Characterization of a temperature-sensitive mutant of Salmonella typhimurium defective in apolipoprotein N-acyltransferase.
On screening 440 temperature-sensitive (ts) mutants of Salmonella typhimurium, a mutant strain SE5312 which accumulated apolipoprotein (ALP) at 42 degrees C was identified. In vitro assay of apolipoprotein N-acyltransferase activity indicated that the mutant cell envelope contained reduced activity as compared to the wild-type strain. Transduction with a Mud-P22 mapping set placed the ts mutation to 14-17 min region of the S. typhimurium chromosome. P22 transduction using transposon insertions in this region revealed a linkage of the ts mutation to cobD (6%), nag (8%), and corC68 (99%). The ts phenotype was complemented by a 2.3-kilobase EcoRI subclone derived from lambda-phage 170 of Kohara's bank of Escherichia coli. Restriction enzyme analysis of the cloned DNA revealed that this 2.3-kilobase EcoRI fragment included the copper transport (cutE) gene in E. coli. The mutant strain SE5312 was copper-sensitive at 30 degrees C, and the complementing clone conferred copper resistance and restored the ALP N-acyltransferase activity in the mutant cell. Wild-type strain of S. typhimurium harboring this clone exhibited elevated levels of ALP N-acyltransferase activity. These results suggest that the cloned gene encodes the ALP N-acyltransferase. Upon shift to the non-permissive temperature, the viability of the mutant cells decreased, and the mutant cells assumed anomalous morphology. Temperature-resistant revertants could be readily isolated, and a subset of tr revertants contained no detectable lipoprotein. A lpp::Tn10 derivative of the mutant SE5312 was also temperature-resistant. These observations suggest that ALP N-acyltransferase is essential for the growth and viability of S. typhimurium, and this requirement is decreased in the absence of major outer membrane lipoprotein.
Gupta
S D
SD
Department of Microbiology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799.
Gan
K
K
Schmid
M B
MB
Wu
H C
HC
D000215:Acylation; D000217:Acyltransferases / Q000235:genetics* / Q000378:metabolism; D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D001053:Apolipoproteins / Q000378:metabolism; D003001:Cloning, Molecular; D003300:Copper / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005798:Genes, Bacterial; D007700:Kinetics; D008074:Lipoproteins / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D015183:Restriction Mapping; D012486:Salmonella typhimurium / Q000201:enzymology / Q000235:genetics* / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D001053:Apolipoproteins; D008074:Lipoproteins; D003300:Copper; D000217:Acyltransferases; C068902:apolipoprotein N-acyltransferase