Temperature-Sensitive Mutants of
            <i>Escherichia coli</i>
            Requiring Saturated and Unsaturated Fatty Acids for Growth: Isolation and Properties

Harder, Mark E.; Beacham, Ifor R.; Cronan, John E.; Beacham, Kathryn; Honegger, Joy L.; Silbert, David F.

doi:10.1073/pnas.69.11.3105

Cited by 69 publications

(53 citation statements)

References 15 publications

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“…Concerning fatty acid synthesis, Repaske et al (24) reported that oleate can eliminate the lag period in growth due to the absence of bicarbonate in the medium and CO 2 in the aerating gas in some species of bacteria. Harder et al (13) have shown that palmitate and oleate provide for growth in mutants with impaired lipid synthesis. We found that addition of a mixture of these two lipids to either minimal or LB medium had no effect either on the inhibition of growth of BUM012 (⌬cynT) by cyanate or on cyanate decomposition by BUM012 (⌬cynT) (data not shown).…”

Section: Resultsmentioning

confidence: 99%

Role of bicarbonate/CO2 in the inhibition of Escherichia coli growth by cyanate

et al. 1995

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Cyanase is an inducible enzyme in Escherichia coli that catalyzes the reaction of cyanate with bicarbonate to give two CO 2 molecules. The gene for cyanase is part of the cyn operon, which includes cynT and cynS, encoding carbonic anhydrase and cyanase, respectively. Carbonic anhydrase functions to prevent depletion of cellular bicarbonate during cyanate decomposition (the product CO 2 can diffuse out of the cell faster than noncatalyzed hydration back to bicarbonate). Addition of cyanate to the culture medium of a ⌬cynT mutant strain of E. coli (having a nonfunctional carbonic anhydrase) results in depletion of cellular bicarbonate, which leads to inhibition of growth and an inability to catalyze cyanate degradation. These effects can be overcome by aeration with a higher partial CO 2 pressure (M. B. Guilloton, A. F. Lamblin, E. I. Kozliak, M. Gerami-Nejad, C. Tu, D. Silverman, P. M. Anderson, and J. A. Fuchs, J. Bacteriol. 175:1443-1451, 1993). The question considered here is why depletion of bicarbonate/CO 2 due to the action of cyanase on cyanate in a ⌬cynT strain has such an inhibitory effect. Growth of wild-type E. coli in minimal medium under conditions of limited CO 2 was severely inhibited, and this inhibition could be overcome by adding certain Krebs cycle intermediates, indicating that one consequence of limiting CO 2 is inhibition of carboxylation reactions. However, supplementation of the growth medium with metabolites whose syntheses are known to depend on a carboxylation reaction was not effective in overcoming inhibition related to the bicarbonate deficiency induced in the ⌬cynT strain by addition of cyanate. Similar results were obtained with a ⌬cyn strain (since cyanase is absent, this strain does not develop a bicarbonate deficiency when cyanate is added); however, as with the ⌬cynT strain, a higher partial CO 2 pressure in the aerating gas or expression of carbonic anhydrase activity (which contributes to a higher intracellular concentration of bicarbonate/CO 2 ) significantly reduced inhibition of growth. There appears to be competition between cyanate and bicarbonate/CO 2 at some unknown but very important site such that cyanate binding inhibits growth. These results suggest that bicarbonate/CO 2 plays a significant role in the growth of E. coli other than simply as a substrate for carboxylation reactions and that strains with mutations in the cyn operon provide a unique model system for studying aspects of the metabolism of bicarbonate/CO 2 and its regulation in bacteria.Cyanase (EC 4.3.99.1) catalyzes the reaction of cyanate with bicarbonate to give two molecules CO 2 (15):The synthesis of cyanase in Escherichia coli is induced by addition of cyanate to the growth medium (2). The gene for cyanase is part of the cyn operon, which includes three genes in the order cynT, cynS, and cynX, encoding carbonic anhydrase, cyanase, and a hydrophobic protein of unknown function, respectively (11,(30)(31)(32)(33). The function of carbonic anhydrase appears to be to prevent depletion of cellular bicarb...

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Section: Resultsmentioning

confidence: 99%

Role of bicarbonate/CO2 in the inhibition of Escherichia coli growth by cyanate

et al. 1995

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“…Co-expression of these genes in E. coli strain LA1-6, which harbours a temperature-sensitive mutation in one of the carboxyl transferase subunits (accD) (Harder et al, 1972), could not complement the mutation, showing that the enzyme complex formed in , Not detectable. The amount of "%C fixed into acid-stable products was not significantly higher than background levels (10-30 c.p.m., equivalent to 0n02-0n06 mU).…”

Section: In Vitro Reconstitution Of a Pccase Complexmentioning

confidence: 99%

Genetic and biochemical characterization of the α and β components of a propionyl-CoA carboxylase complex of Streptomyces coelicolor A3(2) The GenBank accession numbers for the accA1, aacA2 and pccB sequences determined in this work are AF113603, AF113604 and AF113605, respectively.

