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Deletion of Cg-emb in Corynebacterianeae Leads to a Novel Truncated Cell Wall Arabinogalactan, whereas Inactivation of Cg-ubiA Results in an Arabinan-deficient Mutant with a Cell Wall Galactan Core

2005; Elsevier BV; Volume: 280; Issue: 37 Linguagem: Inglês

10.1074/jbc.m506339200

ISSN

1083-351X

Autores

Luke J. Alderwick, Eva Radmacher, Mathias Seidel, Roland Gande, Paul G. Hitchen, Howard R. Morris, Anne Dell, Hermann Sahm, Lothar Eggeling, Gurdyal S. Besra,

Tópico(s)

Plant-Microbe Interactions and Immunity

Resumo

The cell wall of Mycobacterium tuberculosis has a complex ultrastructure that consists of mycolic acids connected to peptidoglycan via arabinogalactan (AG) and abbreviated as the mAGP complex. The mAGP complex is crucial for the survival and pathogenicity of M. tuberculosis and is the target of several anti-tubercular agents. Apart from sharing a similar mAGP and the availability of the complete genome sequence, Corynebacterium glutamicum has proven useful in the study of orthologous M. tuberculosis genes essential for viability. Here we examined the effects of particular genes involved in AG polymerization by gene deletion in C. glutamicum. The anti-tuberculosis drug ethambutol is thought to target a set of arabinofuranosyltransferases (Emb) that are involved in arabinan polymerization. Deletion of emb in C. glutamicum results in a slow growing mutant with profound morphological changes. Chemical analysis revealed a dramatic reduction of arabinose resulting in a novel truncated AG structure possessing only terminal arabinofuranoside (t-Araf) residues with a corresponding loss of cell wall bound mycolic acids. Treatment of wild-type C. glutamicum with ethambutol and subsequent cell wall analyses resulted in an identical phenotype comparable to the C. glutamicum emb deletion mutant. Additionally, disruption of ubiA in C. glutamicum, the first enzyme involved in the biosynthesis of the sugar donor decaprenol phosphoarabinose (DPA), resulted in a complete loss of cell wall arabinan. Herein, we establish for the first time, (i) that in contrast to M. tuberculosis embA and embB mutants, deletion of C. glutamicum emb leads to a highly truncated AG possessing t-Araf residues, (ii) the exact site of attachment of arabinan chains in AG, and (iii) DPA is the only Araf sugar donor in AG biosynthesis suggesting the presence of a novel enzyme responsible for "priming" the galactan domain for further elaboration by Emb, resulting in the final maturation of the native AG polysaccharide. The cell wall of Mycobacterium tuberculosis has a complex ultrastructure that consists of mycolic acids connected to peptidoglycan via arabinogalactan (AG) and abbreviated as the mAGP complex. The mAGP complex is crucial for the survival and pathogenicity of M. tuberculosis and is the target of several anti-tubercular agents. Apart from sharing a similar mAGP and the availability of the complete genome sequence, Corynebacterium glutamicum has proven useful in the study of orthologous M. tuberculosis genes essential for viability. Here we examined the effects of particular genes involved in AG polymerization by gene deletion in C. glutamicum. The anti-tuberculosis drug ethambutol is thought to target a set of arabinofuranosyltransferases (Emb) that are involved in arabinan polymerization. Deletion of emb in C. glutamicum results in a slow growing mutant with profound morphological changes. Chemical analysis revealed a dramatic reduction of arabinose resulting in a novel truncated AG structure possessing only terminal arabinofuranoside (t-Araf) residues with a corresponding loss of cell wall bound mycolic acids. Treatment of wild-type C. glutamicum with ethambutol and subsequent cell wall analyses