Summary: The use of toxin to attack neighbours and immunity proteins to protect against toxin has been observed in bacterial conflicts, including kin discrimination. Here, we report a novel nuclease‐toxin and its immunity protein function in the colony‐merger incompatibility, a kind of bacterial kin discrimination, in Myxococcus xanthus DK1622. The MXAN_0049 gene was determined to be a genetic determinant for colony‐merger incompatibility, and the incompatibility could be eliminated by deletion of the upstream co‐transcribed MXAN_0050 gene. We demonstrated that the MXAN_0050 protein was a nuclease, and MXAN_0049 protein was able to bind to MXAN_0050 to block nuclease activity in vitro . Expression of MXAN_0050 in Escherichia coli inhibited cellular growth, and the inhibition effect could be recovered by co‐expression of MXAN_0049. We found that deletion of the PAAR‐encoding gene ( MXAN_0044 ) or the type VI secretion system led to the colony‐merger and co‐existence with the Δ MXAN_0049 mutant, suggesting that they were associated with colony‐merger incompatibility. Homologues of the nuclease‐toxin and cognate immunity pair are widely distributed in bacteria. We propose a simplified model to explain the kin discrimination mechanism mediated by the nuclease‐toxin and immunity protein.© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd

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.

Two unrecognizable strains of the same bacterial species form a distinct colony boundary. During growth as colonies, Myxococcus xanthus uses multiple factors to establish cooperation between recognized strains and prevent interactions with unrecognized strains of the same species. Here, ΔMXAN_0049 is a mutant strain deficient in immunity for the paired nuclease gene, MXAN_0050, that has a function in the colony-merger incompatibility of Myxococcus xanthus DK1622. With the aim to investigate the factors involved in boundary formation, a proteome and metabolome study was employed. Visualization of the boundary between DK1622 and ΔMXAN_0049 was done scanning electron microscope (SEM), which displayed the presence of many damaged cells in the boundary. Proteome analysis of the DK1622- boundary disclosed many possible proteins, such as cold shock proteins, cell shape-determining protein MreC, along with a few pathways, such as RNA degradation, phenylalanine, tyrosine and tryptophan biosynthesis, and Type VI secretion system (T6SS), which may play major roles in the boundary formation. Metabolomics studies revealed various secondary metabolites that were significantly produced during boundary formation. Overall, the results concluded that multiple factors participated in the boundary formation in M. xanthus, leading to cellular damage that is helpful in solving the mystery of the boundary formation mechanism.

End pit lakes (EPLs) have been proposed as a method of reclaiming oil sands fluid fine tailings (FFT), which consist primarily of process-affected water and clay- and silt-sized particles. Base Mine Lake (BML) is the first full-scale demonstration EPL and contains thick deposits of FFT capped with water. Because of the fine-grained nature of FFT, turbidity generation and mitigation in BML are issues that may be detrimental to the development of an aquatic ecosystem in the water cap. Laboratory mixing experiments were conducted to investigate the effect of mudline biofilms made up of microbial communities indigenous to FFT on mitigating turbidity in EPLs. Four mixing speeds were tested (80, 120, 160, and 200 rpm), all of which are above the threshold velocity required to initiate erosion of FFT in BML. These mixing speeds were selected to evaluate (i) the effectiveness of biofilms in mitigating turbidity and (ii) the mixing speed required to ‘break’ the biofilms. The impact of biofilm age (10 weeks versus 20 weeks old) on turbidity mitigation was also evaluated. Diverse microbial communities in the biofilms included photoautotrophs, namely cyanobacteria and Chlorophyta (green algae), as well as a number of heterotrophs such as Gammaproteobacteria, Desulfobulbia, and Anaerolineae. Biofilms reduced surface water turbidity by up to 99%, depending on the biofilm age and mixing speed. Lifting and layering in the older biofilms resulted in weaker attachment to the FFT; as such, younger biofilms performed better than older biofilms. However, older biofilms still reduced turbidity by 69% to 95%, depending on the mixing speed. These results indicate that biostabilization is a promising mechanism for turbidity mitigation in EPLs.