Evidence of Autoinduction Heterogeneity via Expression of the Agr System of Listeria monocytogenes at the Single-Cell Level

Abstract

To investigate if the primary function of the Agr system of Listeria monocytogenes is to monitor cell density, we followed Agr expression in batch cultures, in which the autoinducer concentration was uniform, and in biofilms. Expression was heterogeneous, suggesting that the primary function of Agr is not to monitor population density.Quorum sensing (QS) is the mechanism by which bacteria secrete signaling molecules called autoinducers that are sensed by neighboring cells in a population (30). The binding of these autoinducers to cognate receptors results in transcriptional regulation of gene expression. So far, for the species Listeria monocytogenes, one QS system, mediated by the agrBDCA operon, has been described (2, 7). Deletion of agrD or agrA results in impairment of major adaptive strategies, such as biofilm development (22, 23) and virulence (2, 21).Historically, the term QS was coined to illustrate that accumulation of autoinducers enables a coordinated control of gene expression resulting in a population-wide phenotype switch when the population reaches a threshold or quorum (6,8,18). However, recent reports indicate that adaptive functions of QS can be diverse and are not limited to population density sensing (20).For example, phenotypic heterogeneity of QS-regulated traits was reported in biofilms. Several subpopulations with distinct phenotypes organize Bacillus subtilis biofilms (13,14). Extracellular DNA release during the sessile growth of Enterococcus faecalis is directed by a fratricidal mechanism triggered by a quorum-responsive subpopulation (26). Heterogeneity was also observed in QS-regulated bioluminescence of Vibrio harveyi (1).Recent reports showed that confocal laser scanning microscopy (CLSM) associated with fluorescent reporter fusions may be used to trace the spatiotemporal expression of specific genes at a single-cell level within the overall biofilm structure (9, 12). When we traced Agr expression in biofilms, we detected green fluorescent protein (GFP) mainly in a network of elongated chains reminiscent of scaffoldings that surrounded densely populated microcolonies (22). This heterogeneous expression was surprising; indeed, maximum expression was expected within microcolonies, where the autoinducer concentration is maximum (19).Thus, the question of whether the function of this QS system was primarily to monitor population density arose. In order to test this hypothesis, a P agr -gfp fusion was integrated upstream of the agr locus of the L. monocytogenes EGD-e genetic background. This construct was designed to develop Agr expression reporters without affecting expression of the downstream agrBDCA operon (22).We followed GFP fluorescence by flow cytometry and microscopy during growth in batch homogenized liquid cultures, which represents environmental conditions prone to facilitate responses to cell density (confined cultures and no diffusion). Cells were collected by centrifugation (10 min at 8,000 ϫ g), washed, and diluted in 150 mM filtered NaCl solution before flow cytometry a...

Evidence of Autoinducer-Dependent and -Independent Heterogeneous Gene Expression in Sinorhizobium fredii NGR234

“…These findings support the notion that NGR234 can distinguish between very high and very low concentrations of specific AI molecules, and it suggests that phenotypic heterogeneity is a well-controlled process that depends on a more or less defined concentration range of the AI molecules. In this respect, our data are in line with earlier reports on the QS-dependent expression of phenotypic heterogeneity in other microbes, such as V. harveyi (7,8,41) and L. monocytogenes (9). Possible candidate sensor proteins or regulators in NGR234 that are involved in intracellular AI measurements are TraR, NgrR, and any of the other four identified individual LuxR proteins encoded by the bacterial genome.…”

“…This phenomenon has been studied widely in a number of model organisms, such as Bacillus subtilis (36,37), E. coli (38)(39)(40), L. monocytogenes (9), and V. harveyi (7,8,10,41). In light of these reports, we wanted to know if Gram-negative rhizobacteria also show heterogeneity during the expression of AI-dependent and AI-independent genes.…”

“…Anetzberger and colleagues reported on the heterologous expression of the QS-regulated bioluminescence of Vibrio harveyi and the heterogeneous expression of QS-responsive genes (7,8). Other examples for QS-dependent heterogeneity include the Arg QS system of Listeria monocytogenes (9), the bioluminescence and AI system of V. fischeri (10), and the QS-dependent DNA release of Enterococcus faecalis (11). Thus, it appears that QS-dependent gene expression can lead to high levels of phenotypic heterogeneity in an otherwise isogenic population.…”

Negative regulation of bacterial quorum sensing tunes public goods cooperation

“…Here, tuning could be important because QS-controlled products may sometimes-but not always-confer a fitness advantage by nonspecifically inhibiting competing microbes (Hibbing et al, 2010). As a bet-hedging strategy, tuning may complement cell-to-cell variability of QS gene expression in isogenic populations (Anetzberger et al, 2009;Garmyn et al, 2011;Perez and Hagen, 2010). Such phenotypic heterogeneity can be caused by random fluctuations in the levels of regulatory proteins, and may also serve to enhance population fitness in dynamic and unpredictable environments (Veening et al, 2008).…”

You are what you talk: quorum sensing induces individual morphologies and cell division modes in Dinoroseobacter shibae

“…Recently, a high variability of other QS-regulated traits like secretion systems and exoproteolysis was demonstrated for the same organism (Anetzberger et al, 2012). Heterogeneous expression of QS genes and controlled traits have also been shown for Listeria monocytogenes (Garmyn et al, 2011) and Vibrio fischeri, the model organism for coordinated gene expression through communication (Perez and Hagen, 2010;Perez et al, 2011). This paradigm shift is by far not restricted to QS.…”