Genetic analysis of wax ester and triacylglycerol biosynthesis in Acinetobacter calcoaceticus strain...

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" Genetic analysis of wax ester and triacylglycerol biosynthesis in Acinetobacter calcoaceticus strain BD413 "
S. E. Reiser

Document Type	:	Latin Dissertation
Language of Document	:	English
Record Number	:	1113358
Doc. No	:	TLpq304252517
Main Entry	:	S. E. Reiser
Title & Author	:	Genetic analysis of wax ester and triacylglycerol biosynthesis in Acinetobacter calcoaceticus strain BD413\ S. E. Reiser
College	:	Michigan State University
Date	:	1996
student score	:	1996
Degree	:	Ph.D.
Page No	:	194
Abstract	:	The phenomena I have investigated is the accumulation of wax esters and triacylglycerol in the form of intracellular inclusions in the gram negative aerobic bacterium Acinetobacter calcoaceticus strain BD413. This strain of A. calcoaceticus accumulates both wax esters and triacylglycerol as a means of carbon storage when it undergoes nutrient starvation. Mutants of strain BD413 were induced using both chemical and transposon mutagenesis methods. By screening colonies for wax accumulation by staining with the lipophilic dye, Sudan black B, followed by TLC, a total of 8 transposon mutants and 21 chemically induced mutants were isolated. These mutants were separated into 3 general categories: waxtag, waxtag and waxtag. Nutritional supplementation experiments on the waxtag mutant, Wow15, indicated that this mutants genetic lesion was an inability to catalyze the conversion of acyl-CoA to fatty aldehyde. Complementation of this mutant with a cosmid from a cosmid genomic library allowed the identification of an open reading frame encoding the wow15 gene. The open reading frame shared considerable similarity to an open reading frame described in Mycobacterium tuberculosis called ORF2. It is believed that ORF2 acts as a usd\betausd-keto reductase involved in mycolic acid biosynthesis based on its similarity to a known usd\betausd-keto reductase. By expressing the identified open reading frame from A. calcoaceticus in E. coli, and assaying for the encoded protein's activity, the genes enzymatic activity was identified. The gene product was observed to catalyze the formation of fatty alcohol from acyl-CoA via a fatty aldehyde intermediate. The isolated gene has been named acr1 for acyl-CoA reductase. A second aspect of the research described herein concerns mutants isolated following transposon mutagenesis with mini-Tn10PttKm. Flanking sequence surrounding a transposon insertion in strain 11-C7, a waxtag mutant, was cloned by inverse PCR (IPCR). DNA sequencing of this DNA allowed the identification of an open reading frame that shares considerable homology to glnE, glutamate-ammonia-ligase adenylyltransferase, from E. coli. This gene is involved in the regulation of glutamine synthetase (GS), an important enzyme involved in amino acid biosynthesis. Examination of the open reading frames found near the disrupted glnE gene indicated that one of these had significant homology to a branched-chain-amino-acid transaminase and a third open reading frame that did not share homology to anything in GenBank, release 92.0. It seems likely that the putative mutation in this line disrupts the ability of the mutant to respond properly to nitrogen concentrations surrounding it.
Subject	:	Biochemistry
	:	Biochemistry
	:	Biological sciences
	:	Microbiology
	:	Molecular biology
	:	Pure sciences