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

Sven Ef Borgos,Svein Valla,Sergio Bordel,Per Bruheim,Trond E Ellingsen,Helga Ertesvåg,Øyvind Jakobsen,Jens Nielsen,Håvard Sletta

doi:10.1186/1752-0509-7-19

Abstract

BackgroundAlginate is an industrially important polysaccharide, currently produced commercially by harvesting of marine brown sea-weeds. The polymer is also synthesized as an exo-polysaccharide by bacteria belonging to the genera Pseudomonas and Azotobacter, and these organisms may represent an alternative alginate source in the future. The current work describes an attempt to rationally develop a biological system tuned for very high levels of alginate production, based on a fundamental understanding of the system through metabolic modeling supported by transcriptomics studies and carefully controlled fermentations.ResultsAlginate biosynthesis in Pseudomonas fluorescens was studied in a genomics perspective, using an alginate over-producing strain carrying a mutation in the anti-sigma factor gene mucA. Cells were cultivated in chemostats under nitrogen limitation on fructose or glycerol as carbon sources, and cell mass, growth rate, sugar uptake, alginate and CO2 production were monitored. In addition a genome scale metabolic model was constructed and samples were collected for transcriptome analyses. The analyses show that polymer production operates in a close to optimal way with respect to stoichiometric utilization of the carbon source and that the cells increase the uptake of carbon source to compensate for the additional needs following from alginate synthesis. The transcriptome studies show that in the presence of the mucA mutation, the alg operon is upregulated together with genes involved in energy generation, genes on both sides of the succinate node of the TCA cycle and genes encoding ribosomal and other translation-related proteins. Strains expressing a functional MucA protein (no alginate production) synthesize cellular biomass in an inefficient way, apparently due to a cycle that involves oxidation of NADPH without ATP production. The results of this study indicate that the most efficient way of using a mucA mutant as a cell factory for alginate production would be to use non-growing conditions and nitrogen deprivation.ConclusionsThe insights gained in this study should be very useful for a future efficient production of microbial alginates.

Highlights

Alginate is an industrially important polysaccharide, currently produced commercially by harvesting of marine brown sea-weeds
MucA acts as an anti-sigma factor, binding and sequestering the alternative sigma factor σ22, encoded by the algU gene, that is essential for alginate production [8]. σ22 sigma factors are members of the ECF family of transcription factors that are known to respond to membrane stresses, and a recent microarray analysis [9] found that AlgU is a global stress response sigma factor, inducing several systems apart from alginate biosynthesis
The recently published genome sequence of SBW25 [21] shows that it contains all genes necessary for alginate biosynthesis, and from previous work, we have demonstrated the potential for high levels of alginate production in P. fluorescens [22]

Summary

Introduction

Alginate is an industrially important polysaccharide, currently produced commercially by harvesting of marine brown sea-weeds. Several species of Pseudomonas are notable and well-studied in their capability to form biofilms [1], aggregates of cells that adhere to each other and to surfaces, embedded in an extracellular polymeric matrix Formation of such biofilms can have serious clinical consequences, as seen in infections by the opportunistic human pathogen P. aeruginosa. One striking feature that is present in the majority of P. aeruginosa infections of the CF lung, is the so-called mucoid conversion of the pathogen, yielding a phenotype that produces large amounts of the exopolysaccharide alginate. This phenotype correlates with the ability of P. aeruginosa to persist in the lungs of CF patients [4] and is a general marker of poor survival for these patients [5]. The metabolic features controlled by the AlgU-MucA system are not well studied, but a very recent metabolic footprinting study concluded that MucA modulates osmotic stress tolerance [10]

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