Alginate Biosynthesis Factories in Pseudomonas fluorescens: Localization and Correlation with Alginate Production Level

Abstract

Pseudomonas fluorescens is able to produce the medically and industrially important exopolysaccharide alginate. The proteins involved in alginate biosynthesis and secretion form a multiprotein complex spanning the inner and outer membranes. In the present study, we developed a method by which the porin AlgE was detected by immunogold labeling and transmission electron microscopy. Localization of the AlgE protein was found to depend on the presence of other proteins in the multiprotein complex. No correlation was found between the number of alginate factories and the alginate production level, nor were the numbers of these factories affected in an algC mutant that is unable to produce the precursor needed for alginate biosynthesis. Precursor availability and growth phase thus seem to be the main determinants for the alginate production rate in our strain. Clustering analysis demonstrated that the alginate multiprotein complexes were not distributed randomly over the entire outer cell membrane surface.

Identification of a New Phosphatase Enzyme Potentially Involved in the Sugar Phosphate Stress Response in Pseudomonas fluorescens

“…For alginate-producing strains it was found that strain Pf201ΔalgC⌬zwf-1::TnKB60 grew more slowly than its parent, Pf201ΔalgC::TnKB60, but the difference was less pronounced than that between the corresponding nonalginate-producing strains NCIMB10525 and NCIMB10525Δzwf-1. We suggested that the observed growth defect was partly due to accumulation of G6P or F6P and that alginate production would drain away some of these hexose phosphates, thus diminishing the stress (19).…”

“…1), and therefore alginate production competes with the central carbon metabolism for this vital metabolite (23,24). As a consequence, alginate-producing P. fluorescens strains usually reach a lower cell density than nonproducers due to F6P drainage for alginate biosynthesis (19,25). Overproduction of a hexose phosphate phosphatase would also be expected to have a negative effect on growth, since it would introduce a futile cycle and deplete the cell of important metabolites.…”

“…In our earlier study (19), we found that mutant NCIMB10525Δzwf-1⌬zwf-2, which does not produce any active G6PD enzymes, hardly grew on fructose. The effect was less severe in the single mutant NCIMB10525Δzwf-1.…”

“…In a previous study (19), we found that the non-alginate-producing P. fluorescens zwf-1 zwf-2 double mutant grown on fructose exhibited a severe growth defect. In the zwf-1 single mutant, the growth defect was less pronounced, likely because the PP pathway still operates in the normal direction and provides part of the metabolites needed for growth (Fig.…”

“…The enzyme was eluted at approximately 0.1 M imidazole. An aliquot of 0.9 g of the purified Spp protein was electrophoretically separated on SDS-polyacrylamide gels according to Laemmli (33) and then subjected to Western blot analysis as described previously (19).…”