resulted in an identical phenotype comparable to the C. glutamicum emb deletion mutant. Additionally, disruption of ubiA in C. glutamicum, the first enzyme involved in the biosynthesis of the sugar donor decaprenol phosphoarabinose (DPA), resulted in a complete loss of cell wall arabinan. Herein, we establish for the first time, (i) that in contrast to M. tuberculosis embA and embB mutants, deletion of C. glutamicum emb leads to a highly truncated AG possessing t-Araf residues, (ii) the exact site of attachment of arabinan chains in AG, and (iii) DPA is the only Araf sugar donor in AG biosynthesis suggesting the presence of a novel enzyme responsible for "priming" the galactan domain for further elaboration by Emb, resulting in the final maturation of the native AG polysaccharide. The Corynebacterianeae represent a distinct and unusual group within Gram-positive bacteria, with the most prominent members being the human pathogens Mycobacterium tuberculosis and Mycobacterium leprae (1Bloom B.R. Murray C.J. Science. 1992; 257: 1055-1064Crossref PubMed Scopus (1245) Google Scholar). In addition, the human pathogen Corynebacterium diphtheriae is the causal agent of diphtheria, and serious economic losses occur from the infection of animals by corynebacterial strains, such as Corynebacterium pseudotuberculosis and Corynebacterium matruchotii (2Coyle M.B. Lipsky B.A. Clin. Microbiol. Rev. 1990; 3: 227-246Crossref PubMed Scopus (284) Google Scholar, 3Funke G. von Graevenitz A. Clarridge 3rd, J.E. Bernard K.A. Clin. Microbiol. Rev. 1997; 10: 125-159Crossref PubMed Google Scholar). Furthermore, non-pathogenic bacteria belong to this taxon, such as Corynebacterium glutamicum, which is used in the industrial production of amino acids (4Sahm H. Eggeling L. de Graaf A.A. Biol. Chem. 2000; 381: 899-910Crossref PubMed Scopus (128) Google Scholar). A common feature to all these bacteria is that they possess an unusual cell wall matrix composed of mycolic acids, arabinogalactan, and peptidoglycan and is often referred to as the mycolyl-arabinogalactan-peptidoglycan (mAGP) 6The abbreviations used are: mAGP, mycolyl arabinogalactan peptidoglycan; AG, arabinogalactan; Ara, arabinose; CMAME, corynomycolic acid methyl ester; DPA, decaprenol phosphoarabinose; EMB, ethambutol; Gal, galactose; GC, gas chromatography; GC/MS, gas chromatography/mass spectrometry; GlcNAc, N-acetyl-galactosamine; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; Rha, rhamnose; OD, optimal density. complex (5McNeil M. Daffe M. Brennan P.J. J. Biol. Chem. 1990; 265: 18200-18206Abstract Full Text PDF PubMed Google Scholar, 6Besra G.S. Khoo K.H. McNeil M.R. Dell A. Morris H.R. Brennan P.J. Biochemistry. 1995; 34: 4257-4266Crossref PubMed Scopus (210) Google Scholar, 7Daffe M. Brennan P.J. McNeil M. J. Biol. Chem. 1990; 265: 6734-6743Abstract Full Text PDF PubMed Google Scholar, 8McNeil M. Daffe M. Brennan P.J. J. Biol. Chem. 1991; 266: 13217-13223Abstract Full Text PDF PubMed Google Scholar, 9Dover L.G. Cerdeno-Tarraga A.M. Pallen M.J. Parkhill J. Besra G.S. FEMS Microbiol. Rev. 2004; 28: 225-250Crossref PubMed Scopus (96) Google Scholar). Arabinogalactan (AG) plays a crucial role in covalently anchoring the outer lipid layer to peptidoglycan. Synthesis of AG begins with the formation of the linker unit through the transfer of GlcNAc-1-P and Rha from their respective sugar nucleotides (UDP-GlcNAc and dTDP-Rha) to form polyprenol-P-P-GlcNAc and polyprenol-P-P-GlcNAc-Rha lipid intermediates (10Mikusova K. Mikus M. Besra G.S. Hancock I. Brennan P.J. J. Biol. Chem. 1996; 271: 7820-7828Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 11Mikusova K. Yagi T. Stern R. McNeil M.R. Besra G.S. Crick D.C. Brennan P.J. J. Biol. Chem. 2000; 275: 33890-33897Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). The