A Sulfoglycolytic Entner-Doudoroff Pathway in Rhizobium leguminosarum bv. trifolii SRDI565

Abstract

Rhizobia are nitrogen-fixing bacteria that engage in symbiotic relationships with plant hosts but can also persist as free-living bacteria in the soil and rhizosphere. Here, we show that free-living Rhizobium leguminosarum SRDI565 can grow on the sulfosugar sulfoquinovose (SQ) or the related glycoside SQ-glycerol using a sulfoglycolytic Entner-Doudoroff (sulfo-ED) pathway, resulting in production of sulfolactate (SL) as the major metabolic end product. Comparative proteomics supports the involvement of a sulfo-ED operon encoding an ABC transporter, sulfo-ED enzymes, and an SL exporter. Consistent with an oligotrophic lifestyle, proteomics data revealed little change in expression of the sulfo-ED proteins during growth on SQ versus mannitol, a result confirmed through biochemical assay of sulfoquinovosidase activity in cell lysates. Metabolomics analysis showed that growth on SQ involves gluconeogenesis to satisfy metabolic requirements for glucose-6-phosphate and fructose-6-phosphate. Metabolomics analysis also revealed the unexpected production of small amounts of sulfofructose and 2,3-dihydroxypropanesulfonate, which are proposed to arise from promiscuous activities of the glycolytic enzyme phosphoglucose isomerase and a nonspecific aldehyde reductase, respectively. The discovery of a rhizobium isolate with the ability to degrade SQ builds our knowledge of how these important symbiotic bacteria persist within soil. IMPORTANCE Sulfonate sulfur is a major form of organic sulfur in soils but requires biomineralization before it can be utilized by plants. Very little is known about the biochemical processes used to mobilize sulfonate sulfur. We show that a rhizobial isolate from soil, Rhizobium leguminosarum SRDI565, possesses the ability to degrade the abundant phototroph-derived carbohydrate sulfonate SQ through a sulfoglycolytic Entner-Doudoroff pathway. Proteomics and metabolomics demonstrated the utilization of this pathway during growth on SQ and provided evidence for gluconeogenesis. Unexpectedly, off-cycle sulfoglycolytic species were also detected, pointing to the complexity of metabolic processes within cells under conditions of sulfoglycolysis. Thus, rhizobial metabolism of the abundant sulfosugar SQ may contribute to persistence of the bacteria in the soil and to mobilization of sulfur in the pedosphere.

Environmental and Intestinal Phylum Firmicutes Bacteria Metabolize the Plant Sugar Sulfoquinovose via a 6-Deoxy-6-sulfofructose Transaldolase Pathway

“… Denger et al., 2014 ; Felux et al., 2015 ) for cleaving SQG to SQ and glycerol ( Speciale et al., 2016 ), and every SQ-degrading strain examined thus far by proteomics strongly co-induced this sulfoquinovosidase (Figures 2 and 6 B; refs. Denger et al., 2014 ; Felux et al., 2015 ; Burrichter et al., 2018 ; Li et al., 2020 ). Furthermore, E. coli is able to grow with SQG ( Abayakoon et al., 2018 , and our own observation) as well as P. putida SQ1, B. aryabhattai SOS1 (our own observations), and SQ-degrading Rhizobium leguminosarum SRDI565 ( Li et al., 2020 ).…”

“… Denger et al., 2014 ; Felux et al., 2015 ; Burrichter et al., 2018 ; Li et al., 2020 ). Furthermore, E. coli is able to grow with SQG ( Abayakoon et al., 2018 , and our own observation) as well as P. putida SQ1, B. aryabhattai SOS1 (our own observations), and SQ-degrading Rhizobium leguminosarum SRDI565 ( Li et al., 2020 ). We suggest that for the SFT pathway, the glycerol acquired from SQG cleavage may directly feed into generation of the acceptor molecule GAP for the SF transaldolase, through glycerol kinase and glycerol-3-phosphate dehydrogenase, some of which are co-encoded in the predicted gene clusters (see Figure 5 ).…”

The Molecular Basis of Sulfosugar Selectivity in Sulfoglycolysis

“…The orthogonal packing of eight β strands within the lid domains of the two subunits form a 'β-clasp' dimerization motif, a distinctive feature previously seen in members of the ribokinase superfamily that includes tagatose-6-phosphate kinase (TPK) and the minor isozyme from the glycolytic pathway, F6P kinase (PfkB). [14][15][16] Close hydrophobic contacts and reciprocal interactions are observed at the dimer interface where residues from the opposite subunit in a βclasp motif protrude into the active site, which is likely to be crucial for both catalysis and structural stability. An ordered sequential Bi-Bi mechanism is reported for ribokinases, with sugar binding interactions driving domain closure that precedes ATP binding.…”

“…Sulfoglycolytic cells must therefore utilize the traditional gluconeogenesis pathway to satisfy demands for cell wall biosynthesis and the PPP. 16 Since these pathways must operate in tandem, it is presumably important that the sulfoglycolytic enzymes exhibit high selectivity for sulfosugar intermediates and limited activity on the analogous glycolytic intermediates to ensure the chemical unidirectionality of each pathway and avoid futile cycles.…”

The Molecular Basis of Sulfosugar Selectivity in Sulfoglycolysis

“…The orthogonal packing of eight β strands within the lid domains of the two subunits form a 'βclasp' dimerization motif, a distinctive feature previously seen in members of the ribokinase superfamily that includes tagatose-6-phosphate kinase (TPK) and the minor isozyme from glycolytic pathway, F6P kinase (PfkB). [14][15][16] Close hydrophobic contacts and reciprocal interactions are observed at the dimer interface where residues from the opposite subunit in a β-clasp motif protrude into the active site, which is likely to be crucial for both catalysis and structural stability. (Figure 4e,f).…”