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  1. Free, publicly-accessible full text available March 1, 2023
  2. Brun, Yves V. (Ed.)
    ABSTRACT The alphaproteobacterium Sinorhizobium meliloti secretes two acidic exopolysaccharides (EPSs), succinoglycan (EPSI) and galactoglucan (EPSII), which differentially enable it to adapt to a changing environment. Succinoglycan is essential for invasion of plant hosts and, thus, for the formation of nitrogen-fixing root nodules. Galactoglucan is critical for population-based behaviors such as swarming and biofilm formation and can facilitate invasion in the absence of succinoglycan on some host plants. The biosynthesis of galactoglucan is not as completely understood as that of succinoglycan. We devised a pipeline to identify putative pyruvyltransferase and acetyltransferase genes, construct genomic deletions in strains engineered to produce either succinoglycan or galactoglucan, and analyze EPS from mutant bacterial strains. EPS samples were examined by 13 C cross-polarization magic-angle spinning (CPMAS) solid-state nuclear magnetic resonance (NMR). CPMAS NMR is uniquely suited to defining chemical composition in complex samples and enables the detection and quantification of distinct EPS functional groups. Galactoglucan was isolated from mutant strains with deletions in five candidate acyl/acetyltransferase genes ( exoZ , exoH , SMb20810 , SMb21188 , and SMa1016 ) and a putative pyruvyltransferase ( wgaE or SMb21322 ). Most samples were similar in composition to wild-type EPSII by CPMAS NMR analysis. However, galactoglucan produced frommore »a strain lacking wgaE exhibited a significant reduction in pyruvylation. Pyruvylation was restored through the ectopic expression of plasmid-borne wgaE . Our work has thus identified WgaE as a galactoglucan pyruvyltransferase. This exemplifies how the systematic combination of genetic analyses and solid-state NMR detection is a rapid means to identify genes responsible for modification of rhizobial exopolysaccharides. IMPORTANCE Nitrogen-fixing bacteria are crucial for geochemical cycles and global nitrogen nutrition. Symbioses between legumes and rhizobial bacteria establish root nodules, where bacteria convert dinitrogen to ammonia for plant utilization. Secreted exopolysaccharides (EPSs) produced by Sinorhizobium meliloti (succinoglycan and galactoglucan) play important roles in soil and plant environments. The biosynthesis of galactoglucan is not as well characterized as that of succinoglycan. We employed solid-state nuclear magnetic resonance (NMR) to examine intact EPS from wild-type and mutant S. meliloti strains. NMR analysis of EPS isolated from a wgaE gene mutant revealed a novel pyruvyltransferase that modifies galactoglucan. Few EPS pyruvyltransferases have been characterized. Our work provides insight into the biosynthesis of an important S. meliloti EPS and expands the knowledge of enzymes that modify polysaccharides.« less
  3. ABSTRACT The ability of vancomycin-arginine (V-r) to extend the spectrum of activity of glycopeptides to Gram-negative bacteria was investigated. Its MIC towards Escherichia coli , including β-lactamase expressing Ambler classes A, B, and D, was 8 to 16 μg/ml. Addition of 8 times the MIC of V-r to E. coli was acutely bactericidal and associated with a low frequency of resistance (<2.32 × 10 −10 ). In vivo , V-r markedly reduced E. coli burden by >7 log 10 CFU/g in a thigh muscle model. These data warrant further development of V-r in combatting E. coli , including resistant forms.