John E. Cronan

Triclosan Resistance of Pseudomonas aeruginosa PAO1 Is Due to FabV, a Triclosan-Resistant Enoyl-Acyl Carrier Protein Reductase

ABSTRACT Triclosan, a very widely used biocide, specifically inhibits fatty acid synthesis by inhibition of enoyl-acyl carrier protein (ACP) reductase. Escherichia coli FabI is the prototypical triclosan-sensitive enoyl-ACP reductase, and E. coli is extremely sensitive to the biocide. However, other bacteria are resistant to triclosan, because they encode triclosan-resistant enoyl-ACP reductase isozymes. In contrast, the triclosan resistance of Pseudomonas aeruginosa PAO1 has been attributed to active efflux of the compound (R. Chuanchuen, R. R. Karkhoff-Schweizer, and H. P. Schweizer, Am. J. Infect. Control 31:124-127, 2003). We report that P. aeruginosa contains two enoyl-ACP reductase isozymes, the previously characterized FabI homologue plus a homologue of FabV, a triclosan-resistant enoyl-ACP reductase recently demonstrated in Vibrio cholerae. By deletion of the genes encoding P. aeruginosa FabI and FabV, we demonstrated that FabV confers triclosan resistance on P. aeruginosa. Upon deletion of the fabV gene, the mutant strain became extremely sensitive to triclosan (>2,000-fold more sensitive than the wild-type strain), whereas the mutant strain lacking FabI remained completely resistant to the biocide.

The virion of the lipid-containing bacteriophage PR4

Abstract Bacteriophage PR4 is a lipid-containing phage that infects Escherichia coli K-12. We have examined the molecular composition of homogeneous preparations of this phage with the following results. The genome is a linear double-stranded DNA molecule of 14,500 base pairs ( M r 9.6 × 10 6 ) having a GC content of 46%. The phage lipids are 98% (by weight) phospholipids and 2% neutral lipids. The phospholipids consist of 56% phosphatidylethanolamine, 37% phosphatidylglycerol, and 4.6% cardiolipin, by weight. The relative phosphatidylglycerol content of the phage phospholipids is twice that found in the host cell. The fatty acid compositions of the viral and cellular phospholipids are identical. The phage capsid contains 14 proteins ranging in molecular weight from 9000 to 60,000. The major capsid protein, comprising 80% of the total virion protein, has a molecular weight of 37,200. The purified phage contains 73% protein, 13% lipid, and 14% DNA (by weight) and has an isoelectric point of pH 4.2.

Studies on the biosynthesis of cyclopropane fatty acids in Escherichia coli

Abstract By use of various mutants either defective in unsaturated fatty acid biosynthesis or altered in the level of S-adenosylmethionine we have tested the effects of changes in the intracellular concentrations of these substrates on in vivo cyclopropane fatty acid synthesis. A 1.5-fold change in unsaturated fatty acid content produced an equivalent change in cyclopropane fatty acid content. A 20-fold difference in S-adenosylmethionine content had little or no effect on cyclopropane fatty acid formation. Studies with unsaturated fatty acid auxotrophs showed that unsaturated fatty acid synthesis is not required for cyclopropane fatty acid synthesis. Other studies demonstrated that cyclopropane fatty acid content had little effect on phospholipid stability. Finally, we have shown by three methods (gas-liquid chromatography, proton and 15C nuclear magnetic resonance spectra) that the cyclopropane ring of these Escherichia coli acids has the cis configuration.

A mutant of Escherichia coli auxotrophic for organic phosphates: Evidence for two defects in inorganic phosphate transport

SummaryAn inorganic phosphate transport mutant has been isolated as a sn-glycerol-3-phosphate auxotroph and characterized genetically. Two lesions are responsible for the transport defect. One lesion, pst, is located at minute 74 of the E. coli genetic map while the other lesion, pit, is located at minute 68. All “K10” strains that were examined carry the pit lesion. Evidence is presented that the glycerol phosphate and hexose phosphate transport systems. The mapping data indicate that the genetic distance between malA and xyl is greater than that now allowed.

Mutants of Escherichia coli with temperature-sensitive lesions in membrane phospholipid synthesis: Genetic analysis of glycerol-3-phosphate acyltransferase mutants

SummaryEscherichia coli mutants possessing temperature-sensitive lesions in glycerol-3-phosphate acyltransferase, the enzyme catalysing the first step in phospholipid biosynthesis, have been characterized genetically. By recombinational and complementation tests, the mutants have been found to map in a single locus, called plsA, which is cotransducibile with the purE locus and lies between the purE and proC loci at minute 13 on the E. coli genetic map.

