Match!

Characterization of a Pseudomonas aeruginosa Fatty Acid Biosynthetic Gene Cluster: Purification of Acyl Carrier Protein (ACP) and Malonyl-Coenzyme A:ACP Transacylase (FabD)

Published on Sep 1, 1999in Journal of Bacteriology3.23
Alecksandr J. Kutchma2
Estimated H-index: 2
(CSU: Colorado State University),
Tung T. Hoang9
Estimated H-index: 9
(CSU: Colorado State University),
Herbert P. Schweizer45
Estimated H-index: 45
(CSU: Colorado State University)
Abstract
A DNA fragment containing the Pseudomonas aeruginosa fabD (encoding malonyl-coenzyme A [CoA]:acyl carrier protein [ACP] transacylase), fabG (encoding β-ketoacyl-ACP reductase), acpP (encoding ACP), and fabF (encoding β-ketoacyl-ACP synthase II) genes was cloned and sequenced. This fab gene cluster is delimited by the plsX (encoding a poorly understood enzyme of phospholipid metabolism) and pabC (encoding 4-amino-4-deoxychorismate lyase) genes; the fabF and pabC genes seem to be translationally coupled. The fabH gene (encoding β-ketoacyl-ACP synthase III), which in most gram-negative bacteria is located between plsX and fabD, is absent from this gene cluster. A chromosomal temperature-sensitive fabD mutant was obtained by site-directed mutagenesis that resulted in a W258Q change. A chromosomal fabF insertion mutant was generated, and the resulting mutant strain contained substantially reduced levels of cis-vaccenic acid. Multiple attempts aimed at disruption of the chromosomal fabG gene were unsuccessful. We purified FabD as a hexahistidine fusion protein (H6-FabD) and ACP in its native form via an ACP-intein-chitin binding domain fusion protein, using a novel expression and purification scheme that should be applicable to ACP from other bacteria. Matrix-assisted laser desorption–ionization spectroscopy, native polyacrylamide electrophoresis, and amino-terminal sequencing revealed that (i) most of the purified ACP was properly modified with its 4′-phosphopantetheine functional group, (ii) it was not acylated, and (iii) the amino-terminal methionine was removed. In an in vitro system, purified ACP functioned as acyl acceptor and H6-FabD exhibited malonyl-CoA:ACP transacylase activity.
  • References (44)
  • Citations (52)
References44
Newest
Published on May 1, 2001in Current protocols in molecular biology
Stanley Tabor28
Estimated H-index: 28
(Harvard University)
This unit describes the expression of genes by placing them under the control of the bacteriophage T7 RNA polymerase. T7 RNA polymerase is a very active enzyme: it synthesizes RNA at a rate several times that of E. coli RNA polymerase and it terminates transcription less frequently; in fact, its transcription can circumnavigate a plasmid, resulting in RNA several times the plasmid length in size. T7 RNA polymerase is also highly selective for initiation at its own promoter sequences and is resis...
Published on Sep 1, 1999in Journal of Bacteriology3.23
Tung T. Hoang9
Estimated H-index: 9
(CSU: Colorado State University),
Herbert P. Schweizer45
Estimated H-index: 45
(CSU: Colorado State University)
The Pseudomonas aeruginosa fabI structural gene, encoding enoyl-acyl carrier protein (ACP) reductase, was cloned and sequenced. Nucleotide sequence analysis revealed that fabI is probably the last gene in a transcriptional unit that includes a gene encoding an ATP-binding protein of an ABC transporter of unknown function. The FabI protein was similar in size and primary sequence to other bacterial enoyl-ACP reductases, and it contained signature motifs for the FAD-dependent pyridine nucleotide r...
