Cross-induction of the L-fucose system by L-rhamnose in Escherichia coli.

Dissimilation of L-fucose as a carbon and energy source by Escherichia coli involves a permease, an isomerase, a kinase, and an aldolase encoded by the fuc regulon at minute 60.2. Utilization of L-rhamnose involves a similar set of proteins encoded by the rha operon at minute 87.7. Both pathways lead to the formation of L-lactaldehyde and dihydroxyacetone phosphate. A common NAD-linked oxidoreductase encoded by fucO serves to reduce L-lactaldehyde to L-1,2-propanediol under anaerobic growth conditions, irrespective of whether the aldehyde is derived from fucose or rhamnose. In this study it was shown that anaerobic growth on rhamnose induces expression of not only the fucO gene but also the entire fuc regulon. Rhamnose is unable to induce the fuc genes in mutants defective in rhaA (encoding L-rhamnose isomerase), rhaB (encoding L-rhamnulose kinase), rhaD (encoding L-rhamnulose 1-phosphate aldolase), rhaR (encoding the positive regulator for the rha structural genes), or fucR (encoding the positive for the fuc regulon). Thus, cross-induction of the L-fucose enzymes by rhamnose requires formation of L-lactaldehyde; either the aldehyde itself or the L-fuculose 1-phosphate (known to be an effector) formed from it then interacts with the fucR-encoded protein to induce the fuc regulon.     

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.

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 the fuc 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-controlling region shared by the operons fucAO 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 of fucAO and noninducibility of fucPIK. The DNA of the fucAO operon and a part of the adjacent fucP was sequenced.     

A mutant crp allele that differentially activates the operons of the fuc regulon in Escherichia coli.

L-Fucose is used by Escherichia coli through an inducible pathway mediated by a fucP-encoded permease, a fucI-encoded isomerase, a fucK-encoded kinase, and a fucA-encoded aldolase. The adolase catalyzes the formation of dihydroxyacetone phosphate and L-lactaldehyde. Anaerobically, lactaldehyde is converted by a fucO-encoded oxidoreductase to L-1,2-propanediol, which is excreted. The fuc genes belong to a regulon comprising four linked operons: fucO, fucA, fucPIK, and fucR. The positive regulator encoded by fucR responds to fuculose 1-phosphate as the effector. Mutants serially selected for aerobic growth on propanediol became constitutive in fucO and fucA [fucO(Con) fucA(Con)], but noninducible in fucPIK [fucPIK(Non)]. An external suppressor mutation that restored growth on fucose caused constitutive expression of fucPIK. Results from this study indicate that this suppressor mutation occurred in crp, which encodes the cyclic AMP-binding (or receptor) protein. When the suppressor allele (crp-201) was transduced into wild-type strains, the recipient became fucose negative and fucose sensitive (with glycerol as the carbon and energy source) because of impaired expression of fucA. The fucPIK operon became hyperinducible. The growth rate on maltose was significantly reduced, but growth on L-rhamnose, D-galactose, L-arabinose, glycerol, or glycerol 3-phosphate was close to normal. Lysogenization of fuc+ crp-201 cells by a lambda bacteriophage bearing crp+ restored normal growth ability on fucose. In contrast, lysogenization of [fucO(Con)fucA(Con)fucPIK(Non)crp-201] cells by the same phage retarded their growth on fucose.     

Constitutive activation of L-fucose genes by an unlinked mutation in Escherichia coli.

Wild-type Escherichia coli cannot grow on L-1,2-propanediol; mutants that can do so have increased basal activity of an NAD-linked L-1,2-propanediol oxidoreductase. This enzyme belongs to the L-fucose system and functions normally as L-lactaldehyde reductase during fermentation of the methylpentose. In wild-type cells, the activity of this enzyme is fully induced only anaerobically. Continued aerobic selection for mutants with an improved growth rate on L-1,2-propanediol inevitably leads to full constitutive expression of the oxidoreductase activity. When this occurs, L-fuculose 1-phosphate aldolase concomitantly becomes constitutive, whereas L-fucose permease, L-fucose isomerase, and L-fuculose kinase become noninducible. It is shown in this study that the noninducibility of the three proteins can be changed by two different kinds of suppressor mutations: one mapping external to and the other within the fuc gene cluster. Both mutations result in constitutive synthesis of the permease, the isomerase, and the kinase, without affecting synthesis of the oxidoreductase and the aldolase. Since expression of the fuc structural genes is activated by a protein specified by the regulator gene fucR, and since all the known genes of the fuc system are clustered at minute 60.2 of the chromosome, the external gene in which the suppressor mutation can occur probably has an unrelated function in the wild-type strain. The internal suppressor mutation might be either in fucR or in the promoter region of the genes encoding the permease, the isomerase, and the kinase, if these genes belong to the same operon.     

