Bacterial Metabolism During Biofilm Growth Investigated by 13C Tracing

Abstract

This study investigated the metabolism of Pseudomonas aeruginosa PAO1 during its biofilm development via microscopy imaging, gene expression analysis, and 13C-labeling. First, dynamic labeling was employed to investigate glucose utilization rate in fresh biofilms (thickness 40∼60 micrometer). The labeling turnover time of glucose-6-P indicated biofilm metabolism was substantially slower than planktonic cells. Second, PAO1 was cultured in continuous tubular biofilm reactors or shake flasks. Then 13C-metabolic flux analysis of PAO1 was performed based on the isotopomer patterns of proteinogenic amino acids. The results showed that PAO1 biofilm cells during growth conserved the flux features as their planktonic mode. (1) Glucose could be degraded by two cyclic routes (the TCA cycle and the Entner-Doudoroff-Embden-Meyerhof-Parnas loop) that facilitated NAD(P)H supplies. (2) Anaplerotic pathways (including pyruvate shunt) increased flux plasticity. (3) Biofilm growth phenotype did not require significant intracellular flux rewiring (variations between biofilm and planktonic flux network, normalized by glucose uptake rate as 100%, were less than 20%). (4) Transcription analysis indicated that key catabolic genes in fresh biofilm cells had expression levels comparable to planktonic cells. Finally, PAO1, Shewanella oneidensis (as the comparing group), and their c-di-GMP transconjugants (with different biofilm formation capabilities) were 13C-labeled under biofilm reactors or planktonic conditions. Analysis of amino acid labeling variances from different cultures indicated Shewanella flux network was more flexibly changed than PAO1 during its biofilm formation.

Fluxomic Analysis Reveals Central Carbon Metabolism Adaptation for Diazotroph Azotobacter vinelandii Ammonium Excretion

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The Wsp intermembrane complex mediates metabolic control of the swim‐attach decision of Pseudomonas putida

“…On the background of the above, we have studied the wsp orthologous operon in P. putida by making individual and block-deletion mutants, in order to determine the importance of the similarities between this complex and P. aeruginosa's and to decide how we could take advantage of its function in order to control biofilm formation. We also studied several mutant phenotypes in three different sole carbon sources, as the nature of them influences extracellular polysaccharide (EPS) building blocks used for biofilm formation (Reeves et al, 1996;Ramos et al, 2001;Wan et al, 2018). The results below not only expose the similarities with the P. aeruginosa's Wsp system of reference but also reveal a striking dependence of biofilm formation on growth conditions and metabolic status.…”

Scanning electrochemical microscopy and its potential for studying biofilms and antimicrobial coatings

“…Given the heterogeneity of biofilms [25], metabolite secretion of cells may be different depending on the location in the biofilm, due to the internal gradients of e.g. nutrients, oxygen and pH [26].…”