scholarly journals Acyl carrier protein promotes MukBEF action in Escherichia coli chromosome organization-segregation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Josh P. Prince ◽  
Jani R. Bolla ◽  
Gemma L. M. Fisher ◽  
Jarno Mäkelä ◽  
Marjorie Fournier ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Acyl Carrier Protein ◽  
Coiled Coil ◽  
Acid Synthesis ◽  
Chromosome Organization ◽  
Carrier Protein ◽  
Accessory Proteins ◽  
Structural Maintenance Of Chromosomes ◽  
Smc Proteins

AbstractStructural Maintenance of Chromosomes (SMC) complexes act ubiquitously to compact DNA linearly, thereby facilitating chromosome organization-segregation. SMC proteins have a conserved architecture, with a dimerization hinge and an ATPase head domain separated by a long antiparallel intramolecular coiled-coil. Dimeric SMC proteins interact with essential accessory proteins, kleisins that bridge the two subunits of an SMC dimer, and HAWK/KITE proteins that interact with kleisins. The ATPase activity of the Escherichia coli SMC protein, MukB, which is essential for its in vivo function, requires its interaction with the dimeric kleisin, MukF that in turn interacts with the KITE protein, MukE. Here we demonstrate that, in addition, MukB interacts specifically with Acyl Carrier Protein (AcpP) that has essential functions in fatty acid synthesis. We characterize the AcpP interaction at the joint of the MukB coiled-coil and show that the interaction is necessary for MukB ATPase and for MukBEF function in vivo.

scholarly journals Acyl Carrier Protein is essential for MukBEF action in Escherichia coli chromosome organization-segregation

2021 ◽  
Author(s):  
Josh Prince ◽  
Jani Bolla ◽  
Gemma Fisher ◽  
Jarno Makela ◽  
Majorie Fournier ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Acyl Carrier Protein ◽  
Coiled Coil ◽  
Acid Synthesis ◽  
Chromosome Organization ◽  
Carrier Protein ◽  
Accessory Proteins ◽  
Structural Maintenance Of Chromosomes ◽  
Smc Proteins

Abstract Structural Maintenance of Chromosomes (SMC) complexes contribute ubiquitously to chromosome organization-segregation. SMC proteins have a conserved architecture, with a dimerization hinge and an ATPase head domain separated by a long antiparallel intramolecular coiled-coil. Dimeric SMC proteins interact with essential accessory proteins, kleisins that bridge the two subunits of an SMC dimer, and HAWK/KITE accessory proteins that interact with kleisins. The ATPase activity of the Escherichia coli SMC protein, MukB, is essential for in vivo function and is regulated by interactions with its dimeric kleisin, MukF, and KITE, MukE. Here we demonstrate that, in addition, MukB interacts with Acyl Carrier Protein (AcpP) that has essential functions in fatty acid synthesis. We characterize the AcpP interaction site at the joint of the MukB coiled-coil and show that the interaction is essential for MukB ATPase and for MukBEF function in vivo. Therefore, AcpP is an essential co-factor for MukBEF action in chromosome organization-segregation.

scholarly journals Acyl Carrier Protein is essential for MukBEF action in Escherichia coli chromosome organization-segregation

2021 ◽  
Author(s):  
Josh P Prince ◽  
Jani R Bolla ◽  
Gemma L.M. Fisher ◽  
Jarno Makela ◽  
Carol V. Robinson ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Acyl Carrier Protein ◽  
Coiled Coil ◽  
Acid Synthesis ◽  
Chromosome Organization ◽  
Carrier Protein ◽  
Accessory Proteins ◽  
Structural Maintenance Of Chromosomes ◽  
Smc Proteins

Structural Maintenance of Chromosomes (SMC) complexes contribute ubiquitously to chromosome organization-segregation. SMC proteins have a conserved architecture, with a dimerization hinge and an ATPase head domain separated by a long antiparallel intramolecular coiled-coil. Dimeric SMC proteins interact with essential accessory proteins, kleisins that bridge the two subunits of an SMC dimer, and HAWK/KITE accessory proteins that interact with kleisins. The ATPase activity of the Escherichia coli SMC protein, MukB, is essential for in vivo function and is regulated by interactions with its dimeric kleisin, MukF, and KITE, MukE. Here we demonstrate that, in addition, MukB interacts with Acyl Carrier Protein (AcpP) that has essential functions in fatty acid synthesis. We characterize the AcpP interaction site at the joint of the MukB coiled-coil and show that the interaction is essential for MukB ATPase and for MukBEF function in vivo. Therefore, AcpP is an essential co-factor for MukBEF action in chromosome organization-segregation.

scholarly journals Isolation and Characterization of β-Ketoacyl-Acyl Carrier Protein Reductase (fabG) Mutants of Escherichia coli and Salmonella enterica Serovar Typhimurium

2004 ◽  
Vol 186 (6) ◽  
pp. 1869-1878 ◽  
Author(s):  
Chiou-Yan Lai ◽  
John E. Cronan
Keyword(s):  
Escherichia Coli ◽  
Fatty Acid ◽  
Fatty Acid Synthesis ◽  
Salmonella Enterica ◽  
Acyl Carrier Protein ◽  
Acid Synthesis ◽  
Temperature Sensitive ◽  
Carrier Protein ◽  
Nonpermissive Temperature ◽  
Serovar Typhimurium

