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Competitive Interactions Between Incompatible Mutants of the Social Bacterium Myxococcus xanthus DK1622

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Competitive Interactions Between Incompatible Mutants of the Social Bacterium Myxococcus xanthus DK1622

Ya Gong et al. Front Microbiol.

Abstract

Due to the high similarity in their requirements for space and food, close bacterial relatives may be each other's strongest competitors. Close bacterial relatives often form visible boundaries to separate their swarming colonies, a phenomenon termed colony-merger incompatibility. While bacterial species are known to have many incompatible strains, it is largely unclear which traits lead to multiple incompatibilities and the interactions between multiple incompatible siblings. To investigate the competitive interactions of closely related incompatible strains, we mutated Myxococcus xanthus DK1622, a predatory bacterium with complex social behavior. From 3392 random transposon mutations, we obtained 11 self-identification (SI) deficient mutants that formed unmerged colony boundaries with the ancestral strain. The mutations were at nine loci with unknown functions and formed nine independent SI mutants. Compared with their ancestral strain, most of the SI mutants showed reduced growth, swarming and development abilities, but some remained unchanged from their monocultures. When pairwise mixed with their ancestral strain for co-cultivation, these mutants exhibited improved, reduced or unchanged competitive abilities compared with the ancestral strain. The sporulation efficiencies were affected by the DK1622 partner, ranging from almost complete inhibition to 360% stimulation. The differences in competitive growth between the SI mutants and DK1622 were highly correlated with the differences in their sporulation efficiencies. However, the competitive efficiencies of the mutants in mixture were inconsistent with their growth or sporulation abilities in monocultures. We propose that the colony-merger incompatibility in M. xanthus is associated with multiple independent genetic loci, and the incompatible strains hold competitive interaction abilities, which probably determine the complex relationships between multiple incompatible M. xanthus strains and their co-existence strategies.

Keywords: Myxococcus xanthus; colony-merger incompatibility; competitive interaction; multiple genetic loci; self-identification; transposon-mutation.

Figures

Figure 1
Figure 1
Screening for incompatible mutants in M. xanthus DK1622. (A) Visible boundaries (red arrows) formed between two adjacent colonies of pMiniHimar-lacZ insertion mutants on CTT agar plates supplemented with kanamycin. (B) The colony development between the 11 SI mutants and DK1622. All photos are representative of three biological replicates. Scale bar, 5 mm (A,B).
Figure 2
Figure 2
Characteristics of incompatible mutants. (A) Colony-forming unit (CFU) counts of incompatible mutants and DK1622 after incubation for 48 hr on CTT medium. (B) Colony expansions on CTT plates containing 0.3% agar and 1.5% agar, respectively. The pictures were taken after 5 days of incubation. (C) The developmental aggregation of the strains on TPM medium. The pictures were taken after 72 h of incubation. The bottom of the figure shows the strains used for fruiting body formation. The scale bars in (B,C) represent 1 mm. (D) The relative sporulation production of the strains on TPM medium. Error bars represent the standard deviation from three independent experiments.
Figure 3
Figure 3
Co-development of incompatible mutants and their ancestral strain DK1622 on the TPM medium. (A) Morphologies of the fruiting bodies of the co-cultures. The scale bars represent 1 mm. (B) The log-scale difference between each strain's sporulation ability in mixture and in monoculture. (C) The competitive sporulation differences of co-cultured partners after eliminating the strain's sporulation difference in monoculture. Three dilutions and three replications were performed for each experiment. The error bars represent the standard deviation, and asterisks denote p-values for t-tests of differences from zero: *p < 0.05, **p < 0.01.
Figure 4
Figure 4
Competitive growth abilities of incompatible mutants and DK1622 in paired mixtures. (A) The log-scale difference between each strain's growth ability in mixture and in monoculture. (B) The competitive growth differences of SI mutants and DK1622 in 1:1 mixtures under vegetative growth conditions after eliminating the strain's growth difference in monoculture. Three dilutions and three replications were performed for each assay, and the error bars represent the standard deviations. Asterisks denote the p-values for t-tests of differences from zero: *p < 0.05, **p < 0.01.
Figure 5
Figure 5
The competitive abilities between the incompatible mutants and the wild-type strain under vegetative growth and development conditions. The average CSIDK values during vegetative growth (y-Axis) strongly correlated with the values during co-development (x-Axis; r = 0.89, p < 0.01).
Figure 6
Figure 6
Strain-specific PCR amplification during vegetative growth to assay the presence of partners in mixed cultures among incompatible mutants. The number represents the specific SI mutant; i.e., 01 corresponds to SI01, 02 to SI02, etc. The + symbol indicates that the strain was mixed with an equal volume of TPM buffer as the positive control. The – symbol indicates the blank DK1622 control genome. M, molecular weight markers.

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