intermediates polyprenol-P-P-GlcNAc and polyprenol-P-P-GlcNAc-Rha then serve as acceptors for the sequential addition of galactofuranose (Galf) residues from UDP-Galf (generated from UDP-Galp via Glf (12Weston A. Stern R.J. Lee R.E. Nassau P.M. Monsey D. Martin S.L. Scherman M.S. Besra G.S. Duncan K. McNeil M.R. Tuber Lung Dis. 1997; 78: 123-131Abstract Full Text PDF PubMed Scopus (103) Google Scholar, 13Sanders D.A. Staines A.G. McMahon S.A. McNeil M.R. Whitfield C. Naismith J.H. Nat. Struct. Biol. 2001; 8: 858-863Crossref PubMed Scopus (149) Google Scholar)) to form polyprenol-P-P-GlcNAc-Rha-Gal30 through a novel enzyme designated GlfT (Rv3808c). This latter enzyme expresses two glycosyltransferase activities, a UDP-Galf:β-d-(1→5)-Galf and a UDP-Galf:β-d-(1→6)-Galf, both activities being required for alternating β(1→5) and β(1→6) linkages during galactan polymerization (11Mikusova K. Yagi T. Stern R. McNeil M.R. Besra G.S. Crick D.C. Brennan P.J. J. Biol. Chem. 2000; 275: 33890-33897Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 14Kremer L. Dover L.G. Morehouse C. Hitchin P. Everett M. Morris H.R. Dell A. Brennan P.J. McNeil M.R. Flaherty C. Duncan K. Besra G.S. J. Biol. Chem. 2001; 276: 26430-26440Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Chemical analysis of the mature lipid-linked galactan, synthesized in vitro (11Mikusova K. Yagi T. Stern R. McNeil M.R. Besra G.S. Crick D.C. Brennan P.J. J. Biol. Chem. 2000; 275: 33890-33897Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), suggests that this intermediate then serves as the acceptor for the subsequent addition of arabino-furanose (Araf) residues from the arabinose sugar donor β-d-arabino-furanosyl-1-monophosphoryldecaprenol (DPA) in the formation of the Araf portion (α1→5, α1→3, and β1→2 linkages) of AG (15Xin Y. Lee R.E. Scherman M.S. Khoo K.H. Besra G.S. Brennan P.J. McNeil M. Biochim. Biophys. Acta. 1997; 1335: 231-234Crossref PubMed Scopus (28) Google Scholar, 16Wolucka B.A. McNeil M.R. de Hoffmann E. Chojnacki T. Brennan P.J. J. Biol. Chem. 1994; 269: 23328-23335Abstract Full Text PDF PubMed Google Scholar, 17Lee R.E. Brennan P.J. Besra G.S. Glycobiology. 1997; 7: 1121-1128Crossref PubMed Scopus (93) Google Scholar, 18R. E. Lee K.M. Brennan P.J. Besra G.S. J. Am. Chem. Soc. 1995; 117: 11829-11832Crossref Scopus (149) Google Scholar). The AG-lipid intermediate at some point is mycolylated and transglycosylated to peptidoglycan (19Hancock I.C. Carman S. Besra G.S. Brennan P.J. Waite E. Microbiology. 2002; 148: 3059-3067Crossref PubMed Scopus (28) Google Scholar, 20Yagi T. Mahapatra S. Mikusova K. Crick D.C. Brennan P.J. J. Biol. Chem. 2003; 278: 26497-26504Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Early studies demonstrated that administration of ethambutol (EMB) led to a rapid cessation of mycolic acid transfer to the cell wall and an accumulation of trehalose monomycolate and trehalose dimycolate (21Takayama K. Armstrong E.L. Kunugi K.A. Kilburn J.O. Antimicrob. Agents Chemother. 1979; 16: 240-242Crossref PubMed Scopus (79) Google Scholar). Subsequently, EMB was shown to inhibit specifically AG biosynthesis (22Takayama K. Kilburn J.O. Antimicrob. Agents Chemother. 1989; 33: 1493-1499Crossref PubMed Scopus (284) Google Scholar). The precise molecular target of EMB occupies the emb locus in Mycobacterium avium and M. tuberculosis. The locus consists of embRAB in M. avium (23Belanger A.E. Besra G.S. Ford M.E. Mikusova K. Belisle J.T. Brennan P.J. Inamine J.