Quorum sensing mediates morphology and motility transitions in the model archaeon Haloferax volcanii

Abstract

Quorum sensing (QS) is a population density-dependent mechanism of intercellular communication, whereby microbes secrete and detect signals to regulate behaviors such as virulence and biofilm formation. Although QS is well-studied in bacteria, little is known about cell-cell communication in archaea. The model archaeon Haloferax volcanii can transition from motile rod-shaped cells to non-motile disks as population density increases. In this report, we demonstrate that this transition is induced by a secreted small molecule present in cell-free conditioned medium (CM). The CM also elicits a response from a bacterial QS bioreporter, suggesting the potential for inter-domain crosstalk. To investigate the Hfx. volcanii QS response, we performed quantitative proteomics and detected significant differential abundances of 236 proteins in the presence of CM, including proteins involved in cell structure, motility, glycosylation, and two-component systems. We also demonstrate that a mutant lacking the cell shape regulatory factor DdfA does not undergo shape and motility transitions in the presence of CM, allowing us to identify protein abundance changes in the QS response pathway separate from those involved in shape and motility. In the ∆ddfA strain, only 110 proteins had significant differential abundance, and comparative analysis of these two proteomics experiments enabled us to identify proteins dependent on and independent of DdfA in the QS response pathway. Our study provides the first detailed analysis of QS pathways in any archaeon, strengthening our understanding of archaeal communication as well as providing the framework for studying intra- and interdomain crosstalk.

Importance: Understanding the complex signaling networks in microbial communities has led to many invaluable applications in medicine and industry. Yet, while archaea are ubiquitous and play key roles in nutrient cycling, little is known about the roles of archaeal intra- and interspecies cell-cell communication in environments such as the human, soil, and marine microbiomes. In this study, we established the first robust system for studying quorum sensing in archaea by using the model archaeon Haloferax volcanii. We demonstrated that different behaviors, such as cell shape and motility, are mediated by a signal molecule, and we uncovered key regulatory components of the signaling pathway. This work advances our understanding of microbial communication, shedding light on archaeal intra- and interdomain interactions, and contributes to a more complete picture of the interconnected networks of life on Earth.

Keywords: Haloferax volcanii; archaea; cell shape; intercellular signaling; motility; quorum sensing.

Fig 1

Hfx. volcanii cells are disk-shaped in the early log phase in the presence of conditioned medium (CM). Differential interference contrast (DIC) images of Hfx. volcanii cells from 5 mL cultures grown to OD₆₀₀ 0.05 with no additive (top) and with 50 µL of CM (1% [vol/vol]) added (bottom). Scale bar is 10 µm (a). Cell shape quantification of three biological replicates of each condition to determine the lowest effective volume of CM from an Hfx. volcanii culture grown to OD₆₀₀ 1.5 (b) or of the effect of 1% CM from cultures grown to differing optical densities (c) by using the eccentricity parameter of CellProfiler, which outputs a value between 0 (circle) and 1 (line segment). Eccentricity values that range between 0.5 and 0.9 correspond to disks, while values that range between 0.9 and 1.0 correspond to rods, and each data point represents a single counted cell. The median of each condition denoted by a line. Statistical analysis performed via nested one-way ANOVA with multiple comparisons to the “No additive” condition. ns is P > 0.05, * is P < 0.05, ** is P < 0.01, and **** is P < 0.0001.

Fig 2

CM incorporated into soft agar plates inhibits swimming motility. Motility halos of Hfx. volcanii stab-inoculated onto soft agar plates of Hv-Cab (left) and Hv-Cab supplemented with 25% CM (right). Scale bar denotes 1 cm (a). Quantification of motility halo radius in millimeters of six biological replicates. Error bars depict mean with standard deviation. Statistical significance determined with unpaired parametric two-tailed t-test. **** is P < 0.0001 (b).

