Molecular Mechanisms Of Morphological Differentiation In Paenibacillus Polymyxa SC2

Paenibacillus polymyxa is rod-shaped, endospore forming, Gram-positive bacteria, often isolated from rhizosphere soil of plants. It is widely recognized as a plant-growth-promoting rhizobacteria(PGPR) as a result of its outstanding promoting effect on plant growth and significant inhibition against the growth of pathogenic microorganisms in recent years. P.polymyxa produces various antimicrobial substances, such as polymyxin, circulin, jolipeptin,polypeptins, gatavalin and fusaricidins, some of which are synthesized by non-ribosomal peptide synthetases(NRPS). These features attract more and more researchers.Our lab isolated a P. polymyxa strain, named SC2, previously. It shows great inhibition against various pathogenic microorganisms, including fungus and bacteria. Under the laboratory environment, however, the colony morphology of SC2 shows great instability.Two mutants, named SC2-M1 and SC2-M2 were isolated from the successive cultivation of SC2. Both of them showed significant differences in endospore formation,flagella and motility, colony morphology, exopolysaccharide production, antagonism, biofilm formation, nutrient utilization and growth curve when compared with wild type SC2.Using genome resequencing and transcriptome sequencing strategy, we got a more comprehensive information at a genetic and transcriptomic level. Genome resequencing revealed 14 mutations occurred in SC2-M1 compared with SC2, including 10 SNV, three Indels and one ITX. While nine mutations occurred in SC2-M2 compared with SC2,including seven SNV and two Indels. Only one mutation occurred in both SC2-M1 and SC2-M2 was directly related to sporulation, which was the G>A in position 3,383,926 and located in 632 th nucleotide of spo0 A. This SNV caused a former 211 Arg turned into a 211 His.spo0A is a key regulator gene which play a role in the initial stage of sporulation. Comparison of amino acis sequences of Spo0 A in P. polymyxa SC2 and other endospore-forming strains indicated a high conservation at 211 Arg, and it is happened to be located in the HTH domain which interact with its target DNA sequence.Transcriptome sequencing results revealed that a large number of sporulation-relatedgenes were significantly down-regulated in a transcribe level both in SC2-M1 and SC2-M2,especially the ones which were under the direct control of Spo0 A, such as spoIIAA, spoIIAB,spoIIE, sigE and sigF. Genes related to the initial stage of sporulation, however, such as genes related to phosphorelay including spo0 A, and a series of kinase(kinA-E), exhibited no significant differences at the transcribe level. So functional inactivation which induced by the single amino acid mutation occurred in Spo0 A probably caused the failure to form endospore.Complementation of spo0 A was performed in both SC2-M1 and SC2-M2. Using genome recombination with the help of suicide plasmid, we complemented the mutated211 His to Arg. By the same way, we also substituted the His to Asp and Ala in SC2-M2,respectively. Results exhibited that only 211 Arg could restored the endospore formation ability. This study confirmed the sporulation defeciency was directly caused by the G>A mutation in 632 th nucleotide of spo0 A, further more, it confirmed the function requirement of211 Arg of Spo0 A.Except for the sporulation defeciency which was caused by the SNV located in spo0 A,transcriptomic analysis revealed other key mutations probably caused differences in flagella and motility, exopolysaccharide production, and fusaricidins production, providing a comprehensive analysis of differences exhibited by mutants were induced by mutations located in their genome.Molecular mechanism of sporulation is not only a basic biological question, but also has significance in agricultural applications. This study provided abundant genetic backgroud information of sporlation in P. polymyxa. Further more, database obtained in this study could shed light on the research of functional genomics of P. polymyxa SC2.