Molecular cloning of the Escherichia coli B L-fucose-D-arabinose gene cluster.

To metabolize the uncommon pentose D-arabinose, enteric bacteria often recruit the enzymes of the L-fucose pathway by a regulatory mutation. However, Escherichia coli B can grow on D-arabinose without the requirement of a mutation, using some of the L-fucose enzymes and a D-ribulokinase that is distinct from the L-fuculokinase of the L-fucose pathway. To study this naturally occurring D-arabinose pathway, we cloned and partially characterized the E. coli B L-fucose-D-arabinose gene cluster and compared it with the L-fucose gene cluster of E. coli K-12. The order of the fucA, -P, -I, and -K genes was the same in the two E. coli strains. However, the E. coli B gene cluster contained a 5.2-kb segment located between the fucA and fucP genes that was not present in E. coli K-12. This segment carried the darK gene, which encodes the D-ribulokinase needed for growth on D-arabinose by E. coli B. The darK gene was not homologous with any of the L-fucose genes or with chromosomal DNA from other D-arabinose-utilizing bacteria. D-Ribulokinase and L-fuculokinase were purified to apparent homogeneity and partially characterized. The molecular weights, substrate specificities, and kinetic parameters of these two enzymes were very dissimilar, which together with DNA hybridization analysis, suggested that these enzymes are not related. D-Arabinose metabolism by E. coli B appears to be the result of acquisitive evolution, but the source of the darK gene has not been determined.     

Analysis of lambda insertions in the fucose utilization region of Escherichia coli K-12: use of lambda fuc and lambda argA transducing bacteriophages to partially order the fucose utilization genes

Escherichia coli K-12 strains have deletions for the normal lambda integration site were lysogenized with bacteriophage lambda at a site within the L-fucose utilization system (fuc). The frequency of lambda integration at this site is approximately 2 X 10(-8) to 5 X 10(-7). Studies of the lytic properties of these strains indicated very infrequent cell lysis with a relatively low phage burst size. Transductional ability of the phage lysates was found to be normal, comparable to that found in conventional low-frequency transducing lysates. Two major classes of transducing phage were found. One carried the markers argA and fucA (a fucose utilization gene of unknown function previously referred to as fuc-1) and the gene for D-arabinose utilization (dar+). The other carried only fucC, the gene specifying L-fuculose-1-phosphate aldolase. A minor class of phage was found that carried fucA, but not argA or dar+. Upon consideration of the transductional nature of these phage classes, we are proposing that the gene order for the L-fucose utilization system is dar, fucA, (lambda), fucC.     

Biosynthesis and catabolism of 6-deoxy L-talitol by Klebsiella aerogenes mutants.

A mutant strain of Klebsiella aerogenes was constructed and, when incubated anaerobically with L-fucose and glycerol, synthesized and excreted a novel methyl pentitol, 6-deoxy L-talitol. The mutant was constitutive for the synthesis of L-fucose isomerase but unable to synthesize L-fuculokinase activity. Thus, it could convert the L-fucose to L-fuculose but was incapable of phosphorylating L-fuculose to L-fuculose 1-phosphate. The mutant was also constitutive for the synthesis of ribitol dehydrogenase, and in the presence of sufficient reducing power this latter enzyme catalyzed the reduction of the L-fuculose to 6-deoxy L-talitol. The reducing equivalents required for this reaction were generated by the oxidation of glycerol to dihydroxyacetone with an anaerobic glycerol dehydrogenase. The parent strain of K. aerogenes was unable to utilize the purified 6-deoxy L-talitol as a sole source of carbon and energy for growth; however, mutant could be isolated which had gained this ability. Such mutants were found to be constitutive for the synthesis of ribitol dehydrogenase and were thus capable of oxidizing 6-deoxy L-talitol to L-fuculose. Further metabolism of L-fuculose was shown by mutant analysis to be mediated by the enzymes of the L-fucose catabolic pathway.     

