The Quorum Sensing Auto-Inducer 2 (AI-2) Stimulates Nitrogen Fixation and Favors Ethanol Production over Biomass Accumulation in Zymomonas mobilis

Abstract

Autoinducer 2 (or AI-2) is one of the molecules used by bacteria to trigger the Quorum Sensing (QS) response, which activates expression of genes involved in a series of alternative mechanisms, when cells reach high population densities (including bioluminescence, motility, biofilm formation, stress resistance, and production of public goods, or pathogenicity factors, among others). Contrary to most autoinducers, AI-2 can induce QS responses in both Gram-negative and Gram-positive bacteria, and has been suggested to constitute a trans-specific system of bacterial communication, capable of affecting even bacteria that cannot produce this autoinducer. In this work, we demonstrate that the ethanologenic Gram-negative bacterium Zymomonas mobilis (a non-AI-2 producer) responds to exogenous AI-2 by modulating expression of genes involved in mechanisms typically associated with QS in other bacteria, such as motility, DNA repair, and nitrogen fixation. Interestingly, the metabolism of AI-2-induced Z. mobilis cells seems to favor ethanol production over biomass accumulation, probably as an adaptation to the high-energy demand of N2 fixation. This opens the possibility of employing AI-2 during the industrial production of second-generation ethanol, as a way to boost N2 fixation by these bacteria, which could reduce costs associated with the use of nitrogen-based fertilizers, without compromising ethanol production in industrial plants.

Genome-Wide Analyses of Proteome and Acetylome in Zymomonas mobilis Under N2-Fixing Condition

“…Overall, our results show that Z. mobilis has the same molecular responses to nitrogen fixation as other diazotrophic organisms. A notable finding was that the ethanol-producing protein alcohol dehydrogenase was upregulated, which is consistent with prior studies showing that Z. mobilis can fix nitrogen efficiently without affecting ethanol production ( Kremer et al, 2015 ; Palamae et al, 2020 ; Alencar et al, 2021 ).…”

Metabolic Remodeling during Nitrogen Fixation in Zymomonas mobilis

“…Increasing expression of zinc-dependent ADHI rather than iron-dependent ADHII may be important for maintaining iron homeostasis. Increased ADHI expression may help explain the previously reported increase in specific ethanol production during N 2 fixation ( 17 , 19 , 20 ). However, the fact that no difference in ADHI abundance was detected during continuous N 2 -fixing conditions implies a possible difference in short-term and long-term mechanisms for increasing ethanol production.…”

“…This is in contrast to other environmental inhibitors such as oxygen and lignocellulosic toxins, which have been shown to increase expression of motility-related genes in Z. mobilis ( 48 – 50 ). Previous transcriptomics analysis of Z. mobilis both during adaptation to high glucose concentrations and in the presence of a quorum sensing autoinducer each showed that expression of flagellar genes decreased concomitantly with increased expression of genes from the nif cluster ( 20 , 46 ). Given that the Rnf complex utilizes energy stored in the electrochemical gradient to drive production of reduced ferredoxin (the electron donor to nitrogenase reductase), decreased expression of flagellar proteins (and, presumably, decreased energy consumption by the flagellar motor) may be important for maintaining a robust electrochemical gradient during N 2 -fixing conditions.…”

“…During N 2 fixation, Z. mobilis exhibits increased specific rates of glucose consumption and ethanol production ( 17 , 19 , 20 ). However, the physiological factors that drive this response are not well understood.…”

“…However, little is known about the physiology of Z. mobilis during N 2 fixation. Previous studies have shown that during N 2 fixation, Z. mobilis exhibits a lower growth rate and lower growth yield but a higher biomass-specific ethanol production rate, higher specific glucose consumption rate, and equivalent or slightly higher ethanol yield ( 17 , 19 , 20 ). This is a striking observation considering the already highly catabolic metabolism employed by Z. mobilis during replete ammonium (NH 4 + ) availability ( 21 ).…”