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.
Chugai Biopharmaceuticals, Inc., San Diego, California 92121, USA.
Jonathan A
JA
Mills
Kelly
K
Motichka
Markus
M
Jucker
Henry P
HP
Wu
Brian C
BC
Uhlik
Richard J
RJ
Stern
Michael S
MS
Scherman
Varalakshmi D
VD
Vissa
Fei
F
Pan
Manikuntala
M
Kundu
Yu Fang
YF
Ma
Michael
M
McNeil
D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D002250:Carbon Radioisotopes; D002455:Cell Division; D002470:Cell Survival; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D005685:Galactans / Q000096:biosynthesis*; D006602:Hexosyltransferases / Q000378:metabolism*; D007700:Kinetics; D016296:Mutagenesis; D020102:Mycobacterium smegmatis / Q000201:enzymology* / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D010457:Peptidoglycan / Q000096:biosynthesis*; D016133:Polymerase Chain Reaction
D001426:Bacterial Proteins; D002250:Carbon Radioisotopes; D017931:DNA Primers; D005685:Galactans; D010457:Peptidoglycan; C053856:arabinogalactan-peptidoglycan complex; D006602:Hexosyltransferases; C088697:rhamnosyltransferase 1