The lipid-containing bacteriophage PR4: Effects of altered lipid composition on the virion

Phage PR4 was grown on a variety of Escherichia coli mutants defective in fatty acid and phospholipid metabolism. The composition of the phage lipids was modified by changing the composition of the host membrane phospholipids. The compositions of both the polar and the acyl moieties of the phospholipids were altered. The proportion of saturated fatty acids in the phage phospholipids was increased in increments from 44% of the total fatty acids to 55%, 61% and 69% of the total fatty acids using a host mutant with a temperature-sensitive defect in unsaturated fatty acid biosynthesis. The increase in saturated fatty acids led to a pronounced loss of infectivity when the phage were incubated at temperatures between 2 degrees C and 30 degrees C (temperatures below those at which the phage were grown). The greater the level of saturated fatty acids in the phage phospholipids, the higher the temperature below which the phage were inactivated. Our results strongly suggest that the phage membrane undergoes a lipid phase transition, which can disrupt and inactivate the virion. The phospholipid composition of PR4 was also altered by using host mutants defective in phosphatidylethanolamine and/or cardiolipin synthesis. Phage PR4 grown on wild-type host strains contains 56% phosphatidylethanolamine, 37% phosphatidylglycerol, 4.6% cardiolipin and no detectable phosphatidylserine. However, in response to changes in the host, PR4 preparations were obtained with phospholipid compositions varying from 28% to 60% in phosphatidylethanolamine, from 22% to 39% in phosphatidylglycerol, from 1% to 15% in cardiolipin and containing as much as 35% phosphatidylserine. These changes in phospholipid composition did not affect the infectivity of the phage. Moreover, the increased level of phosphatidylglycerol in the phage relative to the host was not altered by these manipulations. It is concluded that the net charge of the phage membrane phospholipids is not involved in the selection or function of the viral phospholipids. We also present evidence suggesting that the phage and host membranes do not fuse during the course of infection.

Haemophilus influenzae RD lacks a stringently conserved fatty acid biosynthetic enzyme and thermal control of membrane lipid composition

The organization of the fatty acid synthetic genes of Haemophilus influenzae Rd is remarkably similar to that of the paradigm organism, Escherichia coli K-12, except that no homologue of the E. coli fabF gene is present. This finding is unexpected, since fabF is very widely distributed among bacteria and is thought to be the generic 3-ketoacyl-acyl carrier protein (ACP) synthase active on long-chain-length substrates. However, H. influenzae Rd contains a homologue of the E. coli fabB gene, which encodes a 3-ketoacyl-ACP synthase required for unsaturated fatty acid synthesis, and it seemed possible that the H. influenzae FabB homologue might have acquired the functions of FabF. E. coli mutants lacking fabF function are unable to regulate the compositions of membrane phospholipids in response to growth temperature. We report in vivo evidence that the enzyme encoded by the H. influenzae fabB gene has properties essentially identical to those of E. coli FabB and lacks FabF activity. Therefore, H. influenzae grows without FabF function. Moreover, as predicted from studies of the E. coli fabF mutants, H. influenzae is unable to change the fatty acid compositions of its membrane phospholipids with growth temperature. We also demonstrate that the fabB gene of Vibrio cholerae El Tor N16961 does not contain a frameshift mutation as was previously reported.

Nonsense mutants of the lipid-containing bacteriophage PR4

Thirty-three nonsense mutants of phage PR4 representing 12 complementation groups were isolated. One or two mutants of each group were grown on a suppressor-negative (Su-) host and characterized by the following criteria (i) proteins synthesized, (ii) level of phage DNA synthesis, and (iii) ability to assemble particles. We determined the protein and phospholipid compositions of the particles assembled in an Su- host, the presence of DNA in the particles, and the ability of the particles to adsorb to host cells. Finally each complementation group was tested for the ability to lyse an Su- host. We have identified one protein required for DNA synthesis, five proteins required for proper assembly of the protein coat and lipid membrane of the phage, two proteins required for stable insertion of DNA into the virion, a protein required for adsorption, a protein required for attachment of the adsorption protein to the virion, and a phage-encoded lytic enzyme.

Evidence for a direct effect on fatty acid synthesis in relA gene control of membrane phospholipid synthesis

The direct regulation of fatty acid synthesis by the relA gene has been demonstrated in vivo by use of a system in which fatty acid synthesis is uncoupled from membrane phospholipid synthesis.

An efficient biosynthetic method to prepare fatty acyl chains highly enriched with 13C

Abstract A method has been developed for the biosynthetic incorporation of 13C acetate into fatty acyl chains. It combines both high specific enrichment and high utilization of the acetate.