as well as from glycerol-glucose mixtures.

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 alpha(1--3)-linked arabinofuranosyl (Araf) 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, arabinofuranosyltransferase C (aftC). Enzyme assays using the sugar donor beta-d-arabinofuranosyl-1-monophosphoryl-decaprenol (DPA) and a newly synthesized linear alpha(1--5)-linked Ara(5) neoglycolipid acceptor together with chemical identification of products formed, clearly identified AftC as a branching alpha(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.

Phosphorus is an essential component of macromolecules, like DNA, and central metabolic intermediates, such as sugar phosphates, and bacteria possess enzymes and control mechanisms that provide an optimal supply of phosphorus from the environment. UDP-sugar hydrolases and 5′ nucleotidases may play roles in signal transduction, as they do in mammals, in nucleotide salvage, as demonstrated for UshA of Escherichia coli , or in phosphorus metabolism. The Corynebacterium glutamicum gene ushA was found to encode a secreted enzyme which is active as a 5′ nucleotidase and a UDP-sugar hydrolase. This enzyme was synthesized and secreted into the medium when C. glutamicum was starved for inorganic phosphate. UshA was required for growth of C. glutamicum on AMP and UDP-glucose as sole sources of phosphorus. Thus, in contrast to UshA from E. coli , C. glutamicum UshA is an important component of the phosphate starvation response of this species and is necessary to access nucleotides and related compounds as sources of phosphorus.

In its natural environment, Escherichia coli is exposed to short-chain fatty acids, such as acetic acid or propionic acid, which can be utilized as carbon sources but which inhibit growth at higher concentrations. DNA microarray experiments revealed expression changes during exponential growth on complex medium due to the presence of sodium acetate or sodium propionate at a neutral external pH. The adaptive responses to acetate and propionate were similar and involved genes in three categories. First, the RNA levels for chemotaxis and flagellum genes increased. Accordingly, the expression of chromosomal fliC ′-′ lacZ and flhDC ′-′ lacZ fusions and swimming motility increased after adaptation to acetate or propionate. Second, the expression of many genes that are involved in the uptake and utilization of carbon sources decreased, indicating some kind of catabolite repression by acetate and propionate. Third, the expression of some genes of the general stress response increased, but the increases were more pronounced after short-term exposure for this response than for the adaptive response. Adaptation to propionate but not to acetate involved increased expression of threonine and isoleucine biosynthetic genes. The gene expression changes after adaptation to acetate or propionate were not caused solely by uncoupling or osmotic effects but represented specific characteristics of the long-term response of E. coli to either compound.

Summary: The Corynebacterium glutamicum ack and pta genes encoding the acetate-activating enzymes acetate kinase and phosphotransacetylase were isolated, subcloned on a plasmid and re-introduced into Corynebacterium glutamicum. Relative to the wild-type, the recombinant strains showed about tenfold higher specific activities of both enzymes. Sequence analysis of a 3657 bp DNA fragment revealed that the ack and pta genes are contiguous in the corynebacterial chromosome, with pta upstream and the last nucleotide of the pta stop codon (TAA) overlapping the first of the ack start codon (ATG). The predicted gene product of pta consists of 329 amino acids (M r 35242), that of ack consists of 397 amino acids (M r 43098) and the amino acid sequences of the two polypeptides show up to 60% (phosphotransacetylase) and 53% (acetate kinase) identity in comparison with respective enzymes from other organisms. Northern (RNA) blot hybridizations using pta- and ack-specific probes and transcriptional cat fusion experiments revealed that the two genes are transcribed as a 2·5 kb bicistronic mRNA and that the expression of this operon is induced when Corynebacterium glutamicum grows on acetate instead of glucose as a carbon source. Directed inactivation of the chromosomal pta and ack genes led to the absence of detectable phosphotransacetylase and acetate kinase activity in the respective mutants and to their inability to grow on acetate. These data indicate that no isoenzymes of acetate kinase and phosphotransacetylase are present in Corynebacterium glutamicum and that a functional acetate kinase/phosphotransacetylase pathway is essential for growth of this organism on acetate.

NADP(+)-dependent isocitrate dehydrogenase (ICD) is an important enzyme of the intermediary metabolism, as it controls the carbon flux within the citric acid cycle and supplies the cell with 2-oxoglutarate and NADPH for biosynthetic purposes. In the amino acid-producing organism Corynebacterium glutamicum, the specific activity of ICD was independent of the growth substrate and of the growth phase at approximately 1 U/mg, indicating that this enzyme is constitutively formed. The ICD gene, icd, was isolated, subcloned on a plasmid, and introduced into C. glutamicum. Compared with the wild type, the recombinant strains showed up to 10-fold-higher specific ICD activities. The nucleotide sequence of a 3,595-bp DNA fragment containing the icd gene was determined. The predicted gene product of icd consists of 739 amino acids (M(r) = 80.091) and showed 58.5% identity with the monomeric ICD isozyme II from Vibrio sp. strain ABE-1 but no similarity to any known ICD of the dimeric type. Inactivation of the chromosomal icd gene led to glutamate auxotrophy and to the absence of any detectable ICD activity, suggesting that only a single ICD is present in C. glutamicum. From an icd-overexpressing C. glutamicum strain, ICD was purified and biochemically characterized. The native ICD was found to be a monomer; to be specific for NADP+; to be weakly inhibited by oxaloacetate, 2-oxoglutarate, and citrate; and to be severely inhibited by oxaloacetate plus glyoxylate. The data indicate that ICD from C. glutamicum is structurally similar to ICDs from bacteria of the genera Vibrio, Rhodomicrobium, and Azotobacter but different from all other known procaryotic and eucaryotic ICDs.