Rodrı́guez

Gramajo

1999

Microbiology

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Two genes, accA1 and accA2, with nearly identical nucleotide sequences were cloned from Streptomyces coelicolor A3(2). The deduced amino acid sequences of the product of these two genes showed high similarity to BcpA2 of Saccharopolyspora erythraea and other biotin-containing proteins from different organisms assumed to be the α subunit of a propionyl-CoA carboxylase. A gene, pccB, encoding the carboxyl transferase subunit of this enzyme complex was also characterized. Strains disrupted in accA1 did not show any change in acetyl-or propionyl-CoA carboxylase activity, whilst cellfree extracts of a pccB mutant strain contained a reduced level of propionylCoA carboxylase. No mutants in accA2 could be isolated, suggesting that the gene may be essential. Heterologous expression of accA1, accA2 and pccB in Escherichia coli and in vitro reconstitution of enzyme activity confirmed that PccB is the β subunit of a propionyl-CoA carboxylase and that either AccA1 or AccA2 could act as the α component of this enzyme complex. The fact that accA2 mutants appear to be inviable suggests that this gene encodes a biotinylated protein that might be shared with other carboxyl transferases essential for the growth of S. coelicolor.

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“…However, ACP seemed certain to be an essential protein since mutants in several fatty acid synthetic genes that encoded proteins requiring ACP-bound substrates were essential genes (10,24,43,45,46). We and others assumed that conditionally lethal (temperature-sensitive) acpP mutants would be isolated by the general selections that successfully gave strains containing conditionally lethal defects in other genes acting early in (or throughout) the fatty acid synthetic pathway (14,24). However, such mutants were not forthcoming.…”

mentioning

confidence: 99%

Gene-Specific Random Mutagenesis of Escherichia coli In Vivo: Isolation of Temperature-Sensitive Mutations in the Acyl Carrier Protein of Fatty Acid Synthesis

Lay¹,

Cronan

2006

J Bacteriol

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Acyl carrier proteins (ACPs) are very small acidic proteins that play a key role in fatty acid and complex lipid synthesis. Moreover, recent data indicate that the acyl carrier protein of Escherichia coli has a large protein interaction network that extends beyond lipid synthesis. Despite extensive efforts over many years, no temperature-sensitive mutants with mutations in the structural gene (acpP) that encodes ACP have been isolated. We report the isolation of three such mutants by a new approach that utilizes error-prone PCR mutagenesis, overlap extension PCR, and phage Red-mediated homologous recombination and that should be generally applicable. These mutants plus other experiments demonstrate that ACP function is essential for the growth of E. coli. Each of the mutants was efficiently modified with the phosphopantetheinyl moiety essential for the function of ACP in lipid synthesis, and thus lack of function at the nonpermissive temperature cannot be attributed to a lack of prosthetic group attachment. All of the mutant proteins were largely stable at the nonpermissive temperature except the A68T/N73D mutant protein. Fatty acid synthesis in strains that carried the D38V or A68T/N73D mutations was inhibited upon a shift to the nonpermissive temperature and in the latter case declined to a small percentage of the rate of the wild-type strain.Two systems for fatty acid biosynthesis, called types I and II, are found in nature. Type I systems occur in the cytosols of mammalian and plant cells, in some plant plastids, and in a few bacteria. In these systems, the steps of fatty acid biosynthesis are catalyzed by one or two very large polypeptides that contain multiple active sites (47). In contrast, the type II systems which occur in bacteria, mitochondria, most plant plastids, and protozoan apicoplasts produce fatty acids using a series of soluble enzymes in which each protein has a discrete enzymatic activity (38). A key component of both systems is acyl carrier protein (ACP) which constitutes a domain of the type I multifunctional proteins but is a very small, soluble, and highly acidic protein in type II systems. ACP is functional only in fatty acid biosynthesis after it has been posttranslationally modified by covalent attachment of a 4Ј-phosphopantetheinyl (4Ј-PP) moiety. The 4Ј-PP prosthetic group is attached to the hydroxyl group of a centrally located serine residue by the AcpS 4Ј-PP transferase. Acyl intermediates are bound to the 4Ј-PP thiol in a thioester linkage that allows ACP to shuttle intermediates among the fatty acid synthetic enzymes. The thioester linkage also serves to facilitate chemistry at acyl chain ␤-carbons.Escherichia coli contains a single ACP encoded by the acpP gene (35). This was the first ACP discovered (40, 52) and remains the best studied of these proteins. Despite the breadth and depth of studies of E. coli ACP and its acylated derivatives, no mutant strain encoding a mutant ACP has been reported. However, ACP seemed certain to be an essential protein since mutants in several f...

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Temperature-Sensitive Mutants of Escherichia coli Requiring Saturated and Unsaturated Fatty Acids for Growth: Isolation and Properties

Cited by 69 publications

References 15 publications

Role of bicarbonate/CO2 in the inhibition of Escherichia coli growth by cyanate

Role of bicarbonate/CO2 in the inhibition of Escherichia coli growth by cyanate

Genetic and biochemical characterization of the α and β components of a propionyl-CoA carboxylase complex of Streptomyces coelicolor A3(2) The GenBank accession numbers for the accA1, aacA2 and pccB sequences determined in this work are AF113603, AF113604 and AF113605, respectively.

Gene-Specific Random Mutagenesis of Escherichia coli In Vivo: Isolation of Temperature-Sensitive Mutations in the Acyl Carrier Protein of Fatty Acid Synthesis

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