Abstract
Decreased biomass growth in iron (Fe)-limited Pseudomonas is generally attributed to downregulated expression of Fe-requiring proteins accompanied by an increase in siderophore biosynthesis. Here, we applied a stable isotope-assisted metabolomics approach to explore the underlying carbon metabolism in glucose-grown Pseudomonas putida KT2440. Compared to Fe-replete cells, Fe-limited cells exhibited a sixfold reduction in growth rate but the glucose uptake rate was only halved, implying an imbalance between glucose uptake and biomass growth. This imbalance could not be explained by carbon loss via siderophore production, which accounted for only 10% of the carbon-equivalent glucose uptake. In lieu of the classic glycolytic pathway, the Entner-Doudoroff (ED) pathway in Pseudomonas is the principal route for glucose catabolism following glucose oxidation to gluconate. Remarkably, gluconate secretion represented 44% of the glucose uptake in Fe-limited cells but only 2% in Fe-replete cells. Metabolic (13) C flux analysis and intracellular metabolite levels under Fe limitation indicated a decrease in carbon fluxes through the ED pathway and through Fe-containing metabolic enzymes. The secreted siderophore was found to promote dissolution of Fe-bearing minerals to a greater extent than the high extracellular gluconate. In sum, bypasses in the Fe-limited glucose metabolism were achieved to promote Fe availability via siderophore secretion and to reroute excess carbon influx via enhanced gluconate secretion.
Highlights
Environmental aerobic bacteria have to cope with limited iron (Fe) availability during organic carbon utilization
The expression of Fe-containing metabolic enzymes were shown to be decreased in Fe- limited Pseudomonas (Somerville et al 1999; Heim et al, 2003; Palma et al, 2003; Vasil 2007; Bronstein et al, 2008; Kim et al 2010; Lim et al 2012), a genus of aerobic bacteria that are ubiquitous in natural soils and waters
The growth rate obtained with the Fe-replete P. putida cells was 0.60 ± 0.04 h−1, in close agreement with previous values obtained with Fe-replete glucose- grown Pseudomonas fluorescens (Fuhrer et al 2005) and P. putida, (0.49 ± 0.03 h−1 and 0.56 ± 0.02 h−1, respectively) (Fig. 1B)
Summary
Environmental aerobic bacteria have to cope with limited iron (Fe) availability during organic carbon utilization. Based on the aforementioned studies, we hypothesized that the decreased metabolic investment toward biomass growth in glucose-grown P. putida under Fe limitation could be due to (1) a decrease in carbon uptake and subsequent influx toward the ED pathway, (2) a decrease in metabolic flux through Fe-d emanding pathways, (3) overflow secretion of partially oxidized metabolites, and (4) a high metabolic expense for siderophore biosynthesis.
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