A novel exopolysaccharide pathway from a freshwater Sphingomonas isolate

Abstract

Bacteria embellish their cell envelopes with a variety of specialized polysaccharides. Biosynthesis pathways for these glycans are complex, and final products vary greatly in their chemical structures, physical properties and biological activities. This tremendous diversity comes from the ability to arrange complex pools of monosaccharide building blocks into polymers with many possible linkage configurations. Due to the complex chemistry of bacterial glycans, very few biosynthetic pathways have been defined in detail. To better understand the breadth of polysaccharide production in nature we isolated a bacterium from Lake Michigan called Sphingomonas sp. LM7 that is proficient in exopolysaccharide (EPS) production. We identified genes that contribute to EPS biosynthesis in LM7 by screening a transposon mutant library for colonies displaying altered colony morphology. A gene cluster was identified that appears to encode a complete wzy/wzx-dependent polysaccharide assembly pathway. Deleting individual genes in this cluster caused a non-mucoid phenotype and a corresponding loss of EPS secretion, confirming that LM7 assembles a novel wzy/wzx-dependent polysaccharide. We extracted EPS from LM7 cultures and showed that it contains a linear chain of 3- and 4- linked glucose, galactose, and glucuronic acid residues. Finally, we found that the EPS pathway we identified diverges from those of adhesive polysaccharides such as the holdfast that are conserved in higher Alphaproteobacteria. Our approach of characterizing complete biosynthetic pathways holds promise for engineering of polysaccharides with valuable properties.

Keywords: Sphingomonas; biopolymer; polysaccharide; wzy.

Figure 1:. Isolation of Sphingomonas sp. LM7

(A) Sphingomonas sp. LM7 growing on agar plates. The isolate grows as yellow, mucoid colonies. (B) Phylogenetic tree showing select isolates from the Sphingomonadales order of Alphaproteobacteria. The neighbor-joining tree was constructed from 16S rDNA sequences aligned with ClustalX. Bootstrapping values are indicated at the relevant nodes. * indicates canonical sphingan producing strains. ^# highlights the non-canonical sphingan called sanxan produced by Sphingomonas sanxanigenes NX02. Note that the sanxan biosynthesis genes appear distinct from the sphingan or promonan genes (56).

Figure 2:. Identification of two polysaccharide biosynthesis clusters in LM7

(A) Screening for transposon insertion mutants with altered colony morphology. Mutagenized cells were plated on agar plates. Under these conditions, the majority of colonies show the mucoid morphology. The blue carat points to a non-mucoid mutant. (B) Map of the LM7 genome showing the locations of the two gene clusters that influence colony morphology. (C) Phenotypes of ΔwzyA and ΔwzyB mutants. The top image shows growth of LM7 strains on solid medium containing Congo Red dye. Wild type and the ΔwzyB mutant appear mucoid and react with the Congo Red dye, while the ΔwzyA mutant does not. The middle image shows an EPS extraction. Wild-type and ΔwzyB cultures produce secreted matrix. ΔwzyA cells do not secrete this matrix. The bottom image shows density gradient centrifugation of LM7 strains. The ΔwzyB mutant displays increased cell density relative to wild type and ΔwzyA cells.

Figure 3:. The promonan biosynthesis cluster

(A) Map of the prm cluster. Open-reading frames are colored by predicted function. Purple: monosaccharide incorporation; green: assembly/secretion; grey: polysaccharide modification. (B) Morphologies of individual prm mutants on solid medium supplemented with Congo Red. (C) Complementation of prmH, prmJ and prmQ deletion mutants. Non-mucoid phenotypes (ΔprmH and ΔprmJ) and the rugose phenotype (ΔprmQ) can be reverted by introducing the relevant gene in trans.

Figure 4:. Conservation of polysaccharide biosynthesis genes in Alphaproteobacteria

(A) Schematic of Alphaproteobacterial phylogeny showing the relationship among isolates with putative polysaccharide adhesin pathways. (B) Phenotypes associated with holdfast production. fWGA stained shows the loss of holdfast production in the C. crescentus ΔhfsE ΔpssY ΔpssZ and ΔhfsJ mutants. Crystal violet (CV) staining of cultures grown in microtiter plates shows the loss of surface adhesion in the ΔhfsE ΔpssY ΔpssZ and ΔhfsJ mutants. (C) Inferred activities for initiation and subsequent glycosyltransferase enzymes in four polysaccharide pathways. (D) CV staining assay testing the effect of introducing PHPT genes from various bacteria into the C. crescentus ΔhfsE ΔpssY ΔpssZ mutant. All tested genes appear capable of converting UPP to UPP-Glc. (E) CV staining assay testing the effect of introducing wecG-family GT genes from various bacteria into the C. crescentus ΔhfsJ mutant. wecGs from Brucella ovis, Rhizobium leguminosarum and Phaeobacter inhibens can carry of the HfsJ reaction, while wecG from Escherichia coli (negative control) and prmH from Sphingomonas sp. LM7 cannot.