Abstract
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.
Highlights
On screening 440 temperature-sensitive ( t s )mutants ized to theinner membranes of E. coli (3)
Restriction screened a collection of 440 temperature-sensitive' mutants of Salmonella typhimurium obtained by chemical mutagenesis using diethyl sulfate (6) for the accumulation of ALP at 42 "C.Thispaper describes the identification and characterization of such a mutant, strain SE5312
We report the genetic and biochemical evidence which suggest that the ts mutation in the apolipoprotein N-acyltransferase (Int)gene of S. typhimurium is allelic to thecutE gene of E. coli
Summary
Characterizationof a Temperature-sensitive Mutant of Salmonella typhimurium Defective inApolipoprotein N-Acyltransferase". In vitro and in vivo of Salmonella typhimurium, a mutant strain SE5312 substrate specificity experiments revealed that any of the which accumulated apolipoprotein (ALP) at 42 "C was phospholipids could serve asa donor of fatty acid in N - identified. Transduction with a Mud-P22 mapping setplaced the ts mutation to[14,15,16,17] min region of the S. typhimurium chromosome. Restriction screened a collection of 440 temperature-sensitive (ts)' mutants of Salmonella typhimurium obtained by chemical mutagenesis using diethyl sulfate (6) for the accumulation of ALP at 42 "C.Thispaper describes the identification and characterization of such a mutant, strain SE5312. R. Benson (University of Utah, Salt t1tahepnmapdt:p:ATveniLraalPObtuiNlrdiet-eya-rrociefyvsSlaitst.ritatvaynenpsthfo.efimrTtahusheereismueismut,oaeabnsnsstdeeSrnvEtthia5ati3ilso1nfr2oesqrwusatiuhrsgeegmalegsesonrtot wLogmtlnahueBkLnceaoBtscsC-.etaeiGgtaryiarna,erdUewn5pTe%lpr)ae.ltaegLtsreB.oswM((nv89/)vawm)t eiw3nr0eia,msu3asu7els,demadtneodfdooi4urb2mtraa"idCnsiuowPpap2ictl2thei-vmfaereeerlneaatbcteieodollniownngiiintehesLx.0pBT.e2ero%tir-is decreased in the absence of major outer membrane racycline-sensitive transductants in Mud-P22 mapping experiments lipoprotein.
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