ABSTRACT FabG, β-ketoacyl-acyl carrier protein (ACP) reductase, performs the NADPH-dependent reduction of β-ketoacyl-ACP substrates to β-hydroxyacyl-ACP products, the first reductive step in the elongation cycle of fatty acid biosynthesis. We report the first documented fabG mutants and their characterization. By chemical mutagenesis followed by a tritium suicide procedure, we obtained three conditionally lethal temperature-sensitive fabG mutants. The Escherichia coli [fabG (Ts)] mutant contains two point mutations: A154T and E233K. The β-ketoacyl-ACP reductase activity of this mutant was extremely thermolabile, and the rate of fatty acid synthesis measured in vivo was inhibited upon shift to the nonpermissive temperature. Moreover, synthesis of the acyl-ACP intermediates of the pathway was inhibited upon shift of mutant cultures to the nonpermissive temperature, indicating blockage of the synthetic cycle. Similar results were observed for in vitro fatty acid synthesis. Complementation analysis revealed that only the E233K mutation was required to give the temperature-sensitive growth phenotype. In the two Salmonella enterica serovar Typhimurium fabG(Ts) mutants one strain had a single point mutation, S224F, whereas the second strain contained two mutations (M125I and A223T). All of the altered residues of the FabG mutant proteins are located on or near the twofold axes of symmetry at the dimer interfaces in this homotetrameric protein, suggesting that the quaternary structures of the mutant FabG proteins may be disrupted at the nonpermissive temperature.

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

2006 ◽  
Vol 188 (1) ◽  
pp. 287-296 ◽  
Author(s):  
Nicholas R. De Lay ◽  
John E. Cronan
Keyword(s):  
Escherichia Coli ◽  
Fatty Acid ◽  
Fatty Acid Synthesis ◽  
Acyl Carrier Protein ◽  
Lipid Synthesis ◽  
Acid Synthesis ◽  
Temperature Sensitive ◽  
Carrier Protein ◽  
Nonpermissive Temperature ◽  
Overlap Extension

ABSTRACT 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 λ 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.

scholarly journals Scavenging of Cytosolic Octanoic Acid by Mutant LplA Lipoate Ligases Allows Growth of Escherichia coli Strains Lacking the LipB Octanoyltransferase of Lipoic Acid Synthesis

2009 ◽  
Vol 191 (22) ◽  
pp. 6796-6803 ◽  
Author(s):  
Fatemah A. M. Hermes ◽  
John E. Cronan
Keyword(s):  
Escherichia Coli ◽  
Lipoic Acid ◽  
Minimal Medium ◽  
Octanoic Acid ◽  
Acyl Carrier Protein ◽  
Acid Synthesis ◽  
Carrier Protein ◽  
Missense Mutations ◽  
Mutant Strains ◽  
Mutant Genes

ABSTRACT The LipB octanoyltransferase catalyzes the first step of lipoic acid synthesis in Escherichia coli, transfer of the octanoyl moiety from octanoyl-acyl carrier protein to the lipoyl domains of the E2 subunits of the 2-oxoacid dehydrogenases of aerobic metabolism. Strains containing null mutations in lipB are auxotrophic for either lipoic acid or octanoic acid. We report the isolation of two spontaneously arising mutant strains that allow growth of lipB strains on glucose minimal medium; we determined that suppression was caused by single missense mutations within the coding sequence of the gene (lplA) that encodes lipoate-protein ligase. The LplA proteins encoded by the mutant genes have reduced Km values for free octanoic acid and thus are able to scavenge cytosolic octanoic acid for octanoylation of lipoyl domains.

scholarly journals α-proteobacteria synthesize biotin precursor pimeloyl-ACP using BioZ 3-ketoacyl-ACP synthase and lysine catabolism

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Yuanyuan Hu ◽  
John E. Cronan
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Active Site ◽  
Acyl Carrier Protein ◽  
Acid Synthesis ◽  
Side Chain ◽  
Carrier Protein ◽  
Pimelic Acid ◽  
Lysine Degradation ◽  
Lysine Catabolism

Abstract Pimelic acid, a seven carbon α,ω-dicarboxylic acid (heptanedioic acid), is known to provide seven of the ten biotin carbon atoms including all those of the valeryl side chain. Distinct pimelate synthesis pathways were recently elucidated in Escherichia coli and Bacillus subtilis where fatty acid synthesis plus dedicated biotin enzymes produce the pimelate moiety. In contrast, the α-proteobacteria which include important plant and mammalian pathogens plus plant symbionts, lack all of the known pimelate synthesis genes and instead encode bioZ genes. Here we report a pathway in which BioZ proteins catalyze a 3-ketoacyl-acyl carrier protein (ACP) synthase III-like reaction to produce pimeloyl-ACP with five of the seven pimelate carbon atoms being derived from glutaryl-CoA, an intermediate in lysine degradation. Agrobacterium tumefaciens strains either deleted for bioZ or which encode a BioZ active site mutant are biotin auxotrophs, as are strains defective in CaiB which catalyzes glutaryl-CoA synthesis from glutarate and succinyl-CoA.

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