The transport of mannitol in Sinorhizobium meliloti is carried out by a broad-substrate polyol transporter SmoEFGK and is affected by the ability to transport and metabolize fructose

Abstract

The smo locus (sorbitol mannitol oxidation) is found on the chromosome of S. meliloti's tripartite genome. Mutations at the smo locus reduce or abolish the ability of the bacterium to grow on several carbon sources, including sorbitol, mannitol, galactitol, d-arabitol and maltitol. The contribution of the smo locus to the metabolism of these compounds has not been previously investigated. Genetic complementation of mutant strains revealed that smoS is responsible for growth on sorbitol and galactitol, while mtlK restores growth on mannitol and d-arabitol. Dehydrogenase assays demonstrate that SmoS and MtlK are NAD⁺-dependent dehydrogenases catalysing the oxidation of their specific substrates. Transport experiments using a radiolabeled substrate indicate that sorbitol, mannitol and d-arabitol are primarily transported into the cell by the ABC transporter encoded by smoEFGK. Additionally, it was found that a mutation in either frcK, which is found in an operon that encodes the fructose ABC transporter, or a mutation in frk, which encodes fructose kinase, leads to the induction of mannitol transport.

Keywords: ABC transporter; Rhizobium; metabolism; polyol.

Fig. 1.

Locus diagrams of the smo (a) and frc (b) loci. Locus tags appear above the genes and annotation information appears below. Black wedges represent the sites of insertional mutagenesis. Strain designations are listed above these indicators. Note that SMc04251, which is not at the smo locus within the Rm1021 genome, is included in (a) because it was annotated as smoM implying that it was related to sorbitol/mannitol utilization.

Fig. 2.

Non-denaturing PAGE gel of sorbitol inducible dehydrogenase activity. Extracts from wild-type (lane 1), SMc01500 (lane 2), SMc01500 with smoS in trans (lane 3), and SMc01500 with mtlK in trans (lane 4) were grown in glycerol and induced with sorbitol for 6 h. Gels were stained for dehydrogenase activity using an assay reagent containing p-nitroblue tetrazolium, phenazine methosulfate and NAD⁺. The substrate added is listed below each panel.

Fig. 3.

Transport rates of ¹⁴C-mannitol. (a) Rm1021 (wild-type) and SRmD495 (smoK::Tn5-B20) grown in mannitol and glucose, 2 µM labelled mannitol was used to initiate the assay. (b) Uptake of ¹⁴C-mannitol by Rm1021 in competition with unlabeled substrates. Cells were grown on mannitol as a sole carbon source. 2 µM labelled mannitol was competed against either 2 µM or 10 µM unlabeled substrate. Accumulation of label is shown in nmole/mg protein/min. Data are expressed as the mean±sd of three independent replicates.

Fig. 4.

Induction of the smo locus measured by GFP relative fluorescence (RF). Strains were grown in LB broth either with, or without sorbitol (sbt) as described in Methods. GFP was read at wavelengths of 485 (excitation) and 510 (emission). RF=(fluorescence-background)/OD600. Strains used are mtlK::gfp, FL4643; smoC::Nm, mtlK::gfp, SRmD642. Rm1021 was used to control for background fluorescence. The data is shown as the average ±sd of three biological replications.

Fig. 5.

Kinase activity of Frk. Partially purified Frk was assayed for kinase activity using either fructose (fru) or glucose (glu) as substrates. Activity was coupled to the production of NAD⁺ by PK and LDH and was measured at 340 nm for 2 min in a buffer containing 60 mM HEPES pH 7.5, 6 mM MgCl2, 3 mM ATP, 3 mM PEP, 0.3 mM NADH and 1/50 vol PK/LDH mix. Activity is plotted as μmol NAD⁺ produced per milligram protein over time. Data points represent the average ±sd of three biological replications.

Fig. 6.

Transport rates of ¹⁴C-fructose and ¹⁴C-mannitol. (a) Rm1021 in competition with unlabeled substrates. Cells were grown on fructose as a sole carbon source. 2 µM labelled mannitol was competed against either 2 µM or 10 µM unlabeled substrate. (b) Uptake of ¹⁴C-fructose or ¹⁴C-mannitol by various transport mutants. Cells were grown in fructose and glucose, 2 µM radiolabelled substrate was used to initiate the assay. Accumulation of label is shown in nmole/mg protein/min. Data are expressed as the mean±sd of three independent replicates.

Fig. 7.

Pathways for the metabolism of sorbitol, mannitol and d-arabitol. Sorbitol and mannitol are oxidized to fructose by SmoS and MtlK, respectively. Fructose generated may be mutorotated by FrcS before phosphorylation into fructose-6-phosphate by Frk and isomerization by Pgi. d-arabitol becomes d-xylulose, catalysed by MtlK, which is subsequently phosphorylated by XylB into d-xylulose-5-phosphate.