The mechanism of killing of Escherichia coli by a novel beta-lactam antibiotic, an amidino penicillin, has been investigated. This compound converts E. coli to relatively stable spherical forms at low concentration. However, the amidino penicillin caused no alteration in any of those parameters of peptidoglycan synthesis which can be studied. Above 10 μg of the antibiotic per ml the cells began to lyse, and a second mode of killing appeared. Mutants resistant to the amidino penicillin were isolated and several were studied in detail. Three mutant phenotypes were distinguished: (i) spherical shape and hypersensitive to lysis by either amidino penicillin or ampicillin; (ii) spherical shape and normally sensitive to lysis; (iii) rod shape, converted to viable spheres by amidino penicillin and normally sensitive to lysis.

The In the present investigation, mutant strains of S. cerevisiae defective in acyl-CoA synthetase [acid:CoA ligase (AMPforming), EC 6.2.1.3] were isolated and utilized to determine whether fatty acid itself or a metabolite of fatty acid is more directly responsible for the repression of acetyl-CoA carboxylase observed upon addition of fatty acid to the culture medium. Such mutants promised to be useful for this purpose, since acyl-CoA can be regarded as an obligatory intermediate for further metabolism of fatty acid. Studies with these mutants have provided evidence indicating that the repression of acetyl-CoA carboxylase by exogenous fatty acid is mediated by some metabolite of fatty acid rather than by nonesterified fatty acid.MATERIALS AND METHODS Chemicals. Fatty acids were purchased from Nakarai (Kyoto, Japan). [9,10-3H]Palmitic acid was obtained from the Commissariat a l'Energie Atomique (Saclay, France), and [U-'4C]palmitic acid and sodium [1-'4C]acetate from the Radiochemical Centre (Amersham, England). Phosphatidylcholine, phosphatidylethanolamine, and triglyceride were products of Serdary (London, Canada). 1,2-Diglyceride was prepared as described previously (6). CoA and ATPwere obtained from Boehringer (Mannheim, Germany), and Triton X-100 from Rohm and Haas (Philadelphia, Pa.). Silica gel G plates for thin-layer chromatography were purchased from Merck (Rahway, N.J.

We report the complete nucleotide sequence of two genes encoding major peroxisomal polypeptides (PXPs) of Candida tropicalis. One, POX4, encodes PXP-4, which is the most abundant polypeptide in cells grown on oleic acid, and the other, POX5, is the gene for PXP-5. Each of the two polypeptides was found to be the subunit of a distinct long-chain acyl-coenzyme A oxidase: acyl-CoA oxidase II (PXP-4) or acyl-CoA oxidase I (PXP-5). Both the genes had no intron and gave a single open reading frame. The NH2-terminal sequences, except the initiator methionine, and the calculated molecular weights of the deduced polypeptides were consistent with those of the respective PXPs. Well-conserved sequences of 12 and 16 hydrophobic amino acids were present in the middle of the polypeptide, instead of at the NH2 terminus, and may be internal signal sequences for the peroxisomal location of PXPs. Although the two polypeptides were significantly homologous throughout their sequences, the local homologies in two regions out offive were markedly diverged from the average (63%); the homology in the second region was 93%, whereas that in the fourth one was only 24%. The implications of this finding are discussed in respect to the multiplicity of peroxisomal enzymes and the presence of multifunctional proteins in peroxisomes.

