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. 2024 Jul 23;121(30):e2402559121.
doi: 10.1073/pnas.2402559121. Epub 2024 Jul 16.

Cell-cell transfer of adaptation traits benefits kin and actor in a cooperative microbe

Kalpana Subedi  1   2 , Pravas C Roy  1 , Brandon Saiz  2 , Franco Basile  2 , Daniel Wall  1
Affiliations

Cell-cell transfer of adaptation traits benefits kin and actor in a cooperative microbe

Kalpana Subedi et al. Proc Natl Acad Sci U S A. .

Abstract

Microbes face many physical, chemical, and biological insults from their environments. In response, cells adapt, but whether they do so cooperatively is poorly understood. Here, we use a model social bacterium, Myxococcus xanthus, to ask whether adapted traits are transferable to naïve kin. To do so we isolated cells adapted to detergent stresses and tested for trait transfer. In some cases, strain-mixing experiments increased sibling fitness by transferring adaptation traits. This cooperative behavior depended on a kin recognition system called outer membrane exchange (OME) because mutants defective in OME could not transfer adaptation traits. Strikingly, in mixed stressed populations, the transferred trait also benefited the adapted (actor) cells. This apparently occurred by alleviating a detergent-induced stress response in kin that otherwise killed actor cells. Additionally, this adaptation trait when transferred also conferred resistance against a lipoprotein toxin delivered to targeted kin. Based on these and other findings, we propose a model for stress adaptation and how OME in myxobacteria promotes cellular cooperation in response to environmental stresses.

Keywords: Myxococcus xanthus; cooperation; detergent resistance; kin recognition; outer membrane exchange.

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Conflict of interest statement

Competing interests statement:The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
Detergent sensitivity and resistance. Viable CFUs of M. xanthus DK6204 and E. coli K12 MG1655 on agar plates with indicated concentrations of (A) DCA and (B) Tween-20. Chemical structures and MIC values are shown. (C and D) Plating efficiencies of parent (DK6204) and adapted strains on CTT-detergent plates compared to CTT only plates. DCA and Tween-20 used at 0.008% (w/v) and 0.02% (v/v) concentrations, respectively. MICs of adapted isolates also are shown. All assays done in biological triplicates; error bars represent SEM.
Fig. 2.
Fig. 2.
Genetic analyses of the dcaR and twnR loci. (A) Strain plating efficiency on 0.008% DCA compared to no detergent plates. Strains; parent (DK6204), dcaRE128K, dcaRE128K with ectopic dcaR+ copy (complementation), ΔdcaR in-frame deletion, DK6204 backcrossed with 3811-KmR linked to indicated dcaR alleles. (B) dcaR gene cluster and domain organization (see SI Appendix, Fig. S1 for predicted 3D structure and location of substitution). Triangle, linked kanamycin marker. (C) Strain sensitivity assessed by CFU on 0.02% Tween-20 plates. Strains: DK6204, twnR, twnR::mariner (Tn), twnR complementation, and twnR in-frame deletion. (D) twnR gene cluster and domain architecture (SI Appendix, Fig. S2). Location of mutations in C and D shown with bars representing deleted (Δ) regions. Experiments were done in biological triplicate, error bars represent SEM; *P < 0.05; **P = 0.0024; ***P = 0.004; ****P < 0.0001 as determined by the unpaired t test. NS, not statistically significant (P > 0.05). Trans, transporter; HP, hypothetical protein; HK, histidine kinase; RTCA, RNA 3′-terminal phosphate cyclase; LipoP, lipoprotein; PBP, penicillin-binding protein 1C; IDR, intrinsically disordered region.
Fig. 3.
Fig. 3.
Adaptation transfer by OME. (A) Detergent sensitive strains were traA+ or traA::KmR (DW2301 and DW1485, KmR) and mixed with twnR or dcaR donors (KmS) at 1:3 ratios. Cell mixtures preincubated on agar plates for 5 h to allow OME. Harvested cells were then transferred to plates with or without 0.08% Tween-20 for 7 h. Collected mixtures were enumerated on CTT-Kan plates to assess the survivability of sensitive kin. Control monocultures were similarly treated with and without detergent exposure. Schematic of adaptation transfer shown. Note, DW2301 overexpresses traAB; see Discussion. (B) Similar to A, except the survival of sensitive kin (KmR) from strain mixtures plotted as a function of Tween-20 exposure time. All experiments done in four biological replicates. Error bars, SEM.
Fig. 4.
Fig. 4.
Adapted actors benefit from cooperative OME. (A) Colony growth on 0.08% Tween-20 agar. Strains plated as monocultures or mixtures of twnR::Tn with detergent sensitive traAB+ or ΔtraAB strains (DK10410 or DW2270) at 3:1 ratios, 6 × 106 cells plated (total). Cultures first preincubated on agar for 6 h for OME then transferred to Tween-20 plates. Micrographs of colony edges taken at indicated times. Highlighted borders show robust growth. (Scale bar, 200 μm.) (B) Cell survival quantified after 24 h incubation. Cells harvested and KmR actors serially diluted and enumerated on kanamycin plates. Data from biological triplicates. Error bars, SEM. ****P < 0.0001 as determined by the unpaired t test. (C) Model of OME on the survival of actor cells. Top, OME results in cooperation that protects kin and benefits the actors resulting in growth. In the absence of OME, kin, stressed by detergent, triggers a response that kills actors resulting in mutual death.
Fig. 5.
Fig. 5.
SitA3 toxin resistance is a transferable phenotype. (A) Experimental design for OME resistance transfer against SitA3 toxin-producing strain. Actor and target were premixed to allow OME followed by addition of inhibitor strain. (B) Nonmotile twnR strain protects SitA3 intoxication of target strain. Following 6 h preincubation of the motile target with the twnR or parent (DK6204) strains (1:3 ratio), mixtures were harvested and combined with the nonmotile SitA3 inhibitor (DW2458) and plated at a 1:8 ratio (other cells to inhibitor). The Top control panel contains no inhibitor but the same ratio of the motile target cells to nonmotile cells (parent strain). Dotted red lines mark swarm fronts. Micrographs at 24 h; scale bar, 200 μm (Left, low magnification) and 100 μm (Right, high magnification). (C) Viable target cells (DW2301, KmR) following incubation (24 h) with indicated ratios of other cells to inhibitor strain (target and twnR or parent strain ratios were constant as in B). ND, none detected. Experiments done in four biological replicates, error bars, SEM.
Fig. 6.
Fig. 6.
Upregulation of fatty acid β-oxidation pathway by Tween-20 exposure. (A) Volcano plot derived from pairwise comparison between Tween-20 treated and untreated parent strain (DK6204) at 2 h (n = 6). Quantified proteins plotted in accordance with their P-value and fold change [label-free quantification (LFQ) intensity difference]. Proteins above the line are statistically significant (P-value < 0.05). (B) Sequential steps of the fatty acid β-oxidation pathway (box) with up-regulated proteins (green) from A are listed. M. xanthus contains 10 FadD (gray) homologs (48), none of which were up-regulated. Gene names derived from references or predicted roles (47, 49). See SI Appendix, Fig. S9 for a comparison to ΔtwnR response and Dataset S2 for further details.
Fig. 7.
Fig. 7.
Adaptation transfer model. In response to stress adapted actor cell expresses modified OM with altered or overexpressed proteins and/or lipids (green circles/membrane). Homotypic recognition of kin by TraAB receptors results in OME and transfer of adaptation phenotype to sensitive cells. The resulting adapted kin is resistant to the stress and does not antagonize actor cells when confronted with an insult.
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