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 ΔMXAN_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.

Summary Glycosylation of natural products can influence their pharmacological properties, and efficient glycosyltransferases (GTs) are critical for this purpose. The polyketide epothilones are potent anti‐tumour compounds, and YjiC is the only reported GT for the glycosylation of epothilone. In this study, we phylogenetically analysed 8261 GTs deposited in CAZy database and revealed that YjiC locates in a subbranch of the Macrolide I group, forming the YjiC‐subbranch with 160 GT sequences. We demonstrated that the YjiC‐subbranch GTs are normally efficient in epothilone glycosylation, but some showed low glycosylation activities. Sequence alignment of YjiC‐subbranch showed that the 66th and 77th amino acid residues, which were close to the catalytic cavity in molecular docking model, were conserved in five high‐active GTs (Q66 and P77) but changed in two low‐efficient GTs. Site‐directed residues swapping at the two positions in the two low‐active GTs (BssGT and BamGT) and the high‐active GT BsGT‐1 demonstrated that the two amino acid residues played an important role in the catalytic efficiency of epothilone glycosylation. This study highlights that the potent GTs for appointed compounds are phylogenetically grouped with conserved residues for the catalytic efficiency.

Background Bacterial predation is an important selective force in microbial community structure and dynamics. However, only a limited number of predatory bacteria have been reported, and their predatory strategies and evolutionary adaptations remain elusive. We recently isolated a novel group of bacterial predators, Bradymonabacteria, representative of the novel order Bradymonadales in δ- Proteobacteria . Compared with those of other bacterial predators (e.g., Myxococcales and Bdellovibrionales ), the predatory and living strategies of Bradymonadales are still largely unknown. Results Based on individual coculture of Bradymonabacteria with 281 prey bacteria, Bradymonabacteria preyed on diverse bacteria but had a high preference for Bacteroidetes . Genomic analysis of 13 recently sequenced Bradymonabacteria indicated that these bacteria had conspicuous metabolic deficiencies, but they could synthesize many polymers, such as polyphosphate and polyhydroxyalkanoates. Dual transcriptome analysis of cocultures of Bradymonabacteria and prey suggested a potential contact-dependent predation mechanism. Comparative genomic analysis with 24 other bacterial predators indicated that Bradymonabacteria had different predatory and living strategies. Furthermore, we identified Bradymonadales from 1552 publicly available 16S rRNA amplicon sequencing samples, indicating that Bradymonadales was widely distributed and highly abundant in saline environments. Phylogenetic analysis showed that there may be six subgroups in this order; each subgroup occupied a different habitat. Conclusions Bradymonabacteria have unique living strategies that are transitional between the “obligate” and the so-called facultative predators. Thus, we propose a framework to categorize the current bacterial predators into 3 groups: (i) obligate predators (completely prey-dependent), (ii) facultative predators (facultatively prey-dependent), and (iii) opportunistic predators (prey-independent). Our findings provide an ecological and evolutionary framework for Bradymonadales and highlight their potential ecological roles in saline environments.

BackgroundThe CRISPR/Cas9 system is a powerful tool for genome editing, in which the sgRNA binds and guides the Cas9 protein for the sequence-specific cleavage. The protocol is employable in different organisms, but is often limited by cell damage due to the endonuclease activity of the introduced Cas9 and the potential off-target DNA cleavage from incorrect guide by the 20 nt spacer.ResultsIn this study, after resolving some critical limits, we have established an efficient CRISPR/Cas9 system for the deletion of large genome fragments related to the biosynthesis of secondary metabolites in Myxococcus xanthus cells. We revealed that the high expression of a codon-optimized cas9 gene in M. xanthus was cytotoxic, and developed a temporally high expression strategy to reduce the cell damage from high expressions of Cas9. We optimized the deletion protocol by using the tRNA–sgRNA–tRNA chimeric structure to ensure correct sgRNA sequence. We found that, in addition to the position-dependent nucleotide preference, the free energy of a 20 nt spacer was a key factor for the deletion efficiency.ConclusionsBy using the developed protocol, we achieved the CRISPR/Cas9-induced deletion of large biosynthetic gene clusters for secondary metabolites in M. xanthus DK1622 and its epothilone-producing mutant. The findings and the proposals described in this paper were suggested to be workable in other organisms, for example, other Gram negative bacteria with high GC content.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0758-x) contains supplementary material, which is available to authorized users.

Background: Bacterial predation is an important selective force in microbial community structure and dynamics. However, only a limited number of predatory bacteria have been reported, and their predatory strategies and evolutionary adaptations remain elusive. We recently isolated a novel group of bacterial predators, Bradymonabacteria, representative of the novel order Bradymonadales in δ- Proteobacteria . Compared with those of other bacterial predators (e.g., Myxococcales and Bdellovibrionales ), the predatory and living strategies of Bradymonadales are still largely unknown. Results: Based on individual coculture of Bradymonabacteria with 281 prey bacteria, Bradymonabacteria preyed on diverse bacteria but had a high preference for Bacteroidetes . Genomic analysis of 13 recently sequenced Bradymonabacteria indicated that these bacteria had conspicuous metabolic deficiencies, but they could synthesize many polymers, such as polyphosphate and polyhydroxyalkanoates. Dual-transcriptome analysis of cocultures of Bradymonabacteria and prey suggested a potential contact-dependent predation mechanism. Comparative genomic analysis with 24 other bacterial predators indicated that Bradymonabacteria had different predatory and living strategies. Furthermore, we identified Bradymonadales from 1552 publicly available 16S rRNA amplicon sequencing samples, indicating that Bradymonadales was widely distributed and highly abundant in saline environments. Phylogenetic analysis showed that there may be six subgroups in this order; each subgroup occupied a different habitat. Conclusions: Bradymonabacteria have unique living strategies that differ from those of so-called “obligate” or “facultative” predators. Thus, we propose a framework to categorize the current bacterial predators into 3 groups: (i) highly prey-dependent predators, (ii) facultatively prey-dependent predators, and (iii) prey-independent predators. Our findings provide an ecological and evolutionary framework for Bradymonadales and highlight their potential ecological roles in saline environments.