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11919-11924Crossref PubMed Scopus (401) Google Scholar) and embCAB in M. tuberculosis (24Telenti A. Philipp W.J. Sreevatsan S. Bernasconi C. Stockbauer K.E. Wieles B. Musser J.M. Jacobs Jr., W.R. Nat. Med. 1997; 3: 567-570Crossref PubMed Scopus (382) Google Scholar). To further define the role of EmbCAB proteins in arabinan biosynthesis, embA, embB, and embC genes were inactivated individually in Mycobacterium smegmatis (25Escuyer V.E. Lety M.A. Torrelles J.B. Khoo K.H. Tang J.B. Rithner C.D. Frehel C. McNeil M.R. Brennan P.J. Chatterjee D. J. Biol. Chem. 2001; 276: 48854-48862Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 26Zhang N. Torrelles J.B. McNeil M.R. Escuyer V.E. Khoo K.H. Brennan P.J. Chatterjee D. Mol. Microbiol. 2003; 50: 69-76Crossref PubMed Scopus (113) Google Scholar). Although all three mutants were viable, only the crucial terminal Ara6 motif, which is the template for mycolylation in AG, was altered in both embA and embB mutants with the remaining AG structure intact (25Escuyer V.E. Lety M.A. Torrelles J.B. Khoo K.H. Tang J.B. Rithner C.D. Frehel C. McNeil M.R. Brennan P.J. Chatterjee D. J. Biol. Chem. 2001; 276: 48854-48862Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). This suggested that both EmbA and EmbB are involved in the formation of the terminal Ara6 motif in AG, and EmbC in the formation of arabinan in lipoarabinomannan (26Zhang N. Torrelles J.B. McNeil M.R. Escuyer V.E. Khoo K.H. Brennan P.J. Chatterjee D. Mol. Microbiol. 2003; 50: 69-76Crossref PubMed Scopus (113) Google Scholar). Our preliminary attempts to obtain deletion mutants of embA and embB in M. tuberculosis or embAB in M. smegmatis have proved unsuccessful, 7W. N. Maughan, unpublished results. presumably due to the essentiality of cell wall mAGP (27Pan F. Jackson M. Ma Y. McNeil M. J. Bacteriol. 2001; 183: 3991-3998Crossref PubMed Scopus (212) Google Scholar, 28Mills J.A. Motichka K. Jucker M. Wu H.P. Uhlik B.C. Stern R.J. Scherman M.S. Vissa V.D. Pan F. Kundu M. Ma Y.F. McNeil M. J. Biol. Chem. 2004; 279: 43540-43546Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 29Vilcheze C. Morbidoni H.R. Weisbrod T.R. Iwamoto H. Kuo M. Sacchettini J.C. Jacobs Jr., W.R. J. Bacteriol. 2000; 182: 4059-4067Crossref PubMed Scopus (244) Google Scholar). In the present study we have established through comparative genomic analyses the first biochemical and molecular description of compete ablation of cell wall arabinan biosynthesis in a non-mycobacterial spp., and we highlight the inherent usefulness of examining related spp. to probe complex biosynthetic pathways. Strains and Culture Conditions—C. glutamicum ATCC 13032 (the wild-type strain, and referred for the remainder of the text as C. glutamicum) and Escherichia coli DH5αmcr were grown in Luria-Bertani (LB) broth (Difco) at 30 °C and 37 °C, respectively. The mutants generated in this study were grown on BHIS (5 g of Tryptone, 5 g of NaCl, 2.5 g of yeast extract, 18.5 g of brain heart infusion (Difco), and 90.1 g of sorbitol per liter). Kanamycin and ampicillin were used at a concentration of 50 μg/ml. The minimal medium CGXII was used for C. glutamicum (30Eggeling L. Bott M. Handbook of Corynebacterium glutamicum. Taylor Francis Group, CRC Press, Boca Raton, FL2005: 535-566Crossref Google Scholar). Samples for lipid analyses were prepared by harvesting cells at an optical density (OD) of 10–15, followed by a saline wash and freeze drying. Cultivation of C. glutamicumΔemb for lipid and cell wall analysis required two pre-cultures: Firstly, a 5-ml BHIS culture was grown for 8 h, which was then used to inoculate a 50-ml BHIS culture for 15 h. This was then used to inoculate a 100-ml BHIS culture to OD 1, which was harvested after reaching an OD 3. Construction of Plasmids—The vectors used for deletion and inactivation were as follows: pK19mobsacBΔemb (NCgl0184, embC), pCg::ubiA (NCgl2781, Rv3806c), with the gene numbers of the C. glutamicum and M. tuberculosis orthologs added in parentheses. The