Journal of Bacteriology, February 2002, p . 861-863, Vol . 184, No . 3

NADPH-Dependent L-Sorbose Reductase Is Responsible for L-Sorbose Assimilation in Gluconobacter suboxydans IFO 3291

Masako Shinjoh,^* Masaaki Tazoe, and Tatsuo Hoshino

Department of Applied Microbiology, Nippon Roche Research Center, Kamakura 247, Japan

Received 22 June 2001/ Accepted 31 October 2001

 
Plasmid pSUP202 and plasmid pSUP2021 (9) were used as a suicide vector and a vector for Tn5 mutagenesis in Gluconobacter strains . Recombinant DNA technique and conjugal mating were done as previously reported (8) . No . 5 medium containing 80 g of D-sorbitol per liter (2) and SL-SCM medium containing (per liter) 3 g of yeast extract, 3 g of beef extract, 3 g of corn steep liquor, 10 g of peptone, 1 g of urea, 1 g of KH₂PO₄, 0.2 g of MgSO₄ · 7H₂O, 1 g of CaCO₃ (production grade), and 20 g of D-sorbitol were used for cultivating the Gluconobacter strains .

A Tn5 mutant defective in L-sorbose reductase activity was obtained from a derivative of G . melanogenus IFO 3293 through Tn5 mutagenesis with P1::Tn5 (3) and was designated strain 26-9A . The mutant was selected as a D-sorbitol nonproducer from L-sorbose under a resting cell system (2) and was confirmed to not grow on L-sorbose (No . 5 medium) . We confirmed the SR deficiency of strain 26-9A by a photometric enzyme assay (11), with strain IFO 3293 as the positive control . Strains IFO 3293 and 26-9A showed 0.20 and <0.01 U of SR activity per mg of cytosol protein, respectively .

After confirming that the Tn5 insertion in 26-9A caused SR deficiency (below 0.01 U/mg of cytosol protein) by reconstructing the Tn5 mutant with the DNA fragment containing Tn5, we determined the nucleotide sequence of the Tn5-inserted region . The region encoded a polypeptide belonging to the mannitol dehydrogenase superfamily including mannitol-2-dehydrogenase of Rhodobacter sphaeroides (accession number P33216 [5]), mannonate oxidoreductase of Escherichia coli (P39160), and mannitol-1-phosphate 5-dehydrogenases of, for example, Enterococcus faecalis (P27543) . The mannitol dehydrogenase (MDH) (EC 1.1.1.67) is a mannitol-fructose oxidoreductase . The SR enzyme of the Gluconobacter strain is a sorbitol-sorbose/mannitol-fructose oxidoreductase (11) . The amino acid sequences deduced from the SR nucleotide sequences around the Tn5 insertion point were aligned with those belonging to the MDH superfamily (data not shown) . In the SR sequence, the MDH signature of PS00974 in the protein motif database PROSITE was found . The corresponding sequence from strain 26-9A, FPNGMVDRITP, and the other region showing a high homology, MTITEGGY, were selected for designing the set of primers for PCR .

We cloned the partial SR gene of G . suboxydans IFO 3291 (ca . 300 bp) through PCR amplification with the primers 5'-ATGAC(C/G)AT(C/T)AC(C/G)GA(A/G)GG(A/C/T)GG(A/C/T)TA and 5'-CG(A/G)TC(A/C/G)ACCAT(A/G/T)CC(A/G)TT(A/G/C)GGGAA and then obtained the complete gene with the PCR product as the probe in an 8.0-kb EcoRV fragment . The complete nucleotide sequence of the open reading frame (ORF) was 1,455 bp (accession number AB063188), encoding 485 amino acids; the calculated M_r of 53,541 agrees with that of the purified enzyme (60,000 [11]) . Computer analysis of the sequence including the upstream and downstream regions showed that there are two ORFs, DnaJ-like protein and ferredoxin, downstream in the direction opposite to that of the SR gene (as shown in Fig . 2), suggesting that there are no ORFs included in an operon with the SR gene .