We have sequenced the nucleotides of the gene POX18 that encodes PXP-18, a major peroxisomal polypeptide inducible by oleic acid in the yeast Candida tropicalis. POX18 had a single open reading frame of 127 amino acids. Some 33% of the amino acid sequence of the predicted basic polypeptide (13805 Da), was identical to that of the nonspecific lipid-transfer protein (sterol carrier protein 2) from rat liver. PXP-18, purified to near homogeneity from isolated peroxisomes, had an amino-terminal sequence identical to that of the predicted polypeptide except for the initiator methionine, and had nonspecific lipid-transfer activity comparable to that of its mammalian equivalents. Unexpectedly, PXP-18 lacked the cysteine residue thought to be essential for the activity of this protein in mammals. RNA blot analysis showed that the POX18 gene was expressed exclusively in cells grown on oleic acid, suggesting that PXP-18 has a role in the /I-oxidation of long-chain fatty acids. PXP-18 modulated acylcoenzyme A oxidase activity at low pH.Peroxisomal proteins are synthesized on free polysomes and imported post-translationally into the organelle, but most of them have no cleavable targeting (topogenic) sequence at the amino-terminus (for review, see [l]). The targeting sequences of five peroxisomal proteins from higher eucaryotes have been found at the carboxy-terminus and shown to contain a tripeptide, Ser-Lys/His-Leu, at or near the end of the sequence [2 -41. We have cloned genes encoding peroxisomal proteins of the yeast Candida tropicalis [5] and sequenced three genes for the subunits (PXP-2, PXP-4 and PXP-5) of acylCoA oxidase isozymes [6, 71 and the gene for catalase (PXP-9) [8]. None of the polypeptides encoded by these genes has the Ser-Lys/His-Leu sequence in their carboxy-terminal regions. The targeting information of PXP-4, which has a molecular mass of 78554 Da, was found in various locations by both in vitro [9] and in vivo [lo] experiments. Thus, the entire protein molecule must be systematically analysed if the yeast peroxisomal targeting sequence is to be identified. Since a small protein is more convenient for this purpose, we chose a 16-kDa protein, PXP-18, which is encoded by the gene POX18 and induced markedly by oleic acid [5]. Its function is not known.

The development of peroxisomes in the cells of Candida tropicalis grown on oleic acid was accompanied by a markedly high expression of peroxisomal proteins. On the basis of this finding, the nuclear DNA library of this yeast was screened by differential hybridization, and 102 clones of oleic acid-inducible sequences were isolated. Seven coding regions were found to form clusters in three stretches of the genomic DNA. Five of the regions were identified as genes for peroxisomal polypeptides (PXPs). The coding sequence for PXP-2 hybrid selected an additional mRNA for PXP-4, the subunit of long-chain acyl coenzyme A oxidase, which was the most abundant PXP. PXP-2 and PXP-4 were close in apparent molecular weight and generated similar peptides when digested with a protease. The gene for PXP-4 was adjacent to that for PXP-2 on the genome and also hybridized to the mRNA coding for PXP-5. These and other similar results suggest that the genes for the peroxisomal proteins of this organism arose by duplication of a few ancestral genes.The enzyme composition of peroxisomes, intracellular compartments generating and degrading hydrogen peroxide, varies widely in contrast to their ubiquitous distribution. The peroxisomes of methanol-assimilating yeasts contain only a few enzymes (10, 40), whereas the equivalents of fatty acidutilizing yeasts have, as in the case of hepatic peroxisomes, more than a dozen enzymes in addition to a n-oxidation system for long-chain fatty acids (1,6,12,20,28,39). In yeast cells, unlike in hepatocytes, fatty acids are oxidized solely in peroxisomes (20), which have a novel long-chain acyl coenzyme A synthetase whose product is exclusively utilized for 3-oxidation but not for lipid synthesis (17,18,28).Candida tropicalis pK233 is a typical yeast strain which assimilates fatty acids or alkanes as the sole source of carbon and energy. The cells grown on glucose contain few peroxisomes, whereas those grown on oleic acid or n-alkanes (C10-C13) develop large numbers of the organelles (30). The peroxisomes of this yeast strain have been purified to near homogeneity (16), and the induction of many associated enzymes has been reported (39). C. tropicalis is thus a suitable unicellular system for studying the biogenesis of peroxisomes and its regulation. In this regard, special attention was directed to reports (29, 31) suggesting the presence of DNA in the peroxisomes of this organism. Extensive searches for the proposed DNA, however, provided no evidence for this and led us to conclude that the biogenesis of peroxisomes must be entirely controlled by nuclear genes (16).We report here that the development of peroxisomes in cells grown on oleic acid is accompanied by a markedly high expression of peroxisomal proteins. On the basis of this finding, we isolated oleic acid-inducible DNA sequences by differential hybridization. At least five oleate-inducible sequences were identified as genes encoding peroxisomal polypeptides (PXPs) and were found to form clusters in the genomic DNA fragments. Genes for the per...