Mannuronan C‐5 epimerases and cellular differentiation of Azotobacter vinelandii

Abstract

Differentiation in Azotobacter vinelandii involves the encystment of the vegetative cell under adverse environmental circumstances and the germination of the resting cell into the vegetative state when growth conditions are satisfactory again. Morphologically, the encystment process involves the development of a protective coat around the resting cell. This coat partly consists of multiple layers of alginate, which is a copolymer of beta-D-mannuronic acid (M) and alpha-L-guluronic acid (G). Alginate contributes to coat rigidity by virtue of a high content of GG blocks. Such block structures are generated through a family of mannuronan C-5 epimerases that convert M to G after polymerization. Results from immunodetection and light microscopy, using stains that distinguish between different cyst components and types, indicate a correlation between cyst coat organization and the amount and appearance of mannuronan C-5 epimerases in the extracellular medium and attached to the cells. Specific roles of individual members of the epimerase family are indicated. Calcium and magnesium ions appear to have different roles in the structural organization of the cyst coat. Also reported is a new gene sharing strong sequence homology with parts of the epimerase-encoded R-modules. This gene is located within the epimerase gene cluster of Azotobacter vinelandii.

Identification and Characterization of anAzotobacter vinelandiiType I Secretion System Responsible for Export of the AlgE-Type Mannuronan C-5-Epimerases

“…In the 48-h sample one major band could be detected in the wild-type samples, while in the 176-h sample several MEs were detected. These results are consistent with those obtained in previously reported similar experiments (28). In contrast, no MEs were detected in the eex mutant samples.…”

Sphagnan - a pectin-like polymer isolated fromSphagnummoss can inhibit the growth of some typical food spoilage and food poisoning bacteria by lowering the pH

“…However, in the case of calcium it is often not essential for the growth of many micro-organisms, and this mechanism would only apply to micro-organisms that have a strict growth requirement for calcium. It was previously claimed that strains of A. vinelandii are dependent on calcium for growth (Norris and Jensen 1958), but it has later been shown that some strains of this species, including strain E isolated from base poor soil, are able to grow in the absence of calcium (Vermani et al 1995(Vermani et al , 1997Høidal et al 2000). Finally, in our test system it is not expected that polyelectrolyte complexation with positively charged amines at low pH makes a contribution towards the antimicrobial effect of sphagnan because the ionic strength in the low-buffering medium is probably too high (0AE17 mol l )1 from NaCl) for such interactions to be thermodynamically favourable (Ballance et al 2008).…”

Mannuronan C-5 Epimerases Suited for Tailoring of Specific Alginate Structures Obtained by High-Throughput Screening of an Epimerase Mutant Library

“…Random mutations were introduced into the recombined library by either using the obtained mannuronan was selectively enriched to 59% with 13 C at carbon position C-1 15,27 .…”

“…To reduce the viscosity of the alginate samples prior to NMR measurements the samples were depolymerised by mild acid hydrolysis to a final average DP n ~30 residues Protein concentrations were determined with a Nanodrop ND-1000 to ensure similar enzyme concentration in the epimerisation reaction. 500 µl 13 C-1-enriched polyM stock solution was preheated in the NMR instrument and 1D proton and carbon spectra were recorded to ensure that the sample has not undergone any degradation or contamination prior to the timeresolved NMR experiment. 50 µl enzyme solution was added to preheated substrate and mixed by inverting the sample 2-3 times.…”

“…The polymer is first produced as mannuronan (polyM) and subsequently the block structure results from the activity of a family of mannuronan C-5 epimerases catalysing non-random epimerization of β-D-mannuronic acid to α-L-guluronic acid at the polymer level 9 . Azotobacter vinelandii encodes a family of seven secreted mannuronan C-5 epimerases, AlgE1-AlgE7, involved in the cellular differentiation of the bacterium [10][11][12][13] . These enzymes display a modular structure being composed of one or two catalytic A-modules and from one to seven regulatory Rmodules.…”