 
Homology search with the nucleotide sequence of the SR gene was done with the Blastx program; recently published sequences of genes encoding NADP-SLDH of G . oxydans strain G624 (AB028937[6]) and NAD-MDH sequences, including that from Pseudomonas aeruginosa (AE004660 [10]), were found in addition to that from R . sphaeroides . The amino acid sequence of SR showed identities of 84.5, 42.6, and 39.6%, respective to the order of the nucleotide sequences described above . SR from G . suboxydans IFO 3291 should be an ortholog of NADP-SLDH from G . oxydans G624 .

An SR gene disruptant was constructed with pSUP202-SR::Km, which has a Km^r gene cassette from pUC4K (Amersham Pharmacia Biotech, Uppsala, Sweden) as an EcoRI fragment in an EcoRI site of the cloned SR gene . The gene disruption was confirmed by Southern blot hybridization and was designated SR3 . In a spectrophotometric assay, strains SR3 and IFO 3291 showed SR activities of 0.02 (negligible) and 0.92 U/mg of cytosol protein, respectively . L-Sorbose assimilation and growth profiles of strains SR3 and IFO 3291 were evaluated with the SL-SCM medium (Table 1) . Both converted D-sorbitol nearly stoichiometrically in 9 to 12 h; IFO 3291 consumed L-sorbose accompanying further cell growth, while SR3 neither consumed it nor grew further . The reason why SR3 consumed D-sorbitol faster, resulting in faster growth and faster production of L-sorbose than IFO 3291, is unknown . Sugar and sugar alcohol assimilation abilities of both strains were further confirmed by using a minimal agar medium containing 2.5% L-sorbose, D-sorbitol, or D-fructose together with 1% Casamino Acids . Strain IFO 3291 grew on all the carbon sources, while SR3 grew on D-sorbitol and D-fructose but not on L-sorbose; this result agrees with the pathway shown in Fig . 1 .

 
A database search of MedLine with the keyword sorbose gave us one sorbose assimilation pathway via L-sorbose-1-phosphate in E . coli, Klebsiella pneumoniae, and Lactobacillus casei; there were no references describing L-sorbose assimilation via D-sorbitol, with the exception of studies by us and coworkers (2, 7, 11) . Adachi et al . (1) supposed from its enzymatic properties that the physiological role of NADPH-SR of G . melanogenus IFO 3294 would be assimilation of L-sorbose . In this report, we first genetically confirmed that SR triggers the L-sorbose assimilation via D-sorbitol, not via L-sorbose-1-phosphate . In addition, we revealed that the disruptant SR3 showed strong D-sorbitol dehydrogenase activity to produce L-sorbose comparable to that of strain IFO 3291 (Table 1), suggesting that SR does not function as a main D-sorbitol-oxidizing enzyme in vivo . This result is reasonable, because the pH of a cytosol is usually around 7, optimal for SR to function as a reductase rather than a dehydrogenase . Considering the existence of NADPH-dependent SR (a dimer with an M_r of 60,000) in G . melanogenus IFO 3294 (1), this SR could also exist in strains IFO 3291 and IFO 3293 and in their derivatives and could function weakly as the second pathway in assimilating L-sorbose for maintenance of life . On the other hand, NADP-SLDH of G . oxydans G624 (with a calculated M_r of 53,642) should be a synonym of SR of G . suboxydans IFO 3291 (with a calculated M_r of 53,541) . In summary, in vivo SR should mainly be responsible for L-sorbose assimilation, not for L-sorbose production . Since L-sorbose is hard for most microorganisms to assimilate, whereas D-sorbitol is easily assimilated, this deposit (D-sorbitol to L-sorbose) and withdrawal (L-sorbose to D-sorbitol) system of sugars and sugar alcohols is a clever strategy adopted by Gluconobacter strains to survive in mixed populations of the microbial world .

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