Structure and Synthesis of a Lipid‐Containing Bacteriophage

Abstract

Enzymes of glycerophosphatide biosynthesis and degradation have been characterized using membrane fractions of Pseudomonas BAL-31, the host cell of the lipid-containing bacteriophage PM2. Inner and outer membrane fractions were investigated. Whereas the outer membrane contained more phosphatidylethanolamine and less phosphatidylglycerol than the inner membrane, the fatty acid composition was almost the same for both membranes. The pathway for glycerophosphatide biosynthesis was the same as that for Escherichia coli and Bacillus megaterium. The biosynthesis of phosphatidylserine and phosphatidylglycerol was shown to proceed via CDP-diglyceride as a common intermediate. Phospholipase A hydrolyzed phosphatidylethanolamine more efficiently than phosphatidylglycerol. All of the enzymatic activities were stimulated by Triton X-100. Phosphatidylserine synthetase was stimulated by Na2S04 and inhibited by MgZf. Phosphatidylserine decarboxylase was completely inhibited by NH,OH. Phosphatidylglyceror synthetase was stimulated by MgZ + . Phospholipase A was stimulated by Ca2 + . Phosphatidylserine decarboxylase and phosphatidylglycerol synthetase were localized in the inner membrane fraction. Phosphatidylserine synthetase and phospholipase A were found in both outer and inner membrane fractions.Effects of virus infection on these enzymes were studied. Phosphatidylglycerol synthetase and phospholipase A activity against phosphatidylethanolamine were activated early post-infection. Phosphatidylserine synthetase and decarboxylase remained unaltered. At later times post-infection, phosphatidylglycerol synthetase and phosphatidylserine decarboxylase were inactivated and phosphatidylserine synthetase was activated. There was little alteration in phospholipase A activity against phosphatidylglycerol after infection.The marine pseudomonad, Pseudomonas BAL-31, is of special interest because it is the host cell of the lipid-containing bacteriophage PM2 [l]. The virus matures at the periphery of the host bacterium [2] and has a lipid bilayer in its membrane structure [3]. The phospholipid composition of the virus is very different from that of its host cell [4]. The origin of the viral phospholipid has been investigated ; one-third of the viral lipids are derived from lipids synthesized before infection and two-thirds from lipids synthesized during viral replication [5].Although a number of aspects of phospholipid synthesis in bacteria have been studied, there have been only two comprehensive studies of phospholipid synthesis; those on Escherichia coli and Bacillus megaterium [6,7]. As part of our general study of the structure and synthesis of bacteriophage PM2, we hereby report on the localization of the enzymes of phospholipid synthesis and degradation and the effects of viral infection on these enzymes. The biosynthetic pathway for phosphatidylethanolamine and phosphatidylglycThis is paper number XXII in the series. Enzymes. Phospholipase A (EC 3.1.1.32); fl-gdlactosidase (EC 3.2

Intracellular Distribution of Enzymes of Phospholipid Metabolism in Several Gram-Negative Bacteria

“…1. Enzymes capable of synthesizing some of these same phospholipids have been reported in Bacillus PP (15), Bacillus licheniformis (12), Salmonella typhimurium (1), Pseudomonas BAL 31 (21), and Micrococcus cerificans (14). Eucaryotic cells have similar pathways (22) leading to all of these phospholipids except for phosphatidylserine and phosphatidylethanolamine.…”

“…However, the phosphatidylserine synthase from E. coli was unexpectedly found tightly associated with the ribosomal fraction of cells broken by several techniques (18). This latter enzymatic activity has been reported to be membrane associated in both Pseudomonas BAL 31 (21) and Bacillus PP (15). Therefore, the phosphatidylserine synthase of E. coli is the only reported enzyme involved in phospholipid biosynthesis that is not found tightly associated with a membrane structure.…”

“…1) have been shown to be tightly associated with the cell membrane ofE. coli (25), S. typhimurium (1), Bacillus PP (15), and Pseudomonas BAL 31 (21). However, the phosphatidylserine synthase from E. coli was unexpectedly found tightly associated with the ribosomal fraction of cells broken by several techniques (18).…”

Phosphatidylglycerophosphate synthease and phosphatidylserine synthase activites in Clostridium perfringens

“…The study of this group of enzymes is important because their action is involved in structural as well as functional properties of cell membranes. The phospholipid biosynthetic enzymes have been extensively studied in several gram-negative bacteria (3,19,23,28). These enzymes have been investigated in only three species of gram-positive bacilli (14,16,21), and only acyltransferase activities have been reported for the gram-positive anaerobe Clostridium butyricum (11).…”

Phospholipid biosynthesis in some anaerobic bacteria

“…phosphatidylethanolamine + C02 Reactions 2 and 4 have also been shown to utilize dCDP-diglyceride. These reactions have also been found in other facultative and aerobic bacteria (5,19,21,26). Because no comparable work had been done with anaerobic bacteria, we carried out a study of phospholipid-synthesizing enzymes in Clostridium butyricum and found that reactions 1 through 4 were present (25); however, phosphatidylserine decarboxylase activity was barely detectable (25).…”