An exopolysaccharide pathway from a freshwater Sphingomonas isolate.

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. As part of an initiative to characterize novel polysaccharide biosynthesis enzymes, 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 the role of this gene cluster in polysaccharide production. We extracted EPS from LM7 cultures and determined that it contains a linear chain of 3- and 4-linked glucose, galactose, and glucuronic acid residues. Finally, we show that the EPS pathway in Sphingomonas sp. LM7 diverges from that of sphingan-family EPSs and adhesive polysaccharides such as the holdfast that are present in other Alphaproteobacteria. Our approach of characterizing complete biosynthetic pathways holds promise for engineering polysaccharides with valuable properties. IMPORTANCE: Bacteria produce complex polysaccharides that serve a range of biological functions. These polymers often have properties that make them attractive for industrial applications, but they remain woefully underutilized. In this work, we studied a novel polysaccharide called promonan that is produced by Sphingomonas sp. LM7, a bacterium we isolated from Lake Michigan. We extracted promonan from LM7 cultures and identified which sugars are present in the polymer. We also identified the genes responsible for polysaccharide production. Comparing the promonan genes to those of other bacteria showed that promonan is distinct from previously characterized polysaccharides. We conclude by discussing how the promonan pathway could be used to produce new polysaccharides through genetic engineering.

The multifaceted role of c-di-AMP signaling in the regulation of Porphyromonas gingivalis lipopolysaccharide structure and function.

Background: This study unveils the intricate functional association between cyclic di-3',5'-adenylic acid (c-di-AMP) signaling, cellular bioenergetics, and the regulation of lipopolysaccharide (LPS) profile in Porphyromonas gingivalis, a Gram-negative obligate anaerobe considered as a keystone pathogen involved in the pathogenesis of chronic periodontitis. Previous research has identified variations in P. gingivalis LPS profile as a major virulence factor, yet the underlying mechanism of its modulation has remained elusive. Methods: We employed a comprehensive methodological approach, combining two mutants exhibiting varying levels of c-di-AMP compared to the wild type, alongside an optimized analytical methodology that combines conventional mass spectrometry techniques with a novel approach known as FLATn. Results: We demonstrate that c-di-AMP acts as a metabolic nexus, connecting bioenergetic status to nuanced shifts in fatty acid and glycosyl profiles within P. gingivalis LPS. Notably, the predicted regulator gene cdaR, serving as a potent regulator of c-di-AMP synthesis, was found essential for producing N-acetylgalactosamine and an unidentified glycolipid class associated with the LPS profile. Conclusion: The multifaceted roles of c-di-AMP in bacterial physiology are underscored, emphasizing its significance in orchestrating adaptive responses to stimuli. Furthermore, our findings illuminate the significance of LPS variations and c-di-AMP signaling in determining the biological activities and immunostimulatory potential of P. gingivalis LPS, promoting a pathoadaptive strategy. The study expands the understanding of c-di-AMP pathways in Gram-negative species, laying a foundation for future investigations into the mechanisms governing variations in LPS structure at the molecular level and their implications for host-pathogen interactions.

A novel exopolysaccharide pathway from a freshwater Sphingomonas isolate.

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.

Characterization of the need for galactofuranose during the Neurospora crassa life cycle.

Galactofuranose is a constituent of the cell walls of filamentous fungi. The galactofuranose can be found as a component of N-linked oligosaccharides, in O-linked oligosaccharides, in GPI-anchored galactomannan, and in free galactomannan. The Neurospora genome contains a single UDP-galactose mutase gene (ugm-1/NCU01824) and two UDP-galactofuranose translocases used to import UDP-galactofuranose into the lumen of the Golgi apparatus (ugt-1/NCU01826 and ugt-2/NCU01456). Our results demonstrate that loss of galactofuranose synthesis or its translocation into the lumen of the secretory pathway affects the morphology and growth rate of the vegetative hyphae, the production of conidia (asexual spores), and dramatically affects the sexual stages of the life cycle. In mutants that are unable to make galactofuranose or transport it into the lumen of the Golgi apparatus, ascospore development is aborted soon after fertilization and perithecium maturation is aborted prior to the formation of the neck and ostiole. The Neurospora genome contains three genes encoding possible galactofuranosyltransferases from the GT31 family of glycosyltransferases (gfs-1/NCU05878, gfs-2/NCU07762, and gfs-3/NCU02213) which might be involved in generating galactofuranose-containing oligosaccharide structures. Analysis of triple KO mutants in GT31 glycosyltransferases shows that these mutants have normal morphology, suggesting that these genes do not encode vital galactofuranosyltransferases.

Genetic Characterization of the Acidic and Neutral Glycosphingolipid Biosynthetic Pathways in Neurospora crassa.

Fungal glycosphingolipids (GSLs) are important membrane components which play a key role in vesicle trafficking. To assess the importance of GSLs in the fungal life cycle, we performed a mutant phenotypic study of the acidic and neutral GSL biosynthetic pathways in Neurospora crassa. GSL biosynthesis begins with two reactions leading up to the formation of dihydrosphingosine. The first of these reactions is catalyzed by serine palmitoyltransferase and generates 3-keto dihydrosphinganine. In N. crassa, this reaction is catalyzed by GSL-1 and GSL-2 and is required for viability. The second reaction is carried out by GSL-3, a 3-keto dihydrosphinoganine reductase to generate dihydrosphingosine, which is used for the synthesis of neutral and acidic GSLs. We found that deletion mutations in the acidic GSL pathway leading up to the formation of mannosylinositol-phosphoceramide are lethal, indicating that acidic GSLs are essential for viability in N. crassa. Once mannosylinositol-phosphoceramide is made, it is further modified by GSL-5, an inositol-phosphoceramide-B C26 hydroxylase, which adds a hydroxyl group to the amide-linked fatty acid. GSL-5 is not required for viability but gives a clear mutant phenotype affecting all stages of the life cycle. Our results show that the synthesis of mannosylinositol-phosphoceramide is required for viability and that the modification of the amide-linked fatty acid is important for acidic GSL functionality. We also examined the neutral GSL biosynthetic pathway and identified the presence of glucosylceramide. The deletion of neutral GSL biosynthetic genes affected hyphal morphology, vegetative growth rate, conidiation, and female development. Our results indicate that the synthesis of neutral GSLs is essential for normal growth and development of N. crassa.