plasmid used for overexpression was pEKEx2emb. To enable deletion of gene cross-over PCR was applied to generate the fragments carrying fused sequences adjacent to the gene in question. The resulting fragments were ligated with pK19mobsacB, and the final plasmids were confirmed by sequencing. For emb deletion, the primers used were emb_start_in 5′-CCC ATC CAC TAA ACT TAA ACA CTC AAC TAC ATC TGA CAC GTT GAT C-3′, emb_start_out 5′-GCT TGG TGA GTT CGG AAA CAG GA-3′, emb_end_in 5′-TGT TTA AGT TTA GTG GAT GGG CTC TGG AAT CCA GGG CAT ATG AAG-3′, and emb_end_out 5′-TTC CAT GAG CAG CTG GCG ATA AC-3′. For the second PCR the primer pair emb_start_out and emb_end_out was used again. The resulting fragment was ligated with SmaI-cleaved pK19mobsacB to generate pK19mobsacBΔemb. For inactivation of ubiA an internal fragment of 321 bp was amplified (pubiA-for: ATC TTC AAC CAG CGC ACG ATC; pubiA-rev: AAT ATC GAT CAC TGG CAT GTG C), which was made blunt and ligated into the SmaI site of the non-replicative vector pK18mob to yield pCg::ubiA. Genomic Mutations—To enable chromosomal inactivation of ubiA, pCg::ubiA was introduced into C. glutamicum by electroporation. Selection for resistance to kanamycin yielded clones whose correct disruption of ubiA was confirmed with different primer pairs annealing in the vector and the bacterial chromosome. Southern Blot Analysis—Genomic DNA was extracted from C. glutamicumΔemb and the wild-type strain and cleaved with EcoRV. The resulting fragments were separated on a 1% agarose gel and blotted onto a Nytran NY13N nitrocellulose membrane, with subsequent washings according to standard protocols. Detection was carried out with a fragment generated by PCR with primers pEmbΔ1 (5′-GTG GTT TAG GGG GTC TGT TGG G-3′) and pEmbΔ2 (5′-GGC AGC GTG CCG ATC ATC GCC-3′) as probe that was labeled with digoxigenin (DIG labeling and detection kit, Roche Applied Science). Extraction and Analysis of Cell Wall Bound Mycolic Acids from C. glutamicum Strains—Cells were grown as described above, harvested, washed, and freeze-dried. Cells (100 mg) were extracted by two consecutive extractions with 2 ml of CHCl3/CH3OH/H2O (10:10:3, v/v) for 3 h at 50 °C. The bound lipids from the delipidated extracts or purified cell walls (see below) were released by the addition of 2 ml of 5% aqueous solution of tetrabutylammonium hydroxide, followed by overnight incubation at 100 °C. After cooling, water (2 ml), CH2Cl2 (4 ml), and CH3I (500 μl) were added and mixed thoroughly for 30 min. The lower organic phase was recovered following centrifugation and washed three times with water (4 ml), dried, and resuspended in diethyl ether (4 ml). After centrifugation the clear supernatant was again dried and resuspended in CH2Cl2 (100 μl). An aliquot (10 μl) from each strain was subjected to TLC using silica gel plates (5735 silica gel 60F254, Merck), and developed in petroleum ether/acetone (95:5, v/v) and charred using 5% molybdophosphoric acid in ethanol at 100 °C to reveal corynomycolic acid methyl esters (CMAMES) and compared with known standards (31Gande R. Gibson K.J. Brown A.K. Krumbach K. Dover L.G. Sahm H. Shioyama S. Oikawa T. Besra G.S. Eggeling L. J. Biol. Chem. 2004; 279: 44847-44857Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Isolation of the mAGP Complex—The thawed bacterial cells were resuspended in phosphate-buffered saline containing 2% Triton X-100 (pH 7.2), disrupted by sonication and centrifuged at 27,000 - g (6Besra G.S. Khoo K.H. McNeil M.R. Dell A. Morris H.R. Brennan P.J. Biochemistry. 