Constitutive activation of the fucAO operon and silencing of the divergently transcribed fucPIK operon by an IS5 element in Escherichia coli mutants selected for growth on L-1,2-propanediol

Abstract

L-1,2-Propanediol is an irretrievable end product of L-fucose fermentation by Escherichia coli. Selection for increased aerobic growth rate on propanediol results in the escalation of basal synthesis of the NAD+-linked oxidoreductase encoded by fucO, a member of thefuc regulon for the utilization of L-fucose. In general, when fucO becomes constitutively expressed, two other simultaneous changes occur: the fucA gene encoding fuculose-1-phosphate aldolase becomes constitutively expressed and the fucPIK operon encoding fucose permease, fucose isomerase, and fuculose kinase becomes noninducible. In the present study, we show that fucO and fucA form an operon which is divergently transcribed from the adjacent fucPIK operon. In propanediol-positive and fucose-negative mutants the cis-controliing region shared by the operonsfucAO and fucPIK is lengthened by 1.2 kilobases. DNA hybridization identified the insertion element to be IS5. This element, always oriented in the same direction with the left end (the BglII end) proximal to fucA, apparently causes constitutive expression offucAO and noninducibility of fucPIK. The DNA of the fucAO operon and a part of the adjacent fucP was sequenced.Following the discovery that a genetic derepression of ribitol dehydrogenase in Klebsiella pneumoniae conferred a growth ability on xylitol (27, 33), we tried to find other models of experimental evolution for assessing the importance of regulatory mutations in the acquisition of novel functions (for a review, see reference 30). During this search, we discovered that Escherichia coli can give rise to mutants that utilize L-1,2-propanediol aerobically as a sole source of carbon and energy. Serial selection on the compound resulted in the emergence of a mutant (ECL3) which grew on the novel carbon and energy source at a rate close to that on glycerol. This mutant produced constitutively an NAD+-linked oxidoreductase active on L-1,2-propanediol (46). The proximity of the locus specifying this enzyme activity and the fuc locus (at min 60) specifying the growth ability on L-fucose led us to ask whether there was a link between the metabolism of the two compounds. When the propanediol-positive mutant was tested with fucose as a carbon and energy source, growth no longer occurred (13).Previous work, together with our further studies (13), revealed the biochemical connection between the two metabolic pathways (Fig. 1). The dissimilation of fucose by wild-type E. coli requires the sequential action of fucose permease (encoded byfucP), fucose isomerase (encoded by fucf), fucose kinase (encoded by fucK), and fuculose-lphosphate aldolase (encoded by fucA). The last enzyme catalyzes the formation of dihydroxyacetone phosphate and L-lactaldehyde. Under anaerobic conditions, the aldehyde is reduced to L-1,2-propanediol by an NAD+-linked oxidoreductase (encoded by fucO) and excreted into the medium. Under aerobic conditions, the aldehyde is oxidized to Llactate and then to pyruvate which enters the general metabolic pool. The inducer of the fucose syst...

Evolution of an Escherichia coli Protein with Increased Resistance to Oxidative Stress

“…It is remarkable that the relatively conservative hydrophobic amino acid substitutions, Ile 7 3 Leu and/or Leu 8 3 Val, near the NAD-binding sequence Gly 15 -Arg-Gly-Ala-Val-Gly 20 of FucO (15,16,47) could confer significant protective effects against MCO damage. This resistance, however, is achieved at a price of decrease in protein stability.…”

“…Strain ECL421 was isolated from another wild-type clone after a similar selection for 140 doublings (32). In both mutants, an IS5 insertion occurred at precisely the same location in the region between the two diverging operons, causing constitutive activation of fucAO and noninducibility of fucPIK (15,33). 2 Since strains ECL3 and ECL421 were isolated after prolonged selection on propanediol, mutations in the coding region of fucO conferring resistance to oxidative damage might also have taken place.…”

“…To determine the exact nature of the mutations and their phenotypic consequences, the fucO alleles of the wild-type and the two mutant strains were amplified by polymerase chain reaction for gene cloning and sequencing. For comparison, the fucO alleles of three other derivative strains were included in the procedure as follows: ECL430, selected from ECL3 for improved propanediol-scavenging power (35); ECL56, a Fuc ϩ revertant of ECL3 generated by an unlinked mutation (33,36); and ECL459, the corresponding Fuc ϩ revertant of ECL421 (15). A single nucleotide substitution, an A to C change converting the N-terminal Ile 7 to Leu 7 , was found in the fucO of strains ECL3, ECL56, and ECL430.…”

“…, and ECL459 -The oligonucleotide primers 5Ј-CGGATCCG-GCATTATCACATCAG and 5Ј-CGAATTCAAGAGTAATTTCGTAAAGC flanking the coding region of fucO (15,16) were synthesized and used to amplify by polymerase chain reaction the gene of each strain. A sample of each amplified product was digested with restriction enzymes to give five overlapping fragments that were then subcloned into pBluescript vectors (Stratagene, La Jolla, CA).…”

Sequencing and characterization of the sdaB gene from Escherichia coli K‐12

“…In the sequence presented here, we see that the putative stop site of sduB is followed by 921 bp of intergenic DNA, after which we find the coding region of the fucose operon, with the fuc0 gene reading in the opposite direction from the sduB gene ( Fig. 1 ; Chen et al, 1989). Since the fucose operon was mapped at 60.2min (Bachmann, 1990;Chakrabarti et al, 1984), this confirms the position determined by conventional mapping and suggests that there is no protein-coding information between sduB and fuc.…”