Published on Sep 1, 1998in Journal of Bacteriology3.23
Jesús Campos-García18
Estimated H-index: 18
(UNAM: National Autonomous University of Mexico),
Alma Delia Caro3
Estimated H-index: 3
(UNAM: National Autonomous University of Mexico)
+ 3 AuthorsGloria Sobero27
Estimated H-index: 27
(UNAM: National Autonomous University of Mexico)
A Pseudomonas aeruginosa gene homologous to the fabG gene, which encodes the NADPH-dependent β-ketoacyl-acyl carrier protein (ACP) reductase required for fatty acid synthesis, was identified. The insertional mutation of this fabG homolog (herein called rhlG) produced no apparent effect on the growth rate and total lipid content of P. aeruginosa cells, but the production of rhamnolipids was completely abrogated. These results suggest that the synthetic pathway for the fatty acid moiety of rhamnol...
Published on Sep 1, 1998in Journal of Bacteriology3.23
Guido Epple1
Estimated H-index: 1
,
Koen M. G. M. van der Drift8
Estimated H-index: 8
+ 1 AuthorsOtto Geiger29
Estimated H-index: 29
Rhizobial capsular polysaccharides (RKPs) play an important role in the development of a nitrogen-fixing symbiosis with the plant host and in Sinorhizobium meliloti AK631 functional rkpABCDEF genes are required for the production of RKPs. After cloning the rkpF gene, we overexpressed and purified the derived protein product (RkpF) in Escherichia coli. Like acyl carrier protein (ACP), the RkpF protein can be labeled in vivo with radioactive β-alanine added to the growth medium. If homogeneous Rkp...
Published on May 1, 1998in Gene2.64
Tung T. Hoang9
Estimated H-index: 9
(CSU: Colorado State University),
RoxAnn R. Karkhoff-Schweizer10
Estimated H-index: 10
(CSU: Colorado State University)
+ 1 AuthorsHerbert P. Schweizer45
Estimated H-index: 45
(CSU: Colorado State University)
Abstract An improved method for gene replacement in Pseudomonas aeruginosa was developed. The method employs several new gene replacement vectors that incorporate (1) the counterselectable sacB marker, (2) a lacZα -allele for blue–white screening, (3) the pUC18/19 vectors multiple cloning site with 10 unique restriction sites, (4) an oriT for conjugation-mediated plasmid transfer and (5) carbenicillin, gentamicin (Gm) and tetracycline selectable markers. A cassette was constructed that contains ...
Published on May 1, 1998in Journal of Bacteriology3.23
Dale L. Val6
Estimated H-index: 6
,
John E. Cronan73
Estimated H-index: 73
(UIUC: University of Illinois at Urbana–Champaign)
Many gram-negative bacteria synthesize N-acyl homoserine lactone autoinducer molecules as quorum-sensing signals which act as cell density-dependent regulators of gene expression. We have investigated the in vivo source of the acyl chain and homoserine lactone components of the autoinducer synthesized by the LuxI homolog, TraI. In Escherichia coli, synthesis of N-(3-oxooctanoyl)homoserine lactone by TraI was unaffected in a fadD mutant blocked in β-oxidative fatty acid degradation. Also, conditi...
Published on Jan 1, 1998
John M. Walker1
Estimated H-index: 1
Protein Protocols on CD-ROM provides instant access to over 2500 time-tested state-of-the-art protein methods and supporting protocols that are unfailingly reproducible and easily accessible through CD-ROM technology. Unprecedented in scope and depth of
Published on Jan 1, 1998in Emerging Infectious Diseases7.18
Christian van Delden29
Estimated H-index: 29
,
Barbara H. Iglewski72
Estimated H-index: 72
(UR: University of Rochester)
Pseudomonas aeruginosa is a bacterium responsible for severe nosocomial infections, life-threatening infections in immunocompromised persons, and chronic infections in cystic fibrosis patients. The bacterium’s virulence depends on a large number of cell-associated and extracellular factors. Cell-to-cell signaling systems control the expression and allow a coordinated, cell-density‐dependent production of many extracellular virulence factors. We discuss the possible role of cell-to-cell signaling...
