Sub-population Census For Myxobacteria In Soil And Molecular Strategy Research For Their Territoriality

The order of Myxococcales is located in δ group of Proteobacteria, including three suborders, seven families, twenty genera and about fifty species. Due to formation of spores resistant to high heat, desiccation and UV, myxobacteria have adaptability to a wide range of environments, such as soils spreading all over the global. Besides of soil, myxobacteria have also been isolated from samples collected in dung of herbivores, rotting wood, bark, leaf surface and even ocean. Because of characteristics of myxobacteria, the traditional isolation and purification methods is time-consuming and limited to some particular population of myxobacteria, limiting the knowledge of myxobacterial ecology. Till now, we have no idea about myxobacterial abundance and community structure. As the maturity and popularity of next generation DNA sequencing techniques impelled us to a period of massive data and the establishment of public metagenomic database provided researchers a sharing platform, culture-independent molecular methods have initiated a new era of microbial ecology.Base on the above ecology issues we concerned, this study was carried out as follows:Firstly, myxobacteria strains in normal garden soil sample （named SDU soil, collected from Shandong University） were isolated by traditional methods and the percentage of myxobacteria among bacteria in this soil sample was detected by quantitative PCR. It was suggested that the proportion of myxobacteria in soil was2.04%～3.42%, but the species of culture strains were rare, predominant in Myxococcus, Corallococcus and Cystobacter with similar16S rRNA gene sequence to type strains, indicating the limitation of the culture-dependent method.Secondly, we surveyed the percentage of myxobacteria in a single soil sample via pyrosequencing on combined universal libraries of the V3-V4and V6-V8hypervariable regions of the16S rRNA gene. Surprisingly, myxobacteria accounted for4.10%of the bacterial community and7.5%of the total operational taxonomic units （OTUs） at the3%similarity level in the soil, containing almost all of the cultivated myxobacterial families or genera. To testify the appearance of myxobacteria in soil niches, we retrieved myxobacteria-related16S rRNA gene sequences of103high-throughput sequencing datasets obtained from public databases. The results indicated that myxobacteria-related sequences were among the predominant groups in these datasets, accounting for0.4%to4.5%of bacterial communities. Based on these results, we can presume that myxobacteria are a predominant terrestrial bacteria group in soil. The correlation was weak between myxobacteria percentage and environmental parameters calculated by Spearman’s rank, indicating the abundance of myxobacteria communities might be influenced by complex biological factors.At last, to conduct a sub-population census of myxobacterial community, two myxobacteria-enriched libraries of Cystobacterineae and Sorangineae suborders were constructed using myxobacteria-semi-specific primers. Although different taxa were predominant or rare, we observed a further division into many lower taxonomic units in myxobacteria-enriched libraries, resulting in low frequencies of each eco-type. These unprecedented results reveal that myxobacteria utilize an egalitarian and reciprocal strategy for community survival in the soil niche.According to the conclusion, the next ecology issue was raised:how is the relationship among the allied species? Myxobacteria are, though unicellular, famous for their complex social behaviours and the cooperation between genetic identical cells maintains throughout the entire life cycle. When cells aggregate after sensing the signal of starvation or move in a group dependent on binding of type VI pili on extracellular matrix, the exchange of signals or proteins on cell membrane is necessary to schedule preciously thousands of individuals. Though these pathways controlling the motility at time and space scales have been studied in detail, study about the interaction between different myxobacteria strains was stagnated on appearance. Exploitation had been reported might occur frequently in Myxococcus strains when mixed together, which was also observed between different Myxococcus spp. strains isolated from SDU soil. What’s more, we further analyzed the inhibition between two different strains in different mixing ratio, for different time and in different forms of co-culture. We found that when the two strains were inoculated in adjacent pair on growth medium, a clear boundary, like Dienes line in Proteus mirabilis, formed between the expanding colonies, while when they were mixed together, one strain was inhibited even killed by the other. The exploitative hierarchy could be converted, which was depended on mixing ratio but independent on direct cellular contact. The following results suggested that these strains could not product antibiotic Myxovirescin A in these strains to confirm it did not play role in the competition between Myxococcus strains.To disclose the involved mechanism of complex competition between different Myxococcus strains, we sought to identify self versus non-self discrimination factors required for boundary formation in model strain M.xanthus DK1622with clear genetic background. The pathways of myxobacterial A-motility, S-motility, production and regulation of exopolysaccharide and development were studied in this strain. We screened3,392inserted mutants generated by transposon plasmid and obtained eleven SI （self identification） mutants that recognize their neighbours as different from self, forming clearly visible boundaries between two colonies and with their parent strain DK1622as well. Though the SI mutants failed to merge with wild type strain, the social phenotypes of pure SI mutants were normal, indicating the cells from genetic identical SI mutant retain the capability to cooperate with each other. Whereas, the exploitative hierarchy was also found in SI mutants when different SI mutants were mixed and inoculated on growth plate, just like different wild Myxococcus strains. Mapping the insertion loci of transposon in eleven SI mutants showed nine loci scattered throughout DK1622genome and none of the inserted genes were reported involved in known pathway or system. The results of quantitative RT-PCR showed that the expressions of other eight SI loci were normal in SI1103（inserted in upstream of MXAN₅062）. These results indicated that there were several independent pathways involved kin discrimination and independent on other social behaviour systems. Three gene loci were identified to be responsible for formation of boundary by deletion and ectopic complementation, ie., MXAN₀049（SI11114）, MXAN₀085（SI1105） and MXAN₁307-MXAN₁309（SI1107, SI1108）. It is interesting that these genes share a sequence similarity with20-30%, and it also seems likely that other species of bacteria have homologous genes encoding self-recognition, which had been verified by replacing MXAN₀049with its homologous LILAB₀8510from M.fulvus HW-1.The Dienes mutual inhibition test has been used as an epidemiological tool to characterize isolates of Proteus mirabilis and Pseudomonas aeruginosa. Recently, by screening the mutants in the same way with us, Gibbs found that two loci were essential for the behaviour, and the two independent pathways were linked by a shared machinery, might type VI secretion system, for export of encoded self-recognition elements. But the biochemical mechanism remained to be identified.The next we focused on gene MXAN₀049for further analyze. As for the function of49, the sequence analysis might give a clue. MXAN₀049had30%identity with IpaC, which has been shown to associate with IpaB to form a translocation pore that enables the secretion of effector proteins by Shigella directly into target cells. However, we found that MXAN₀049was located in cytoplasm. RT-PCR showed that the MXAN₀049gene is part of an operon spanning MXAN₀046to MXAN₀050. What’s more, protein MXAN₀050might interact with MXAN₀049by his-tag pull down. The inhibition also occurred between deletion mutant of MXAN₀049and wild type strain DK1622both on CTT plate and TPM plate, resulting in disappearance or induction of△49. But deletion mutant of MXAN₀050swarmed merging with△49and DK1622. Therefore, we proposed that MXAN₀049and MXAN₀050which shared a similarity of26%with rhs protein were responsible for boundary formation by controlling attack and defense system.Intraspecies territoriality separates incompatible groups of single-celled bacterial species to prevent harm, usually mutual harm, from occurring. We hypothesized that in natural environment after adaptive evolution, different strains of one myxobacterial species localize to separate mini-territories to avoid cannibalism resulting from mixing and thus survive. Accordingly, kin discrimination is important not only for Myxococcus sociality, but also for their ecological functions, as well as speciation.