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A Bifunctional UDP-Sugar 4-Epimerase Supports Biosynthesis of Multiple Cell Surface Polysaccharides in Sinorhizobium meliloti.

ABSTRACT: Sinorhizobium meliloti produces multiple extracellular glycans, including among others, lipopolysaccharides (LPS), and the exopolysaccharides (EPS) succinoglycan (SG) and galactoglucan (GG). These polysaccharides serve cell protective roles. Furthermore, SG and GG promote the interaction of S. meliloti with its host Medicago sativa in root nodule symbiosis. ExoB has been suggested to be the sole enzyme catalyzing synthesis of UDP-galactose in S. meliloti (A. M. Buendia, B. Enenkel, R. Köplin, K. Niehaus, et al. Mol Microbiol 5:1519-1530, 1991, https://doi.org/10.1111/j.1365-2958.1991.tb00799.x). Accordingly, exoB mutants were previously found to be affected in the synthesis of the galactose-containing glycans LPS, SG, and GG and consequently, in symbiosis. Here, we report that the S. meliloti Rm2011 uxs1-uxe-apsS-apsH1-apsE-apsH2 (SMb20458-63) gene cluster directs biosynthesis of an arabinose-containing polysaccharide (APS), which contributes to biofilm formation, and is solely or mainly composed of arabinose. Uxe has previously been identified as UDP-xylose 4-epimerase. Collectively, our data from mutational and overexpression analyses of the APS biosynthesis genes and in vitro enzymatic assays indicate that Uxe functions as UDP-xylose 4- and UDP-glucose 4-epimerase catalyzing UDP-xylose/UDP-arabinose and UDP-glucose/UDP-galactose interconversions, respectively. Overexpression of uxe suppressed the phenotypes of an exoB mutant, evidencing that Uxe can functionally replace ExoB. We suggest that under conditions stimulating expression of the APS biosynthesis operon, Uxe contributes to the synthesis of multiple glycans and thereby to cell protection, biofilm formation, and symbiosis. Furthermore, we show that the C2H2 zinc finger transcriptional regulator MucR counteracts the previously reported CuxR-c-di-GMP-mediated activation of the APS biosynthesis operon. This integrates the c-di-GMP-dependent control of APS production into the opposing regulation of EPS biosynthesis and swimming motility in S. meliloti IMPORTANCE Bacterial extracellular polysaccharides serve important cell protective, structural, and signaling roles. They have particularly attracted attention as adhesives and matrix components promoting biofilm formation, which significantly contributes to resistance against antibiotics. In the root nodule symbiosis between rhizobia and leguminous plants, extracellular polysaccharides have a signaling function. UDP-sugar 4-epimerases are important enzymes in the synthesis of the activated sugar substrates, which are frequently shared between multiple polysaccharide biosynthesis pathways. Thus, these enzymes are potential targets to interfere with these pathways. Our finding of a bifunctional UDP-sugar 4-epimerase in Sinorhizobium meliloti generally advances the knowledge of substrate promiscuity of such enzymes and specifically of the biosynthesis of extracellular polysaccharides involved in biofilm formation and symbiosis in this alphaproteobacterium.

SUBMITTER: Schaper S

PROVIDER: S-EPMC6482925 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Leishmaniases are a set of tropical and sub-tropical diseases caused by protozoan parasites of the genus Leishmania whose severity ranges from self-healing cutaneous lesions to fatal visceral infections. Leishmania parasites synthesise a wide array of cell surface and secreted glycoconjugates that play important roles in infection. These glycoconjugates are particularly abundant in the promastigote form and known to be essential for establishment of infection in the insect midgut and effective transmission to the mammalian host. Since they are rich in galactose, their biosynthesis requires an ample supply of UDP-galactose. This nucleotide-sugar arises from epimerisation of UDP-glucose but also from an uncharacterised galactose salvage pathway. In this study, we evaluated the role of the newly characterised UDP-sugar pyrophosphorylase (USP) of Leishmania major in UDP-galactose biosynthesis. Upon deletion of the USP encoding gene, L. major lost the ability to synthesise UDP-galactose from galactose-1-phosphate but its ability to convert glucose-1-phosphate into UDP-glucose was fully maintained. Thus USP plays a role in UDP-galactose activation but does not significantly contribute to the de novo synthesis of UDP-glucose. Accordingly, USP was shown to be dispensable for growth and glycoconjugate biosynthesis under standard growth conditions. However, in a mutant seriously impaired in the de novo synthesis of UDP-galactose (due to deficiency of the UDP-glucose pyrophosphorylase) addition of extracellular galactose increased biosynthesis of the cell surface lipophosphoglycan. Thus under restrictive conditions, such as those encountered by Leishmania in its natural habitat, galactose salvage by USP may play a substantial role in biosynthesis of the UDP-galactose pool. We hypothesise that USP recycles galactose from the blood meal within the midgut of the insect for synthesis of the promastigote glycocalyx and thereby contributes to successful vector infection.

Project description:Sinorhizobium meliloti is a soil-dwelling alphaproteobacterium that engages in a nitrogen-fixing root nodule symbiosis with leguminous plants. Cell surface polysaccharides are important both for adapting to stresses in the soil and for the development of an effective symbiotic interaction. Among the polysaccharides characterized to date, the acidic exopolysaccharides I (EPS-I; succinoglycan) and II (EPS-II; galactoglucan) are particularly important for protection from abiotic stresses, biofilm formation, root colonization, and infection of plant roots. Previous genetic screens discovered mutants with impaired EPS production, allowing the delineation of EPS biosynthetic pathways. Here we report on a genetic screen to isolate mutants with mucoid colonial morphologies that suggest EPS overproduction. Screening with Tn5-110, which allows the recovery of both null and upregulation mutants, yielded 47 mucoid mutants, most of which overproduce EPS-I; among the 30 unique genes and intergenic regions identified, 14 have not been associated with EPS production previously. We identified a new protein-coding gene, emmD, which may be involved in the regulation of EPS-I production as part of the EmmABC three-component regulatory circuit. We also identified a mutant defective in EPS-I production, motility, and symbiosis, where Tn5-110 was not responsible for the mutant phenotypes; these phenotypes result from a missense mutation in rpoA corresponding to the domain of the RNA polymerase alpha subunit known to interact with transcription regulators.IMPORTANCE The alphaproteobacterium Sinorhizobium meliloti converts dinitrogen to ammonium while inhabiting specialized plant organs termed root nodules. The transformation of S. meliloti from a free-living soil bacterium to a nitrogen-fixing plant symbiont is a complex developmental process requiring close interaction between the two partners. As the interface between the bacterium and its environment, the S. meliloti cell surface plays a critical role in adaptation to varied soil environments and in interaction with plant hosts. We isolated and characterized S. meliloti mutants with increased production of exopolysaccharides, key cell surface components. Our diverse set of mutants suggests roles for exopolysaccharide production in growth, metabolism, cell division, envelope homeostasis, biofilm formation, stress response, motility, and symbiosis.

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A Bifunctional UDP-Sugar 4-Epimerase Supports Biosynthesis of Multiple Cell Surface Polysaccharides in Sinorhizobium meliloti.

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