Published on Sep 1, 1997in Journal of Bacteriology3.23
Tung T. Hoang9
Estimated H-index: 9
(CSU: Colorado State University),
Herbert P. Schweizer45
Estimated H-index: 45
(CSU: Colorado State University)
The Pseudomonas aeruginosa fabA and fabB genes, encoding beta-hydroxyacyl-acyl carrier protein dehydratase and beta-ketoacyl-acyl carrier protein synthase I, respectively, were cloned, sequenced, and expressed in Escherichia coli. Northern analysis demonstrated that fabA and fabB are cotranscribed and most probably form a fabAB operon. The FabA and FabB proteins were similar in size and amino acid composition to their counterparts from Escherichia coli and to the putative homologs from Haemophil...
Published on Mar 1, 1997in Microbiology1.03
Tung T. Hoang14
Estimated H-index: 14
,
Scott Williams1
Estimated H-index: 1
+ 1 AuthorsJoseph S. Lam48
Estimated H-index: 48
asd mutants of Gram-negative and some Gram-positive bacteria have an obligate requirement for diaminopimelic acid (DAP), an essential constituent of the cell wall of these organisms. In environments deprived of DAP, for example mammalian tissues, they will undergo lysis. This was previously exploited to develop vaccine strains of Salmonella typhimurium and cloning vectors containing asd as an in vivo selectable marker. As a first step for development of such systems for Pseudomonas aeruginosa, t...
Cited By52
Newest
Published on May 22, 2019in Frontiers in Microbiology4.26
Qiao-Qiao Guo , Wen-Bin Zhang3
Estimated H-index: 3
+ 5 AuthorsHaihong Wang9
Estimated H-index: 9
Bacterial 3-oxoacyl-ACP reductase (OAR) catalyzes the 3-oxoacyl-ACP reduction step in the fatty acid synthesis pathway. At least 12 genes in the Pseudomonas aeruginosa genome are annotated as OAR-encoding genes. In this study, we characterized the functions of these genes with biochemical and genetic techniques. With the exception of PA2697, which encodes FabG, an essential protein in fatty acid synthesis, only the PA4389 and PA4786 gene products had OAR activity, and the single deletion of thes...
Published on Jun 1, 2018in Biochimie3.36
Vijay Kumar7
Estimated H-index: 7
(IITR: Indian Institute of Technology Roorkee),
Anchal Sharma3
Estimated H-index: 3
(IITR: Indian Institute of Technology Roorkee)
+ 1 AuthorsPravindra Kumar15
Estimated H-index: 15
(IITR: Indian Institute of Technology Roorkee)
Abstract Malonyl-CoA:acyl carrier protein transacylase (FabD), being an essential enzyme of the FAS II pathway, is an attractive target for developing broad-spectrum antibiotics. It performs initiation reaction to form malonyl-ACP, which is a key building block in fatty acid biosynthesis. In this study, we have characterized the FabD from drug-resistant pathogen Moraxella catarrhalis (McFabD). More importantly, we have shown the binding of McFabD with three new compounds from the class of aporph...
Published on Apr 13, 2018in Journal of Biological Chemistry
Richard G. Carroll6
Estimated H-index: 6
(Trinity College, Dublin),
Zbigniew Zasłona7
Estimated H-index: 7
(Trinity College, Dublin)
+ 7 AuthorsLuke A. J. O'Neill93
Estimated H-index: 93
(Trinity College, Dublin)
Different immune activation states require distinct metabolic features and activities in immune cells. For instance, inhibition of fatty acid synthase (FASN), which catalyzes the synthesis of long-chain fatty acids, prevents the proinflammatory response in macrophages; however, the precise role of this enzyme in this response remains poorly defined. Consistent with previous studies, we found here that FASN is essential for lipopolysaccharide-induced, Toll-like receptor (TLR)-mediated macrophage ...
Published on Nov 10, 2017in Frontiers in Microbiology4.26
Jin-Cheng Ma6
Estimated H-index: 6
(SCAU: South China Agricultural University),
Yun-Qi Wu1
Estimated H-index: 1
(Macquarie University)
+ 2 AuthorsHaihong Wang9
Estimated H-index: 9
(SCAU: South China Agricultural University)
The genome of Pseudomonas aeruginosa contains three open reading frames, PA2966, PA1869 and PA3334, which encode putative acyl carrier proteins, AcpP1, AcpP2 and AcpP3, respectively. In this study, we found that, although these apo-ACPs were successfully phosphopantetheinylated by P. aeruginosa phosphopantetheinyl transferase (PcpS) and all holo-forms of these proteins could be acylated by Vibrio harveyi acyl-ACP synthetase (AasS), only AcpP1 could be used as a substrate for the synthesis of fat...
