An improved medium for the direct, positive selection of tetracycline-sensitive clones from a population of tetracycline-resistant strains of Escherichia coli is described. Various genetic techniques have been developed requiring the excision of the tetracyclineresistant transposon TnlO from an insertion site
The structural organization and regulation of the genes involved in short-chain fatty acid degradation in Escherichia coli, referred to as the ato system, have been studied by a combination of classic genetic and recombinant DNA techniques. A plasmid containing a 6.2-kilobase region of the E. coli chromosome was able to complement mutations in the ato structural genes, atoA (acetyl-coenzyme A [CoA]:acetoacetyl [AA]-CoA transferase) and atoB (thiolase II), as well as mutations in the ato regulatory locus, atoC. Complementation studies performed with mutants defective in acetyl-CoA:AA-CoA transferase suggest that two loci, atoD and atoA, are required for the expression of functional AA-CoA transferase. The ato gene products were identified by in vitro transcription and translation and maxicell analysis as proteins of 48, 26.5, 26, and 42 kilodaltons for atoC, atoD, atoA, and atoB, respectively. In vitro and insertional mutagenesis of the ato hybrid plasmid indicated that the ato structural genes were arranged as an operon, with the order of transcription atoD-atoA-atoB. Although transcribed in the same direction as the atoDAB operon, the atoC gene appeared to use a promoter which was distinct from that used by the atoDAB operon. A AatoC plasmid expressed the atoD, atoA, and atoB gene products only in strains containing a functional atoC gene. Although the exact mechanism of control was not evident from these studies, the data suggest that the atoC gene product is an activator which is required for the synthesis or activation of the atoDAB-encoded enzymes.The four-carbon P-keto short-chain fatty acid (SCFA) acetoacetate (AA) can be used by wild-type Escherichia coli as a sole carbon and energy source (7,16,22). The degradation of AA to acetyl coenzyme A (acetyl-CoA) is a two-step reaction (Fig. 1). The activation of AA to acetoacetyl-CoA is catalyzed by acetyl-CoA:AA-CoA transferase and is followed by the subsequent cleavage of AACoA to acetyl-CoA by thiolase II. These enzymes are highly inducible in the presence of AA and are specific for SCFA substrates (22).Earlier studies performed by Pauli and Overath identified the loci responsible for AA degradation as atoA (which encodes AA-CoA transferase) and atoB (which encodes thiolase II) (22). These structural genes are closely linked and are located at the min 47 region of the revised E. coli chromosomal map (22). Studies by Duncombe and Frerman (10) and Sramek and Frerman (30) For BUT or VAL to be metabolized, the ATO enzymes are required as well as the fatty acid degradative enzymes encoded by the fadE and fadB structural genes (Fig. 1).
The expression of the glyoxylate shunt enzymes is required for growth of Escherichia coli on acetate or fatty acids as a sole carbon source. The genes for the two unique enzymes of the glyoxylate shunt, aceA and aceB, are located at 90 min on the E. coli K-12 genetic map. Polar mutations in the aceB gene eliminate aceA gene function, suggesting that these genes constitute an operon and the direction of transcription is from aceB to aceA. Mu d (Ap lac) fusions with the aceA gene have been constructed to study the regulation of the ace operon. Expression of the ace operon is under the transcriptional control of two genes: the iclR gene, which maps near the ace operon, and the fadR gene, which maps at 25 min, and is also involved in the regulation of the fatty acid degradation (fad) regulon. Merodiploid studies demonstrated that both the iclR and fadR genes regulate the glyoxylate shunt in a trans-dominant manner.
A new locus (fadL) that is required for the utilization of long-chain fatty acids has been mapped and partially characterized in an Escherichia coli mutant. The fadL locus has been mapped at 50 min on the chromosome. A mutant bearing a defect in this locus cannot utilize long-chain fatty acids as a sole carbon source. Derivatives of this mutant that can grow on decanoate (termed fadR) are capable of growth on medium-chain but not long-chain fatty acids. It is believed that the fadL mutants is defective in the transport of long-chain fatty acids into the cell for the following reasons: (i) fadR fadL strains can oxidize in vivo decanoate but not oleate; (ii) neither fadL nor fadR fadL strains can incorporate oleate into their membrane lipids; (iii) the activity of the acyl-CoA synthetase (EC 6.2.1.3) in fadR fadL strains is comparable to the acyl-CoA synthetase activity in fadR fadL+ strains; and (iv) in vitro extracts from fadR fadL+ strains. If the above hypothesis is correct, the uptake of long-chain fatty acids by E. coli requires at least two gene products.
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
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