1995; 34: 4257-4266Crossref PubMed Scopus (210) Google Scholar, 7Daffe M. Brennan P.J. McNeil M. J. Biol. Chem. 1990; 265: 6734-6743Abstract Full Text PDF PubMed Google Scholar). The pelleted material was extracted three times with 2% SDS in phosphate-buffered saline at 95 °C for 1 h to remove associated proteins, successively washed with water, 80% (v/v) acetone in water, and acetone, and finally lyophilized to yield a highly purified cell wall preparation (6Besra G.S. Khoo K.H. McNeil M.R. Dell A. Morris H.R. Brennan P.J. Biochemistry. 1995; 34: 4257-4266Crossref PubMed Scopus (210) Google Scholar, 7Daffe M. Brennan P.J. McNeil M. J. Biol. Chem. 1990; 265: 6734-6743Abstract Full Text PDF PubMed Google Scholar). Glycosyl Composition of Cell Walls by Alditol Acetates—Cell wall preparations were hydrolyzed in 250 μl of 2 m trifluoroacetic acid at 120 °C for 2 h as described (6Besra G.S. Khoo K.H. McNeil M.R. Dell A. Morris H.R. Brennan P.J. Biochemistry. 1995; 34: 4257-4266Crossref PubMed Scopus (210) Google Scholar, 7Daffe M. Brennan P.J. McNeil M. J. Biol. Chem. 1990; 265: 6734-6743Abstract Full Text PDF PubMed Google Scholar). Sugar residues were reduced with 50 μl of NaB2H4 (10 mg/ml in ethanol:1 m NH3 (1:1)), and the resultant alditols were per-O-acetylated and examined by gas chromatography (GC) as described previously (6Besra G.S. Khoo K.H. McNeil M.R. Dell A. Morris H.R. Brennan P.J. Biochemistry. 1995; 34: 4257-4266Crossref PubMed Scopus (210) Google Scholar, 7Daffe M. Brennan P.J. McNeil M. J. Biol. Chem. 1990; 265: 6734-6743Abstract Full Text PDF PubMed Google Scholar). Glycosyl Linkage Analysis of Cell Walls—Cell wall preparations (10 mg) were suspended in 0.5 ml of Me2SO (anhydrous) and 100 μl of 4.8 m dimethyl sulfinyl carbanion (6Besra G.S. Khoo K.H. McNeil M.R. Dell A. Morris H.R. Brennan P.J. Biochemistry. 1995; 34: 4257-4266Crossref PubMed Scopus (210) Google Scholar, 7Daffe M. Brennan P.J. McNeil M. J. Biol. Chem. 1990; 265: 6734-6743Abstract Full Text PDF PubMed Google Scholar). The reaction mixture was stirred for 1 h, and then CH3I was slowly added, and the suspension was stirred for a further 1 h; this process was repeated for a total of three times. The reaction mixture was then diluted with an equal volume of water, and the entire contents were dialyzed against water overnight. The resulting per-O-methylated cell wall samples were applied to a C18 Sep-Pak cartridge and purified as described previously (6Besra G.S. Khoo K.H. McNeil M.R. Dell A. Morris H.R. Brennan P.J. Biochemistry. 1995; 34: 4257-4266Crossref PubMed Scopus (210) Google Scholar, 7Daffe M. Brennan P.J. McNeil M. J. Biol. Chem. 1990; 265: 6734-6743Abstract Full Text PDF PubMed Google Scholar). The per-O-methylated cell walls were hydrolyzed using 250 μl of 2 m trifluoroacetic acid at 120 °C for 2 h. The resulting hydrolysate was reduced with NaB2H4, per-O-acetylated, and examined by gas chromatography/mass spectrometry (GC/MS) as described previously (6Besra G.S. Khoo K.H. McNeil M.R. Dell A. Morris H.R. Brennan P.J. Biochemistry. 1995; 34: 4257-4266Crossref PubMed Scopus (210) Google Scholar, 7Daffe M. Brennan P.J. McNeil M. J. Biol. Chem. 1990; 265: 6734-6743Abstract Full Text PDF PubMed Google Scholar). Mass Spectrometry of Per-O-methylated Cell Walls—Per-O-methylated cell walls were prepared as described above. Methanolic-HCl was prepared by bubbling HCl gas into ∼2 ml of methanol until hot to the touch (∼1 molar). The reagent (100 μl) was added to the per-O-methylated cell wall sample, and aliquots were analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) to monitor hydrolysis. The reaction was terminated by drying under nitrogen. MALDI-MS was performed using a PerSeptive Biosystems Voyager DE™ STR mass spectrometer (Applied Biosystems, CA) in the reflectron mode with delayed extraction. Samples were dissolved in methanol, and 1-μl aliquots were loaded onto a metal plate with 1 μl of the matrix 2,5-dihydrobenzoic acid. Sequazyme peptide mass standards were used as