Fig 3

∆ddfA and ∆cirA form motile rod-shaped cells in the presence of CM. Motility halos of wild type (WT), ∆ddfA, and ∆cirA on soft agar plates supplemented with 25% CM (vol/vol). Image of WT on 25% CM reused from Fig. 2a for ease of comparison. Scale bar denotes 1 cm (a, top). DIC microscopy images of WT, ∆ddfA, and ∆cirA cells grown to the early log phase in cultures containing 1% CM. Scale bar denotes 10 µm (a, bottom). Quantification of six replicate motility halo radii in millimeters. Error bars depict mean with standard deviation. Statistical significance determined with ordinary one-way ANOVA with multiple comparisons to the WT condition (b). Cell shape quantification of three biological replicates of WT, ∆ddfA, and ∆cirA grown to OD₆₀₀ 0.05 containing 1% CM, as described in Fig. 1. The median of each condition is denoted by a line. Statistical analysis performed via nested one-way ANOVA with multiple comparisons to the WT. ****, P-value < 0.0001 (c).

Fig 4

Quantitative proteomics analyses indicate proteins with altered abundances in the presence of CM. Volcano plots depicting log₂ fold changes and their corresponding -log₁₀ P-values of protein abundances compared to WT between early and late log phases (a), WT early log regular medium to 1% CM (b), and ∆ddfA early log regular medium to 1% CM (d). Nonsignificant (P-value ≥ 0.05) protein changes correspond to gray circles; significant (P-value < 0.05) and positive protein fold changes correspond to maroon circles (higher abundance in the second condition); significant (P-value < 0.05) and negative protein fold changes correspond to blue circles (higher abundance in the first condition). Raw values can be found in the supplemental material. Interactive HTML files of the volcano plots are available at 10.5281/zenodo.14510577 (a, b, d). Venn diagram of protein overlap with significant differential abundance in the three proteomics comparisons (WT early to late log, WT no CM to 1% CM, and ∆ddfA no CM to 1% CM). A list of proteins represented in each overlapping region can be found in Table S3 (c).

Fig 5

DFS can be extracted from CM and is unaffected by DNase, RNase, or proteinase K. Cell shape quantification of three biological replicates of cultures containing a final concentration of 1% of each of the following: autoclaved CM, flowthrough of CM passing through a 3 kDa centrifugal filter, elution from the filter using Tris buffer, and the Tris buffer as control (a), as well as eluates treated with RQ1 DNase, RNase A, and proteinase K. The “Neg control” condition includes 5 µL RQ1 DNase 10× Reaction Buffer, 5 µL of 50% glycerol, and 5 µL RQ1 DNase Stop Solution to ensure that these additional components do not induce disk formation (b). Cell shape quantification as described in Fig. 1. The line denotes the median value. Statistical analysis performed via nested one-way ANOVA with multiple comparisons to the respective “No additive” or “Neg control” condition. ns is P > 0.05, and **** is P < 0.0001.

Fig 6

Hfx. volcanii CM activates A. tumefaciens bioreporter strain similar to AHL, but DKPs do not induce Hfx. volcanii disk formation. Miller units of cleavage of ortho-nitrophenyl-β-galactoside were measured in the bioreporter strain A. tumefaciens KYC55 (pJZ410)(pJZ384)(pJZ372). “No additive” condition corresponds to basal output of the bioreporter strain, “Media ctrl” condition corresponds to the addition of 200 µL of the Hv-Cab extract, “CM” condition corresponds to 200 µL of the CM extract, and “AHL” condition corresponds to a final concentration 2.5 ng/µL of N-3-oxo-pentanoyl-L-homoserine lactone. Statistical analysis performed via unpaired parametric two-tailed t-test (a). Cell shape quantification of three biological replicates comparing no additive and 1% CM to the addition of DKPs pooled together for final concentration 0.2 µg/mL. Because DKPs were solubilized in ethanol, similar amounts of ethanol (10 µL) were added to the “Neg Ctrl” and “CM” cultures. Cell shape quantification as described in Fig. 1. The line denotes the median value. Statistical analysis performed via nested one-way ANOVA with multiple comparisons to the respective “No additive” or “Neg control” condition (b). ns is P > 0.05, * is P < 0.05, and **** is P < 0.0001.

Fig 7

The initial roadmap for the QS-mediated regulatory network in Hfx. volcanii. A subset of proteins discussed in this study is depicted as increasing or decreasing in abundance (vertical arrows) as a result of direct or indirect regulation (horizontal arrows), resulting in QS-mediated behaviors.