The Azotobacter vinelandii AlgE mannuronan C‐5‐epimerase family is essential for the in vivo control of alginate monomer composition and for functional cyst formation

Abstract

The industrially widely used polysaccharide alginate is a co-polymer of beta-D-mannuronic acid and alpha-L-guluronic acid (G), and the G residues originate from a polymer-level epimerization process catalysed by mannuronan C-5-epimerases. In the genome of the alginate-producing bacterium Azotobacter vinelandii genes encoding one periplasmic (AlgG) and seven secreted such epimerases (AlgE1-7) have been identified. Here we report the generation of a strain (MS163171) in which all the algE genes were inactivated by deletion (algE1-4 and algE6-7) or interruption (algE5). Shake flask-grown MS163171 produced a polymer containing less than 2% G (algG still active), while wild-type alginates contained 25% G. Interestingly, addition of proteases to the MS163171 growth medium resulted in a strong increase in the chain lengths of the alginates produced. MS163171 was found to be unable to form functional cysts, which is a desiccation-resistant differentiated form developed by A. vinelandii under certain environmental conditions. We also generated mutants carrying interruptions in each separate algE gene, and a strain containing algE5 only. Studies of these mutants indicated that single algE gene inactivations, with the exception of algE3, did not affect the fractional G content much. However, for all strains tested the alginate composition varied somewhat as a response to the growth conditions.

Mapping global effects of the anti-sigma factor MucA in Pseudomonas fluorescens SBW25 through genome-scale metabolic modeling

“…Alginate in the medium was quantified as described previously [11]. Since the alginate quantification assay operates on deacetylated alginate, acetylation had to be determined separately.…”

“…An important industrial polymer, alginate is produced in bulk from seaweeds, but bacterial alginates have attracted significant interest over the last years due to the presence of alginate modifying enzymes, i.e. epimerases [11], in the producing organisms. These enzymes, taken together with the possibility to produce very homogeneous alginate from liquid bacterial cultivations, allow precise manipulation of the relative content and sequence of mannuronic and guluronic acid residues in the alginate, which influences the physico-chemical and immunological properties of the purified polymer significantly [12].…”

Genome Sequence ofAzotobacter vinelandii, an Obligate Aerobe Specialized To Support Diverse Anaerobic Metabolic Processes

“…Genes similar to these regulators, however, have not been found in the A. vinelandii genome. Alginate, in addition to its role in respiratory protection, is a major component of the cyst capsule, and alginate with consecutive guluronic residues is essential for the formation of mature cysts (12,80). During encystment, A. vinelandii was shown to incorporate phenolic lipids (alkylresorcinols and alkylpyrones) into the membrane.…”

A small heat-shock protein (Hsp20) regulated by RpoS is essential for cyst desiccation resistance in Azotobacter vinelandii

“…previously to be involved in encystment (Steigedal et al, 2008); a calcium-binding protein encoded within an alginate epimerases gene cluster; two proteins involved in polysaccharide export and biosynthesis; an haemerythrin HHE cation-binding protein, which was detected with two different molecular masses and pIs, suggesting that it undergoes a post-translational modification; and a hypothetical protein. Due to its abundance in cysts, Hsp20 was chosen for further analysis, to investigate its regulation by RpoS and its role in the encystment process.…”