Loss of aldehyde dehydrogenase in an Escherichia coli mutant selected for growth on the rare sugar L-galactose.

Escherichia coli K-12 converts L-fucose to dihydroxyacetone phosphate (C-1 to C-3) and L-lactaldehyde (C-4 to C-6) by a pathway specified by the fuc regulon. Aerobically, L-lactaldehyde serves as a carbon and energy source by the action of an aldehyde dehydrogenase of broad specificity; the product, L-lactate, is then converted to pyruvate. Anaerobically, L-lactaldehyde serves as an electron acceptor to regenerate NAD from NADH by the action of an oxidoreductase; the reduced product, L-12-propanediol, is excreted. A strain selected for growth on L-galactose (a structural analog of L-fucose) acquired a broadened inducer specificity because of an altered fucR gene encoding the activator protein for the fuc regulon (Y. Zhu and E. C. C. Lin, J. Mol. Evol. 23:259-266, 1986). In this study, a second mutation that abolished aldehyde dehydrogenase activity was discovered. The L-fucose pathway converts L-galactose to dihydroxyacetone phosphate and L-glyceraldehyde. Aldehyde dehydrogenase then converts L-glyceraldehyde to L-glycerate, which is toxic. Loss of the dehydrogenase averts the toxicity during growth on L-galactose, but reduces by one-half the aerobic growth yield on L-fucose. When mutant cells induced in the L-fucose system were incubated with radioactive L-fucose, accumulation of radioactivity occurred if the substrate was labeled at C-1 but not if it was labeled C-6. Complete aerobic utilization of carbons 4 through 6 of L-fucose depends not only on an adequate activity of aldehyde dehydrogenase to trap L-lactaldehyde as its anionic acid but also on the lack of L-1,2-propanediol oxidoreductase activity, which converts L-lactaldehyde to a readily excreted alcohol.     

The gene of an archaeal alpha-L-fucosidase is expressed by translational frameshifting

The standard rules of genetic translational decoding are altered in specific genes by different events that are globally termed recoding. In Archaea recoding has been unequivocally determined so far only for termination codon readthrough events. We study here the mechanism of expression of a gene encoding for a alpha-l-fucosidase from the archaeon Sulfolobus solfataricus (fucA1), which is split in two open reading frames separated by a -1 frameshifting. The expression in Escherichia coli of the wild-type split gene led to the production by frameshifting of full-length polypeptides with an efficiency of 5%. Mutations in the regulatory site where the shift takes place demonstrate that the expression in vivo occurs in a programmed way. Further, we identify a full-length product of fucA1 in S.solfataricus extracts, which translate this gene in vitro by following programmed -1 frameshifting. This is the first experimental demonstration that this kind of recoding is present in Archaea.     

Sequence heterogeneity of the ten rRNA operons in Clostridium perfringens

We have cloned and sequenced rRNA operons of Clostridium perfringens strain 13 and analyzed the sequence structure in view of the phylogenesis. The organism had ten copies of rRNA operons all of that comprised of 16S, 23S and 5S rDNAs except for one operon. The operons clustered around the origin of replication, ranging within one-third of the whole genome sequence as it is arranged in a circle. Seven operons were transcribed in clockwise direction, and the remaining three were transcribed in counter clockwise direction assuming that the gyrA was transcribed in clockwise direction. Two of the counter clockwise operons contained tRNA(Ile) genes between the 16S and 23S rDNAs, and the other had a tRNA(Ile) genes between the 16S and 23S rDNAs and a tRNA(Asn) gene in the place of the 5S rDNA. Microheterogeneity was found within the rRNA structural genes and spacer regions. The length of each 16S, 23S and 5S rDNA were almost identical among the ten operons, however, the intergenic spacer region of 16S-23S and 23S-5S were variable in the length depending on loci of the rRNA operons on the chromosome. Nucleotide sequences of the helix 19, helix 19a, helix 20 and helix 21 of 23S rDNA were divergent and the diversity appeared to be correlated with the loci of the rRNA operons on the chromosome.