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Phosphatidylglycerol:prolipoprotein diacylglyceryl transferase (Lgt) is the first enzyme in the posttranslational sequence of reactions resulting in the lipid modification of lipoproteins in bacteria. A previous comparison of the primary sequences of the Lgt enzymes from phylogenetically distant bacterial species revealed several highly conserved amino acid sequences throughout the molecule; the most extensive of these was the region 103HGGLIG108 in the Escherichia coli Lgt (H.-Y. Qi, K. Sankaran, K. Gan, and H. C. Wu, J. Bacteriol. 177:6820-6824, 1995). These studies also revealed that the kinetics of inactivation of E. coli Lgt with diethylpyrocarbonate were consistent with the modification of a single essential histidine or tyrosine residue. The current study was conducted in an attempt to identify this essential amino acid residue in order to further define structure-function relationships in Lgt. Accordingly, all of the histidine residues and seven of the tyrosine residues of E. coli Lgt were altered by site-directed mutagenesis, and the in vitro activities of the altered enzymes, as well the abilities of the respective mutant lgt alleles to complement the temperature-sensitive phenotype of E. coli SK634 defective in Lgt activity, were determined. The data obtained from these studies, in conjunction with additional chemical inactivation studies, support the conclusion that His-103 is essential for Lgt activity. These studies also indicated that Tyr-235 plays an important role in the function of this enzyme. Although other histidine and tyrosine residues were not found to be essential for Lgt activity, alterations of His-196 resulted in a significant reduction of in vitro activity.

Using a combination of biochemical, physical, and genetic techniques, we have shown that the umpA gene of Escherichia coli is allelic with the lgt (phosphatidylglycerol:prolipoprotein diacylglyceryl transferase) of Salmonella typhimurium. These genes are essential for the viability of the respective organism and exhibit 92.8% sequence identity at the amino acid level. In E. coli, lgt and thyA (thymidylate synthase) form an operon. Thymidylate synthase levels are regulated by transcription from the lgt promoter and by translational coupling.

A temperature-sensitive (ts) mutant of Salmonella typhimurium that accumulated unmodified murein prolipoprotein at 42 degrees C but not at 30 degrees C was identified. In vivo and in vitro studies of the biosynthesis of Braun's lipoprotein revealed that this mutant (SE5221) was defective in the glyceryl modification of prolipoprotein. The ts mutation was mapped to 60.6 min of the S. typhimurium chromosome and was linked to argA and cysH. A clone with a 1.4-kilobase S. typhimurium DNA insert that complemented the ts mutation and restored the prolipoprotein modification activity both in vivo and in vitro was isolated. DNA sequencing of the complementing region revealed an open reading frame encoding a protein with 291 amino acids lacking NH2-terminal signal sequence. This open reading frame is immediately 5' to the thyA gene and is allelic to umpA of Escherichia coli. Wild-type strains harboring the cloned gene exhibited elevated levels of prolipoprotein modification activity. At the non-permissive temperature, the mutation affected both growth and viability, and the mutant cells exhibited anomalous cell morphology. The ts phenotype was suppressed by the introduction of a lpp::Tn10 mutation. These results suggest that the cloned gene encodes prolipoprotein glyceryl transferase (lgt), and in the wild-type background, this prolipoprotein modification enzyme is essential for the growth and viability of S. typhimurium.

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.

The structure-function relationship of bacterial prolipoprotein diacylgyceryl transferase (LGT) Has been investigated by a comparison of the primary structures of this enzyme in phylogenetically distant bacterial species, analysis of the sequences of mutant enzymes, and specific chemical modification of the Escherichia coli enzyme. A clone containing the gene for LGT, lgt, of the gram-positive species Staphylococcus aureus was isolated by complementation of the temperature-sensitive lgt mutant of E. coli (strain SK634) defective in LGT activity. In vivo and in vitro assays for prolipoprotein diacylglyceryl modification activity indicated that the complementing clone restored the prolipoprotein modification activity in the mutant strain. Sequence determination of the insert DNA revealed an open reading frame of 837 bp encoding a protein of 279 amino acids with a calculated molecular mass of 31.6 kDa. S. aureus LGT showed 24% identity and 47% similarity with E. coli, Salmonella typhimurium, and Haemophilus influenzae LGT.S. aureus LGT, while 12 amino acids shorter than the E. coli enzyme, had a hydropathic profile and a predicted pI (10.4) similar to those of the E. coli enzyme. Multiple sequence alignment among E. coli, S. typhimurium, H. influenzae, and S. aureus LGT proteins revealed regions of highly conserved amino acid sequences throughout the molecule. Three independent lgt mutant alleles from E. coli SK634, SK635, and SK636 and one lgt allele from S. typhimurium SE5221, all defective in LGT activity at the nonpermissive temperature, were cloned by PCR and sequenced. The mutant alleles were found to contain a single base alteration resulting in the substitution of a conserved amino acid. The longest set of identical amino acids without any gap was H-103-GGLIG-108 in LGT from these four microorganisms. In E. coli lgt mutant SK634, Gly-104 in this region was mutated to Ser, and the mutant organism was temperature sensitive in growth and exhibited low LGT activity in vitro. Diethylpyrocarbonate inactivated the E. coli LGT with a second-order rate constant of 18.6 M-1S-1, and the inactivation of LGT activity was reversed by hydroxylamine at pH 7. The inactivation kinetics were consistent with the modification of a single residue, His or Tyr, essential for LGT activity.