Inactivation of the Mycobacterial Rhamnosyltransferase, Which Is Needed for the Formation of the Arabinogalactan-Peptidoglycan Linker, Leads to Irreversible Loss of Viability

• Published in: The Journal of biological chemistry Vol. 279; no. 42; pp. 43540 - 43546
• Main Authors: Mills, Jonathan A, Motichka, Kelly, Jucker, Markus, Wu, Henry P, Uhlik, Brian C, Stern, Richard J, Scherman, Michael S, Vissa, Varalakshmi D, Pan, Fei, Kundu, Manikuntala, Ma, Yu Fang, McNeil, Michael
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 15.10.2004
• Subjects: Bacterial Proteins - metabolism; Base Sequence; Carbon Radioisotopes; Cell Division; Cell Survival; Cloning, Molecular; DNA Primers; Escherichia coli - genetics; Galactans - biosynthesis; Hexosyltransferases - metabolism; Kinetics; Mutagenesis; Mycobacterium smegmatis; Mycobacterium smegmatis - enzymology; Mycobacterium smegmatis - genetics; Mycobacterium tuberculosis; Mycobacterium tuberculosis - enzymology; Mycobacterium tuberculosis - genetics; Mycobacterium tuberculosis - growth & development; Peptidoglycan - biosynthesis; Polymerase Chain Reaction
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M407782200

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:α- d -GlcNAc-pyrophosphate polyprenol, α-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.