Published on Mar 1, 2017in Frontiers of Chemical Engineering in China2.81
Qingxin Li1
Estimated H-index: 1
(Agency for Science, Technology and Research)
Rhamnolipids are one of the most effective biosurfactants that are of great interest in industrial applications such as enhancing oil recovery, health care, cosmetics, pharmaceutical processes, food processing, detergents for protein folding, and bioremediation due to their unique characteristics such as low toxicity, surface active property to reduce surface/interfacial tensions, and excellent biodegradability. The genes and metabolic pathways for rhamnolipid synthesis have been well elucidated...
Published on Sep 1, 2015in Chemistry and Physics of Lipids2.54
Tatiana Kondakova2
Estimated H-index: 2
(University of Rouen),
François D'Heygère3
Estimated H-index: 3
(CNRS: Centre national de la recherche scientifique)
+ 3 AuthorsCécile Duclairoir Poc4
Estimated H-index: 4
(University of Rouen)
Abstract The genus Pseudomonas is one of the most heterogeneous groups of eubacteria, presents in all major natural environments and in wide range of associations with plants and animals. The wide distribution of these bacteria is due to the use of specific mechanisms to adapt to environmental modifications. Generally, bacterial adaptation is only considered under the aspect of genes and protein expression, but lipids also play a pivotal role in bacterial functioning and homeostasis. This review...
Mallet Pl1
Estimated H-index: 1
,
Sébastien Roy12
Estimated H-index: 12
Alders are well recognized for their ability to colonize the harsh environments created by either natural processes or human activity. They establish symbiosis with the actinomycete Frankia which supplies 70 to 100 % of the plant’s nitrogen requirements. An important challenge facing the oil sands industry in Alberta, Canada, is management of the toxicity of Tailing Sands (TS) that are alkaline, saline and contain Naphthenic Acids (NA).In order to begin to understand how alders, Frankia and thei...
Published on Dec 30, 2013in PLOS ONE2.78
Ana Laura Boechat4
Estimated H-index: 4
(USP: University of São Paulo),
Gilberto Hideo Kaihami6
Estimated H-index: 6
(USP: University of São Paulo)
+ 2 AuthorsRegina L. Baldini12
Estimated H-index: 12
(USP: University of São Paulo)
Extracytoplasmic function (ECF) sigma factors are members of cell-surface signaling systems, abundant in the opportunistic pathogen Pseudomonas aeruginosa. Twenty genes coding for ECF sigma factors are present in P. aeruginosa sequenced genomes, most of them being part of TonB systems related to iron uptake. In this work, poorly characterized sigma factors were overexpressed in strain PA14, in an attempt to understand their role in the bacterium´s physiology. Cultures overexpressing SigX display...
Published on Oct 1, 2013in Mbio6.75
Lei Zhu6
Estimated H-index: 6
(SCAU: South China Agricultural University),
Hongkai Bi6
Estimated H-index: 6
(UIUC: University of Illinois at Urbana–Champaign)
+ 4 AuthorsHaihong Wang9
Estimated H-index: 9
(SCAU: South China Agricultural University)
ABSTRACT Enoyl-acyl carrier protein (enoyl-ACP) reductase catalyzes the last step of the elongation cycle in the synthesis of bacterial fatty acids. The Enterococcus faecalis genome contains two genes annotated as enoyl-ACP reductases, a FabI-type enoyl-ACP reductase and a FabK-type enoyl-ACP reductase. We report that expression of either of the two proteins restores growth of an Escherichia coli fabI temperature-sensitive mutant strain under nonpermissive conditions. In vitro assays demonstrate...
View next paperTHE STRUCTURAL BIOLOGY OF TYPE II FATTY ACID BIOSYNTHESIS