L-sorbose reductase and its transcriptional regulator involved in L-sorbose utilization of Gluconobacter frateurii

Abstract

Upstream of the gene for flavin adenine dinucleotide (FAD)-dependent D-sorbitol dehydrogenase (SLDH), sldSLC, a putative transcriptional regulator was found in Gluconobacter frateurii THD32 (NBRC 101656). In this study, the whole sboR gene and the adjacent gene, sboA, were cloned and analyzed. sboR mutation did not affect FAD-SLDH activity in the membrane fractions. The SboA enzyme expressed and purified from an Escherichia coli transformant showed NADPH-dependent L-sorbose reductase (NADPH-SR) activity, and the enzyme was different from the NADPH-SR previously reported for Gluconobacter suboxydans IFO 3291 in molecular size and amino acid sequence. A mutant defective in sboA showed significantly reduced growth on L-sorbose, indicating that the SboA enzyme is required for efficient growth on L-sorbose. The sboR mutant grew on L-sorbose even better than the wild-type strain did, and higher NADPH-SR activity was detected in cytoplasm fractions. Reverse transcription-PCR experiments indicated that sboRA comprises an operon. These data suggest that sboR is involved in the repression of sboA, but not in the induction of sldSLC, on D-sorbitol and that another activator is required for the induction of these genes by D-sorbitol or L-sorbose.

FIG. 1.

Schematic representation of gene organization, including that of sboRA, in the DNA fragment obtained from G. frateurii THD32. ORFs are represented by arrows indicating their orientation. ORF1 is not complete at its N terminus.

FIG. 2.

SDS-PAGE analysis of purified SboA. Fifteen micrograms of purified protein was applied to a 12.5% (wt/vol) polyacrylamide gel and stained with Coomassie brilliant blue R-250.

FIG. 3.

Growth and enzyme properties of wild-type and mutant strains. (a) Growth profiles of wild-type (diamonds), sboR disruptant (squares), and sboA disruptant (triangles) strains grown on different C sources, including 1% d-sorbitol (left), 0.5% l-sorbose (middle), and 1% glycerol (right). (b) Enzyme activities in soluble fractions from each mutant strain grown on different C sources. NADPH-SR activity (hatched bars) was measured in 50 mM KPB (pH 6.0), and NAD-SLDH activity (gray bars) was measured in 100 mM Tris-HCl (pH 9.0). All assays were performed in triplicate, and the means and standard deviations are shown.

FIG. 4.

Comparison of growth, l-sorbose production, and enzyme activities of wild-type and mutant strains. (a) Wild-type (closed diamonds), sboR mutant (closed squares), sboA mutant (closed triangles), sboR-complemented (closed circles), and sboA-complemented (open squares) strains were cultured on 1% d-sorbitol, and their growth was monitored with a Klett-Summerson photoelectric colorimeter with a red filter. (b) Oxidation products from d-sorbitol in the culture supernatant were measured by the resorcinol test. (c) NADPH-SR activity (hatched bars) and NAD-SLDH activity (gray bars) in the cytoplasmic fractions were measured.

FIG. 5.

Transcriptional organization of sboRA genes and examination of promoter activity in the intergenic region. (a) Schematic representation of the orientation of genes, indicated by large arrows. Thin arrows show the position and direction of the primers used, and the thick bars indicate the regions amplified by the primer pairs. Also shown is the gene region carried by pSA19 for the complementation of sboA in the same or opposite direction from that of the lac promoter, resulting in plasmid pSABsboA or pSAEsboA, respectively. (b) Agarose gel analysis of RT-PCR amplification products. The primer sets used for RT-PCR were sboR2F and sboR2R (lanes 1 and 2), sboAF and sboAR (lane 3), and sboRAF and sboRAR (lanes 4 and 5). Lanes 1 and 5 contain samples in which reverse transcriptase was omitted. (c) NADPH-SR activity in soluble fractions from wild-type and mutant strains grown on d-sorbitol medium (hatched bars) and glycerol (gray bars).