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.

Keywords: AI-2; N2 fixation; Zymomonas mobilis; ethanol production; quorum sensing; transcriptome.

Figure 1

Effects of AI-2 on Z. mobilis growth rate and viability. (A) An initial Z. mobilis culture was established, in 5 mL of liquid MM, from an isolated colony, grown in solid MM. This culture was incubated at 30 °C (without shaking) for 24 h, when aliquots were taken and used to inoculate a flask containing 80 mL of liquid MM, to an OD₆₀₀ = 0.05. This flask was then incubated under the same conditions and cell growth was monitored hourly by OD₆₀₀ readings. When this culture reached mid-exponential growth phase (at t = 14 h), it was subdivided into four vials of 20 mL each. AI-2 was then added to these cultures, at different concentrations (0, 18, 36, and 45 µM). Next, the cultures were re-incubated, as described above, and the OD₆₀₀ continued to be monitored, until all reached stationary phase. OD₆₀₀ readings shown in the graph represent the averages ± SEM from three independent experiments. Aliquots (2 mL) were taken for RNA extraction at the times indicated by the arrows, namely: (i) t = 14 h (before addition of AI-2); (ii) t = 16 h + AI-2 (treated with 45 µM AI- 2); (iii) t = 20 h + AI-2 (treated with 45 µM AI- 2); and (iv) t = 20 h (not treated with AI-2). See text for further details. (B) Representative dot plots showing SYTO 9 and PI fluorescence obtained by flow cytometry in cells grown in the absence of AI-2 (control), treated with 70% isopropyl alcohol (positive control), and treated with 45 µM AI-2, at the same incubation times highlighted in panel (A). (C) Quantification of percentages of live and dead cells. The results are presented as the mean ± SEM of three independent experiments and * represents statistically significant differences in relation to the respective controls (AI-2 untreated cultures), after a two-tailed t-test, using p < 0.05 as a threshold; ns represent statistically non-significant differences.

Figure 2

Gene expression patterns of Z. mobilis cultures, in response to the presence of AI-2 and different growth phases. The gene expression patterns shown above were obtained through RNA-seq experiments, employing samples obtained from the different timepoints depicted in Figure 1. RNA obtained from cells grown for t = 14 h (exponential growth phase) was used as a common reference for transcriptome comparisons with cells grown for t = 20 h (stationary phase), as well as cells induced to early stationary phase, due to the presence of 45 µM AI-2 (t = 16 h + AI-2 and t = 20 h + AI-2). The figure shows the average values for each of the 724 genes identified as modulated during three relative comparisons [log₂(t = 20 h /t = 14 h), Log₂(t = 16 h + AI-2/t = 14 h) and log₂(t = 20 h + AI-2/t = 14 h)] (see Materials and Methods for details). The genes and experimental conditions were subjected to a hierarchical clustering algorithm and visualized with the aid of TMEV [11]. Genes highlighted in Cluster 1 show an expression pattern that differentiates cells treated with AI-2 from those that spontaneously entered stationary phase (see text for details).

Figure 3

Functional distribution of differentially modulated genes. The OMICS Dashboard tool, available through the Pathway Tools package, was used to distribute the 724 genes shown in Figure 2 into the 8 main functional/structural systems/subsystems defined in the ZM4 Pathway Genome Database (PGDB), available at BioCyc (panels A to H) (see text, for details). Total modulation of the different categories is shown by the sum of relative expression (Log2 ratios) for all genes present in each system/subsystem. Details regarding each gene present in each functional/structural category can be found in Supplementary Materials File S3.

Figure 4

Transcriptional profile of genes responsible for N₂ fixation in Z. mobilis, in response to AI-2. The upper panel (A) shows the N₂ fixation locus present in the Z. mobilis chromosome. This locus consists of four operons, which contain genes directly involved in the production/maturation of nitrogenase, as well as those encoding electron donors, involved in the N₂ fixation process. The bottom panel (B) shows the expression pattern of the genes contained in this locus, in the presence or absence of AI-2, according to our RNA-seq data. The top element in panel B shows the expression profile of gene ZMO_RS01195, which is located outside the N₂ fixation locus shown in panel A. This element encodes an RpoN-like sigma factor (Sigma 54), supposedly involved in transcription of N₂-fixation genes, which is also upregulated in response to AI-2.

Figure 5

Acetylene reduction assay (ARA) to measure nitrogenase activity in Z. mobilis, in response to the presence of AI-2. Z. mobilis cells were inoculated in semi-solid MM, either in the absence or in the presence of 45 µM AI-2. After incubation for 48 h at 30 °C, 1 mL of 10% acetylene was injected into the headspace of the flasks, which were incubated for another hour, at 30 °C. The ethylene (C₂H₄) formed inside the bottles (resulting from the action of bacterial nitrogenase) was then detected and quantified by gas chromatography. The cellular biomass present in each flask was estimated by total protein quantification and the nitrogenase activity expressed as nmols of C₂H₄ produced/min/mg of protein. The experiments were carried out in triplicate and the graphs show the mean and SEMs obtained for each condition. The figure also compares the results obtained in an assay conducted in MM medium containing 1 mM (NH₄)₂SO₄ (A) and a similar assay, conducted in MM lacking (NH₄)₂SO₄ (B), since the presence of NH₄⁺ is known to inhibit nitrogenase activity [16].

Figure 6

Ethanol production by Z. mobilis in response to AI-2. Cultures of Z. mobilis were established, as described in Figure 1 and grown for 14 h, until they reached mid exponential growth phase. At this time, AI-2 was added, at 45 µM final concentration, and the cultures were further incubated for additional six hours, until they all reached stationary growth phase. Aliquots (2 mL) were collected at times t = 16 h and t = 20 h for ethanol quantification, using the EnzyChrom^® Ethanol Assay kit, and to quantify total protein accumulated in the bacterial biomass, at these different timepoints (see Materials and Methods for details). Blue bars display the absolute ethanol concentration in each sample (left axis), while the red dots display their relative ethanol production, normalized by biomass accumulation (right axis). The graph shows the average results and their respective SEMs, obtained from three independent experiments. * represents statistically significant differences in relation to the respective controls (AI-2 untreated cultures), after a two-tailed t-test, using p < 0.05 as a threshold.