Identification of apolipoprotein N-acyltransferase (Lnt) in mycobacteria.
Lipoproteins of Gram-negative and Gram-positive bacteria carry a thioether-bound diacylglycerol but differ by a fatty acid amide bound to the alpha-amino group of the universally conserved cysteine. In Escherichia coli the N-terminal acylation is catalyzed by the N-acyltransferase Lnt. Using E. coli Lnt as a query in a BLASTp search, we identified putative lnt genes also in Gram-positive mycobacteria. The Mycobacterium tuberculosis lipoprotein LppX, heterologously expressed in Mycobacterium smegmatis, was N-acylated at the N-terminal cysteine, whereas LppX expressed in a M. smegmatis lnt::aph knock-out mutant was accessible for N-terminal sequencing. Western blot analyses of a truncated and tagged form of LppX indicated a smaller size of about 0.3 kDa in the lnt::aph mutant compared with the parental strain. Matrix-assisted laser desorption ionization time-of-flight/time-of-flight analyses of a trypsin digest of LppX proved the presence of the diacylglycerol modification in both strains, the parental strain and lnt::aph mutant. N-Acylation was found exclusively in the M. smegmatis parental strain. Complementation of the lnt::aph mutant with M. tuberculosis ppm1 restored N-acylation. The substrate for N-acylation is a C16 fatty acid, whereas the two fatty acids of the diacylglycerol residue were identified as C16 and C19:0 fatty acid, the latter most likely tuberculostearic acid. We demonstrate that mycobacterial lipoproteins are triacylated. For the first time to our knowledge, we identify Lnt activity in Gram-positive bacteria and assigned the responsible genes. In M. smegmatis and M. tuberculosis the open reading frames are annotated as MSMEG_3860 and M. tuberculosis ppm1, respectively.
Tschumi
Andreas
A
Institut für Medizinische Mikrobiologie, Universität Zürich, Gloriastrasse 30/32, CH-8006 Zurich, Switzerland.
Nai
Corrado
C
Auchli
Yolanda
Y
Hunziker
Peter
P
Gehrig
Peter
P
Keller
Peter
P
Grau
Thomas
T
Sander
Peter
P
D039903:Actinobacteria / Q000201:enzymology; D000215:Acylation; D000217:Acyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005227:Fatty Acids / Q000378:metabolism; D016680:Genome, Bacterial; D008074:Lipoproteins / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009161:Mycobacterium / Q000201:enzymology* / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D005227:Fatty Acids; D008074:Lipoproteins; D000217:Acyltransferases; C068902:apolipoprotein N-acyltransferase