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Energization of the transport systems for arabinose and comparison with galactose transport in Escherichia coli.

ABSTRACT: 1. Strains of Escherichia coli were obtained containing either the AraE or the AraF transport system for arabinose. AraE+,AraF- strains effected energized accumulation and displayed an arabinose-evoked alkaline pH change indicative of arabinose-H+ symport. In contrast, AraE-,AraF+ strains accumulated arabinose but did not display H+ symport. 2. The ability of different sugars and their derivatives to elicit sugar-H+ symport in AraE+ strains was examined. Only L-arabinose and D-fucose were good substrates, and arabinose was the only inducer. 3. Membrane vesicles prepared from an AraE+,AraF+ strain accumulated the sugar, energized most efficiently by the respiratory substrates ascorbate + phenazine methosulphate. Addition of arabinose or fucose to an anaerobic suspension of membrane vesicles caused an alkaline pH change indicative or sugar-H+ symport on the membrane-bound transport system. 4. Kinetic studies and the effects of arsenate and uncoupling agents in intact cells and membrane vesicles gave further evidence that AraE is a low-affinity membrane-bound sugar-H+ symport system and that AraF is a binding-protein-dependent high-affinity system that does not require a transmembrane protonmotive force for energization. 5. The interpretation of these results is that arabinose transport into E. coli is energized by an electrochemical gradient of protons (AraE system) or by phosphate bond energy (AraF system). 6. In batch cultures the rates of growth and carbon cell yields on arabinose were lower in AraE-,AraF+ strains than in AraE+,AraF- or AraE+,AraF+ strains. The AraF system was more susceptible to catabolite repression than was the AraE system. 7. The properties of the two transport systems for arabinose are compared with those of the genetically and biochemically distinct transport systems for galactose, GalP and MglP. It appears that AraE is analogous to GalP, and AraF to MglP.

SUBMITTER: Daruwalla KR

PROVIDER: S-EPMC1163584 | biostudies-other | 1981 Dec

REPOSITORIES: biostudies-other

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Project description:UnlabelledGlucose is known to inhibit the transport and metabolism of many sugars in Escherichia coli. This mechanism leads to its preferential consumption. Far less is known about the preferential utilization of nonglucose sugars in E. coli. Two exceptions are l-arabinose and d-xylose. Previous studies have shown that l-arabinose inhibits d-xylose metabolism in Escherichia coli. This repression results from l-arabinose-bound AraC binding to the promoter of the d-xylose metabolic genes and inhibiting their expression. This mechanism, however, has not been explored in single cells. Both the l-arabinose and d-xylose utilization systems are known to exhibit a bimodal induction response to their cognate sugar, where mixed populations of cells either expressing the metabolic genes or not are observed at intermediate sugar concentrations. This suggests that l-arabinose can only inhibit d-xylose metabolism in l-arabinose-induced cells. To understand how cross talk between these systems affects their response, we investigated E. coli during growth on mixtures of l-arabinose and d-xylose at single-cell resolution. Our results showed that mixed, multimodal populations of l-arabinose- and d-xylose-induced cells occurred at intermediate sugar concentrations. We also found that d-xylose inhibited the expression of the l-arabinose metabolic genes and that this repression was due to XylR. These results demonstrate that a strict hierarchy does not exist between l-arabinose and d-xylose as previously thought. The results may also aid in the design of E. coli strains capable of simultaneous sugar consumption.ImportanceGlucose, d-xylose, and l-arabinose are the most abundant sugars in plant biomass. Developing efficient fermentation processes that convert these sugars into chemicals and fuels will require strains capable of coutilizing these sugars. Glucose has long been known to repress the expression of the l-arabinose and d-xylose metabolic genes in Escherichia coli. Recent studies found that l-arabinose also represses the expression of the d-xylose metabolic genes. In the present study, we found that d-xylose also represses the expression of the l-arabinose metabolic genes, leading to mixed populations of cells capable of utilizing l-arabinose and d-xylose. These results further our understanding of mixed-sugar utilization and may aid in strain design.

Project description:Cupriavidus necator strain A-04 has shown 16S rRNA gene identity to the well-known industrial strain C. necator H16. Nevertheless, the cell characteristics and polyhydroxyalkanoate (PHA) production ability of C. necator strain A-04 were different from those of C. necator H16. This study aimed to express PHA biosynthesis genes of C. necator strain A-04 in Escherichia coli via an arabinose-inducible expression system. In this study, the PHA biosynthesis operon of C. necator strain A-04, consisting of three genes encoding acetyl-CoA acetyltransferase (phaA A-04, 1182 bp, 40.6 kDa), acetoacetyl-CoA reductase (phaB A-04, 741 bp, 26.4 kDa) and PHB synthase Class I (phaC A-04, 1770 bp), was identified. Sequence analysis of the phaA A-04, phaB A-04, and phaCA-04 genes revealed that phaC A-04 was 99% similar to phaC H16 from C. necator H16. The difference in amino acid residue situated at position 122 of phaC A-04 was proline, whereas that of C. necator H16 was leucine. The intact phaCAB A-04 operon was cloned into the arabinose-inducible araBAD promoter and transformed into E. coli strains Top 10, JM109 and XL-1 blue. The results showed that optimal conditions obtained from shaken flask experiments yielded 6.1 ± 1.1 g/L cell dry mass (CDM), a PHB content of 93.3 ± 0.9% (w/w) and a productivity of 0.24 g/(L⋅h), whereas the wild-type C. necator strain A-04 accumulated 78% (w/w) PHB with a productivity of 0.09 g/(L⋅h). Finally, for the scaled-up studies, fed-batch cultivations by pH-stat control in a 5-L fermenter of E. coli strains XL1-Blue harboring pBAD/Thio-TOPO-phaCAB A-04 and pColdTF-phaCAB A-04 in MR or LB medium, leading to a PHB production of 31.4 ± 0.9 g/L at 54 h with a PHB content of 83.0 ± 3.8% (w/w), a CDM of 37.8 ± 1.2 g/L, a Y P/S value of 0.39 g PHB/g glucose and a productivity of 0.6 g PHB/(L⋅h) using pColdTF-phaCAB A-04 in MR medium. In addition, PHB production was 29.0 ± 1.1 g/L with 60.2 ± 2.3% PHB content in the CDM of 53.1 ± 1.0 g/L, a Y P/S value of 0.21 g PHB/g glucose and a productivity of 0.4 g PHB/(L⋅h) using pBAD/Thio-TOPO-phaCAB A-04 in LB medium. Thus, a relatively high PHB concentration and productivity were achieved, which demonstrated the possibility of industrial production of PHB.

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Energization of the transport systems for arabinose and comparison with galactose transport in Escherichia coli.

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