DNA sequence analysis of the structural gene for Bacillus licheniformis penicillinase has revealed a tetrapeptide sequence of Leu-Ala-Gly-Cys within the NH2-terminal part of the precursor form of penicillinase (pencillin amido-i3-lactamhydrolase, EC 3.5.2.6). The same tetrapeptide occurs in the signal sequence of the prolipoprotein of Escherichia coli, and the cysteine residue in the tetrapeptide of prolipoprotein is modified to form glyceride-cysteine which becomes the NH2 terminus of Braun's lipoprotein. On Penicillinase (penicillin amido-g-lactamhydrolase, EC 3.5.2.6) from Bacillus licheniformis exists in two forms, the exoenzyme which is secreted into the culture medium and the membranebound form which is associated with the membrane (1, 2) presumably via the hydrophobic segment at the NH2 terminus (3). The membrane-bound penicillinase is not a phospholipoprotein because it contains less than 0.02 mol of phosphate per mol of enzyme (3). The structural gene for the penicillinase from B. licheniformis has been cloned on a A-transducing phage (4). The availability of this clone allows study of the expression of this gene both in vitro and in Escherichia coli. In vitro transcription and translation of the penicillinase gene reveals the synthesis of a penicillinase precursor that is larger than the membranebound form (5). The sequence of the NH2-terminal portion of the penicillinase, as deduced from the DNA sequence of the cloned gene, is shown in Fig. 1B (6). The sequence of prepenicillinase includes the tetrapeptide Leu-Ala-Gly-Cys. This is the same tetrapeptide surrounding the site ofposttranslational modification and processing ofprolipoprotein in E. coli (Fig. LA) (1 ,uCi/ ml, 23.5 Ci/mmol), [2-3H]glycerol (50 ,uCi/ml, 6.35 Ci/ 3506The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

The biosynthesis of the diglyceride moiety of murein lipoprotein was studied by fusion of labeled phospholipid vesicles with intact cells of Salmonella typhimurium. Phosphatidylglycerol was found to be an excellent donor for the glyceryl moiety in lipoprotein, whereas phosphatidylethanolamine and cardiolipin were not. The incorporation of radioactivity from monoacyl-phosphatidylglycerol into lipoprotein can be attributed to its conversion to phosphatidylglycerol. The results strongly support our hypothesis that the glyceryl residue covalently linked to murein lipoprotein is derived from the nonacylated glycerol moiety of phosphatidylglycerol. ter (65:25:4, vol/vol), (b) chloroform-methanol-ammonia (70:21:1, vol/vol), and (c) chloroform-methanol-309 on July 31, 2020 by guest

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Globomycin-resistant mutants of Escherichia coli have been isolated and partially characterized. Approximately 2-5% of these mutants synthesize structurally altered Braun's lipoprotein. The majority of these mutants contain unprocessed and unmodified prolipoprotein. One mutant is found to contain modified, processed, but structurally altered lipoprotein. Mutants containing lipid-deficient prolipoprotein or lipoprotein also show increased resistance to globomycin. These results suggest that the inhibition of processing of modified prolipoprotein by globomycin may require fully modified prolipoprotein as the biochemical target of this novel antibiotic. Our failure to isolate mutant containing cleaved but unmodified lipoprotein among globomycin-resistant mutants is consistent with the possibility that modification of prolipoprotein precedes the removal of signal sequence by a unique signal peptidase. Recent evidence indicates that the minor lipoproteins in the cell envelope of E. coli are also synthesized as lipid-containing prolipoproteins and the processing of these prolipoproteins is inhibited by globomycin. These results suggest the existence of modifying enzymes in E. coli which would transfer glyceryl and fatty acyl moieties to cysteine residues located in the proper sequences of the precursor proteins. This speculation is confirmed by our demonstration that Bacillus licheniformis penicillinase synthesized in E. coli as well as in B. licheniformis is a lipoprotein containing glyceride-cysteine at its NH2-terminus.

The biosynthesis of the acyl moieties in murein lipoprotein was studied by fusion of [3H]pahnitate-labeled phospholipid vesicles with intact cells of an fadD mutant ofEscherichia coli. A linear increase in the incorporation of [3H]palmitate radioactivity into both the ester-and amide-linked fatty acids in lipoprotein was observed during a 3-h chase after the fusion. Addition of chloramphenicol completely prevented the incorporation of [3H]palmitate from phospholipids to lipoprotein. These results strongly support our hypothesis that the acyl moieties in phospholipids are the precursors for the fatty acids in murein lipoprotein of E. coli. Among the major glycerophosphatides in E. coli, no specificity was observed regarding the efficacy of the donor. acyl-deficient lipopolysaccharides.