external calibrants (Applied Biosystems, CA). GC and GC/MS of Sugar Composition and Sugar Linkage Analysis— Analysis of alditol acetate sugar derivatives was performed on a CE Instruments ThermoQuest Trace GC 2000. Samples were injected in the splitless mode. The column used was a DB225 (Supelco). The oven was programmed to hold at an isothermal temperature of 275 °C for a run time of 15 min. GC/MS was carried out on a Finnigan Polaris/GCQ Plus™. The column used was a BPX5 (Supelco). Genome Comparison of the emb Locus—M. tuberculosis, M. bovis, M. leprae, and M. avium subsp. paratuberculosis have three emb genes (Fig. 1A), and at least one of these, embB, is suggested to be the target of EMB in mycobacteria (24Telenti A. Philipp W.J. Sreevatsan S. Bernasconi C. Stockbauer K.E. Wieles B. Musser J.M. Jacobs Jr., W.R. Nat. Med. 1997; 3: 567-570Crossref PubMed Scopus (382) Google Scholar, 32Ramaswamy S.V. Amin A.G. Goksel S. Stager C.E. Dou S.J. El Sahly H. Moghazeh S.L. Kreiswirth B.N. Musser J.M. Antimicrob. Agents Chemother. 2000; 44: 326-336Crossref PubMed Scopus (208) Google Scholar, 33Sreevatsan S. Stockbauer K.E. Pan X. Kreiswirth B.N. Moghazeh S.L. Jacobs Jr., W.R. Telenti A. Musser J.M. Antimicrob. Agents Chemother. 1997; 41: 1677-1681Crossref PubMed Google Scholar, 34Ramaswamy S.V. Dou S.J. Rendon A. Yang Z. Cave M.D. Graviss E.A. J. Med. Microbiol. 2004; 53: 107-113Crossref PubMed Scopus (89) Google Scholar). However, C. diphtheriae and C. glutamicum have only one emb gene (35Cerdeno-Tarraga A.M. Efstratiou A. Dover L.G. Holden M.T. Pallen M. Bentley S.D. Besra G.S. Churcher C. James K.D. De Zoysa A. Chillingworth T. Cronin A. Dowd L. Feltwell T. Hamlin N. Holroyd S. Jagels K. Moule S. Quail M.A. Rabbinowitsch E. Rutherford K.M. Thomson N.R. Unwin L. Whitehead S. Barrell B.G. Parkhill J. Nucleic Acids Res. 2003; 31: 6516-6523Crossref PubMed Scopus (251) Google Scholar, 36Kalinowski J. Bathe B. Bartels D. Bischoff N. Bott M. Burkovski A. Dusch N. Eggeling L. Eikmanns B.J. Gaigalat L. Goesmann A. Hartmann M. Huthmacher K. Kramer R. Linke B. McHardy A.C. Meyer F. Mockel B. Pfefferle W. Puhler A. Rey D.A. Ruckert C. Rupp O. Sahm H. Wendisch V.F. Wiegrabe I. Tauch A. J. Biotechnol. 2003; 104: 5-25Crossref PubMed Scopus (769) Google Scholar). This is in accordance with the notion that the genome of Corynebacterium is considered to represent the archetype of Corynebacterianeae and has a low frequency of structural alterations and gene duplications (37Nakamura Y. Nishio Y. Ikeo K. Gojobori T. Gene (Amst.). 2003; 317: 149-155Crossref PubMed Scopus (55) Google Scholar). Interestingly, the single emb of C. glutamicum, Cg-emb, exhibits a higher identity to embC than to embA and embB of Mycobacterium, and increased expression of Cg-emb increases resistance of C. glutamicum toward EMB (38Radmacher E. Stansen K.C. Besra G.S. Alderwick L.J. Maughan W.N. Hollweg G. Sahm H. Wendisch V.F. Eggeling L. Microbiology. 2005; 151: 1359-1368Crossref PubMed Scopus (96) Google Scholar). In M. leprae and M. avium spp. paratuberculosis the paralogous embAB genes are separated by divergently transcribed genes that might indicate a more specific function and a separate regulation in these mycobacteria. The above genomic comparison and the availability of the complete genome sequence of C. glutamicum has proven useful in the study of orthologous M. tuberculosis genes that are essential for viability. Therefore, in this study we examined the effects, in terms of arabinan biosynthesis and utilization of the sugar donor DPA, of firstly, Cg-emb by gene deletion in C. glutamicum, and secondly, disruption of Cg-ubiA (Fig. 1B), an enzyme recently shown to be involved in the biosynthesis of the sugar donor DPA. Construction of C. glutamicumΔemb—In our recent studies on emb of C. glutamicum we placed the chromosomally encoded gene under the control of a tetracycline repressor (38Radmacher E. Stansen K.C. Besra G.S. Alderwick L.J. Maughan W.N. Hollweg G. Sahm H. Wendisch V.F. Eggeling L. Microbiology. 