Volke, D. C. | bioRxiv Science

bioRxiv2021

Extracellular DNA (eDNA) enables early detection of the phenotypic switch of Pseudomonas sp. during biofilm development

The different steps involved in biofilm formation have been the subjects of intensive researches. However, the very early cell decision-making process related to the switch from planktonic to sessile state still remains uncharacterized. Based on the use of Pseudomonas putida KT2440 and derivatives with varying biofilm-forming capabilities, we observed a subpopulation of cells bound to extracellular DNA (eDNA) in the planktonic phase, as indicated by propidium iodide (PI) staining. Strikingly, the size of this eDNA-bound/PI-positive subpopulation correlated with the overall biofilm forming capability of the bacterial population. This finding challenges the conventional view of phenotypic switching and suggests that, in Pseudomonas, biofilm switching is determined collectively based on the quantity of eDNA released in the supernatant. The whole process can be followed based on automated flow cytometry, and the appearance of PI-positive cells was considered as an early-warning indicator for biofilm formation. For this purpose, automated glucose pulsing was used successfully to interfere with the proliferation of PI-positive cells, resulting in a reduction of biofilm formation. This study provides insights into the collective determinants of biofilm switching in Pseudomonas species and introduces a potential strategy for controlling biofilm formation.

microbiology↗

bioRxiv2021

Cofactor specificity of glucose-6-phosphate dehydrogenase isozymes in Pseudomonas putida reveals a general principle underlying glycolytic strategies in bacteria

Glucose-6-phosphate dehydrogenase (G6PDH) is widely distributed in nature and catalyzes the first committing step in the oxidative branch of the pentose phosphate (PP) pathway, feeding either the reductive PP or the Entner-Doudoroff pathway. Besides its role in central carbon metabolism, this dehydrogenase also provides reduced cofactors, thereby affecting redox balance. Although G6PDH is typically considered to display specificity towards nicotinamide adenine dinucleotide phosphate (NADP+), some variants accept nicotinamide NAD+ similarly (or even preferentially). Furthermore, the number of G6PDH isozymes encoded in bacterial genomes varies from none to more than four orthologues. On this background, we systematically analyzed the interplay of the three G6PDH isoforms of the soil bacterium Pseudomonas putida KT2440 from a genomic, genetic and biochemical perspective. P. putida represents an ideal model to tackle this endeavor, as its genome encodes numerous gene orthologues for most dehydrogenases in central carbon metabolism. We show that the three G6PDHs of strain KT2440 have different cofactor specificities, and that the isoforms encoded by zwfA and zwfB carry most of the activity, acting as metabolic gatekeepers for carbon sources that enter at different nodes of the biochemical network. Moreover, we demonstrate how multiplication of G6PDH isoforms is a widespread strategy in bacteria, correlating with the presence of an incomplete Embden-Meyerhof-Parnas pathway. Multiplication of G6PDH isoforms in these species goes hand-in-hand with low NADP+ affinity at least in one G6PDH isozyme. We propose that gene duplication and relaxation in cofactor specificity is an evolutionary strategy towards balancing the relative production of NADPH and NADH. ImportanceProtein families have likely arisen during evolution by gene duplication and divergence followed by neo-functionalization. While this phenomenon is well documented for catabolic activities (typical of environmental bacteria that colonize highly polluted niches), the co-existence of multiple isozymes in central carbon catabolism remains relatively unexplored. We have adopted the metabolically-versatile soil bacterium Pseudomonas putida KT2440 as a model to interrogate the physiological and evolutionary significance of co-existing glucose-6-phosphate dehydrogenase (G6PDH) isozymes. Our results show that each of the three G6PDHs encoded in this bacterium display distinct biochemical properties, especially at the level of cofactor preference, impacting bacterial physiology in a carbon source-dependent fashion. Furthermore, the presence of multiple G6PDHs differing in NAD+- or NADP+-specificity in bacterial species strongly correlates with their predominant metabolic lifestyle. Our findings support the notion that multiplication of genes encoding cofactor-dependent dehydrogenases is a general evolutionary strategy towards achieving redox balance according to the growth conditions.

systems biology↗