Regulation of fatty acid degradation in Escherichia coli: isolation and characterization of strains bearing insertion and temperature-sensitive mutations in gene fadR

Abstract

Transposon TnlO was used to mutagenize the fadR gene in Escherichia coli. Mutants bearing fadR::TnlO insertion mutations were found to (i) utilize the noninducing fatty acid decanoate as sole carbon source, (ii) f8-oxidize fatty acids at constitutive rates, and (iii) contain constitutive levels of the five key ,B-oxidative enzymes. These characteristics were identical to those observed in spontaneous fadR mutants. The constitutive phenotype presented by the fadR: :TnlO mutants was shown to be genetically linked to the associated transposon-encoded drug resistance. These results suggest that the fadR gene product exerts negative control over the fatty acid degradative regulon. The fadR gene of E. coli has been mapped through the use of transposon-mediated fadR insertion mutations. The fadR locus is at 25.5 min on the revised map and cotransduces with purB, hemA, and trp. Three-factor conjugational and transductional crosses indicate that the order of loci in this region of the chromosome is purB-fadR-hemA-trp. Spontaneous fadR mutants were found to map at the same location. Strains that exhibit alterations in the control of the fad regulon in response to changes in temperature were also isolated and characterized. These fadR(Ts) mutants were constitutive for the fad enzymes at elevated temperatures and inducible for these activities at low temperatures. The fadR(Ts) mutations also map at the fadR locus. These results strongly suggest that the fadR gene product is a repressor protein. Wild-type Escherichia coli K-12 is able to grow on long-chain (12 or more carbons) but not on short-(C4 to C5) or medium-(C6 to Cll) chain fatty acids as a sole carbon source (21, 22, 26). Growth in media containing long-chain fatty acids induces the coordinate synthesis of at least five key enzymes involved in the f8-oxidation of fatty acids (21, 22, 26). Mutants unable to grow on fatty acids of any chain length have been obtained and shown to harbor lesions in structural genes for the fi-oxidative enzymes (21, 22) as well as for genes involved in fatty acid activation (11, 15) and transport (11, 19, 20). These fatty acid degradation (fad) lesions map at no fewer than four separate locations on the E. coli chromosome (15, 19, 21). Strains harboring a lesion in one of the structural genes, fadD, lack fatty acyl-coenzyme A synthetase activity and cannot be induced for the other fi-oxidative enzymes (15). These results led Klein et al. (15) to propose that long-chain acyl-coenzyme As serve to induce the fatty acid degradative (fad) system in E. coli. Medium-chain fatty acids can serve as substrates for the ,8-oxidative enzymes, but cannot

Induction of acyl coenzyme A synthetase and hydroxyacyl coenzyme A dehydrogenase during fatty acid degradation in Neurospora crassa

“…In the course of analyzing the effects of fatty acid composition on the properties of the mitochondrial glycerophosphate dehydrogenase (4) of Neurospora, we noted that both wild-type and Cel-strains possess the ability to grow when long-chain fatty acids are present in the medium as the sole carbon and energy source, as earlier suggested from the fatty acid supplementation of Ace-mutants (11). These observations indicated that Neurospora possesses a a-oxidation pathway with some features similar to those previously described for the fatty acid degradation (FAD) system in Escherichia coli (13,14,16). In this report, we describe the intracellular localization and inducibility of the fatty acid oxidation enzymes acyl coenzyme A (CoA) synthetase (EC 6 Growth of cells aid assay of FAD enzymes.…”

Transcriptional regulation of lipid homeostasis in mycobacteria

“…The fact that MabR is the regulator that fine-tunes expression of the genes encoding the FAS-II and possibly the FAS-I systems would also support the essential role of this protein. Interestingly, the essentiality of mabR for M. smegmatis growth is in sharp contrast to the other FA regulatory proteins characterized in bacteria so far, since all of them have been shown to be non-essential for survival of the corresponding microorganism, including FasR of S. coelicolor (Simons et al, 1980;Schujman et al, 2003;Lu and Rock, 2006;Arabolaza et al, 2010).…”

Transcriptional regulation of fatty acid biosynthesis in mycobacteria

“…The fasR gene has been proposed to be essential in M. tuberculosis based on a combination of high density mutagenesis and deep sequencing analysis (Griffin et al, 2011). Interestingly, most of the fatty acid biosynthesis regulators described so far in bacteria are not essential for survival (Simons et al, 1980;Schujman et al, 2003;Lu and Rock, 2006). The only exception is MabR, a novel regulatory protein involved in mycolic acid biosynthesis regulation in mycobacteria (Salzman et al, 2010).…”