2005; 151: 1359-1368Crossref PubMed Scopus (96) Google Scholar) and observed a number of physiological consequences, including reduced growth in presence of repressor (38Radmacher E. Stansen K.C. Besra G.S. Alderwick L.J. Maughan W.N. Hollweg G. Sahm H. Wendisch V.F. Eggeling L. Microbiology. 2005; 151: 1359-1368Crossref PubMed Scopus (96) Google Scholar). These studies encouraged us to test whether it would be possible to obtain a deletion of emb in C. glutamicum. The non-replicative plasmid pk19mobsacBΔemb was constructed carrying sequences adjacent to emb. The vector was introduced into C. glutamicum and in several electroporation assays 3 kanamycin resistance clones were obtained, indicating integration of pk19mobsacBΔemb into the genome by homologous recombination (Fig. 2, A and B). The sacB gene enables for positive selection of a second homologous recombination event that can result either in the original wild-type situation or in clones deleted of emb. More than 200 clones were obtained after 2–4 days and analyzed by PCR, but in all of them the wild-type situation was restored, illustrating a strong disadvantage of emb deletion. Only 3 clones appearing after 10 days were shown by PCR to have emb deleted. A further confirmation of emb deletion in one of the clones chosen was obtained in a Southern blot analysis (Fig. 2C). The chromosomal EcoRV fragment of the wild-type is 7.82 kb in size, whereas that of the emb deletion mutant is reduced to 4.35 kb, which mirrors the absence of emb of 3,438 bp. This confirms the integrity of the gene locus in the emb deletion mutant. In Vitro Growth Analysis of C. glutamicumΔemb—The deletion mutant was transformed with pEKEx2emb (38Radmacher E. Stansen K.C. Besra G.S. Alderwick L.J. Maughan W.N. Hollweg G. Sahm H. Wendisch V.F. Eggeling L. Microbiology. 2005; 151: 1359-1368Crossref PubMed Scopus (96) Google Scholar), and growth was studied on brain-heart-infusion media supplemented with sorbitol for osmotic stabilization (30Eggeling L. Bott M. Handbook of Corynebacterium glutamicum. Taylor Francis Group, CRC Press, Boca Raton, FL2005: 535-566Crossref Google Scholar). Whereas growth of C. glutamicum was completed after 8 h at an OD of 16, C. glutamicumΔemb hardly reached an OD of 2 (Fig. 2D). However, complementation of the deletion mutant with pEKEx2emb restored the wild-type growth phenotype. mRNA transcript quantifications using LightCycler technology confirmed a 5-fold overexpression of emb due to pEKEx2emb when comparing expression of emb with its chromosomal copy (data not shown). M. tuberculosis embC, embA, and embB were cloned into pEKE2, however, although expression and sequence integrity of each clone was confirmed, no complementation of C. glutamicumΔemb was achieved (data not shown). Analysis by light microscopy and electron micrographs showed that when compared with C. glutamicum, C. glutamicumΔemb exhibited profound morphological changes similar to that of C. glutamicum treated with 100 μg/ml EMB (38Radmacher E. Stansen K.C. Besra G.S. Alderwick L.J. Maughan W.N. Hollweg G. Sahm H. Wendisch V.F. Eggeling L. Microbiology. 2005; 151: 1359-1368Crossref PubMed Scopus (96) Google Scholar) (data not shown). Lipid Characterization of Mutants—To relate the phenotypic changes of the C. glutamicumΔemb mutant to its cellular composition, the C. glutamicumΔemb and its Cg-emb complemented strain along with C. glutamicum were analyzed for arabinogalactan-esterified corynomycolic acids. Bound lipids were ana

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