Researches On Genomics And Comparative Genomics Of The Genus Paenibacillus

In 1993,Ash et al re-evaluated the classification status of the genus Bacillus rRNA group 3 and established the new genus Paenibacillus,which belongs to the family Paenibacillaceae within the order Bacillales.At present,the known munbers of the genus Paenibacillus reach up to 244 species.The shared characteristics of the genus Paenibacillus are as follows:Rod-shaped;Gram positive,negative or variable;Motile with peritrichous flagella;Endospore-forming.Many members of Paenibacillus have acquired evolutionarily a close relationship with plants,some of them exhibit specific functions including nitrogen fixation,growth promotion,and disease control.Accordingly,they would have broad application prospects in the agricultural fields.Now,genomics is becaming a popular research direction of microbiology.Our research group had performed some reseaches on genomics of Paenibacillus,but had not yet carried out comparative genomics of this genus.In this study,after genomic annotation of the 50 Paenibacillus species which had been sequenced before March 2016,their comparative genomic analyses were carried out emphasizing on genome composition,gene function,material transport,carbon metabolism,signal transduction,and gene expression regulation.Furthermore,phylogenetic,evolutionary and taxonomic analyses of Paenibacillus were performed using the whole genome.The preliminary results were as follows.1?Analyses of the basic genomic information of the 50 known Paenibacillus species Firstly,the following information was collected for each genome:the first discoverer of the species,whether the sequenced strain was the type strain,where the strain was isolated,Genbank accession numbers of 16s rRNA and genome sequence,sequencing platform and submission institution.Secondly,the repeats,encoding genes,non-encoding RNAs,pseudogenes,gene islands,CRISPRs,prophages,secretory proteins and transmembrane proteins of each genome were predicted by the corresponding softwares,respectively.Finally,circos circular plot of each genome was drawn according to the above-mentioned information.2?Comparative genomic analyses of the 50 known Paenibacillus species Genome components(1)The repetitive sequences in the Paenibacillus genomes consisted of the interspersed repeats and tandem repeats.For the interspersed repeats,the long interspered elements and short interspered elements were predominant,followed by the DNA transposable elements,and the long terminal repeats were rare.The tandem repeats included the minisatellite DNA?microsatellite DNA and low complexity sequence.Notably,the microsatellite DNA showed a significant expansion in each genome.(2)In the 50 genomes,number of coding genes was 2921-8865;number of pseudogenes was 0-66;number of gene islands was 2-23,their total length was 10067 bp-459155 bp;the numbers of secreted proteins and transmembrane proteins were 44-382 and 718-2134,respectively.(3)Non-encoding RNA:copy number of rRNA was different;number of tRNA was 30-104;the highest number of sRNA was up to 29,sRNA was not predicted in 8 genomes.(4)Number of prophage related sequences was 5-318.(5)Number of CRISPR/Cas related sequences was 7-304,coding Casl-8 and Cas10 proteins.The Cas related proteins were not predicted in 16 genomes which had CRISPR.The Cas9 protein was not found in all Paenibacillus genomes,indicating that the Paenibacilus CRISPR/Cas systems were type I and type III.Taken together,the results showed that the genome components,especially the coding genes,of the 50 known Paenibacillus species exhibited significant differences.The possible reasons would be:(1)Gain and loss of different gene families might be quitely distrinct in these genomes during the process of evolution;(2)The sequencing coverage,assembly integrity and prediction accuracy might be the influence factors.Gene function(1)The results of COG cluster analyses demonstrated that the coding genes of the 50 Paenibacillus genomes could be divided into 22 functional groups.(2)KEGG pathway analyses showed that these genomes possessed the maximum number of members of the ABC transport system,the two-component system,amino acid synthesis and carbon metabolism.Moreover,the ABC transport system,PTS system,and two component system could be divided into 8,6 and 10 families,respectively.(3)The related genes of the glycolysis,pentose phosphate pathway and tricarboxylic acid cycle could be all predicted in each genome,indicating that these Paenibacillus species have the complete central carbon metabolism pathways.In addition,10 genomes contained all enzymes for PHB biosynthesis.(4)The genomes had the complete chemotaxis systems except no CheA and CheY genes in the genome of P.dendritiformis,suggesting that P.dendritiformis would be unable to form flagella.(5)Prediction of the related genes indicated that the 50 Paenibacillus species all possessed the c-di-AMP and c-di-GMP second messenger signal transduction systems.The c-di-AMP synthase DisA in 15 genomes only had the DisA_N domain,but it contained DisA N and DisA_linker domains in other genomes.The c-di-GMP degrading enzyme phosphodiesterase which contained EAL domain was present in all genomes,and its copy number was 1-6.The bifunctional gene which contained GGDFF/EAL domains was present in 37 genomes,and its copy number was 1-12.There were 1-2 diguanylate cyclases which contained only GDEF domain in 4 genomes.(5)Regulation of sporulation:The sporulation-specific sigma factors and SpoOA phosphorylation related genes were all existed in each genome,but the copy number of each gene was different.3?Phylogeny,evolution and taxonomy analyses of the Paenibacillus species based on the whole genome(1)Phylogeny and evolution of the Paenibacillus species:The phylogenetic tree of the 50 known species based on whole genome single copy genes and 16S rRNA gene were constructed,respectively.The results showed that the 50 known species could be classified into the 8 evolutionary branches.The majority of members within the evolutionary branches of the 2 evolutionary trees were consistent with each other,although a few of them had changes.The phylogenetic tree based on the whole genome single copy genes could not only reconstruct the phylogenetic relationship of the Paenibacillus species,but also suggest the rnonophyletic origin of the genus Paenibacillus.Collinearity analysis further revealed the very close phylogeny relationships between many species.Cluster analysis of the gene families showed that number of the species-specific gene families in all genome was less than 9%.The results might suggested that:most of the gene families in Paenibacillus were evolutionarily conserved,and the main driving force for the difference in number of genes was the degree of replication and expansion of different genes in different genomes.(2)Taxonomic status of the 50 known Paenibacillus species:Firstly,the pairwise ANI and eDDH values between the 50 known species were calculated.According to the ANI and eDDH values,the taxonomic status of these species was evaluated,respectively.The results showed that the ANI and eDDH values between P.Sonchi and P.riograndensis were all higher than the corresponding thresholds.Therefore,they belonged to synonyms and would be re-classified.The ANI value between P.Peoriae and P.polymyxa was 96.23%,being higher than the threshold,and the eDDH value between them reached up to 69%,being extremely closed to the threshold.Therefore,their classification status also needs to be further evaluated.(3)Taxonomic status of the 61 unnamed Paenibacillus sp.strains:Firstly,the genome sequences of the 61 Paenibacillus sp.strains were downloaded from GenBank,and the pairwise ANI and eDDH valuess between the 111 genomes were calculated,respectively.Then,the taxonomic status of the 61 Paenibacillus sp.strains was evaluated.The results showed that the eDDH values between the 16 Paenibacillus sp.strains and the corresponding closest strains were more than 70%,which exceeded its threshold.Thus,they belonged to same species each two.Additionally,3 Paenibacillus sp.strains could be matched to the known species.The results showed that the ANI values between the 28 Paenibacillus sp.strains and the closest strains were more than 96%,being higher than its threshold.They belonged to the same species each two,and 5 Paenibacillus sp.strains could be matched to the known species.Based on the results,the 61 Paenibacillus sp.strains with genome sequencing could be identified as 35-40 independent species of Paenibacillus.The ANI and eDDH values between J14 and P.odorifer,JCM 16163 and p.naphthalenovorans,FSL H8-237 and P.barengoltzii were both exceeded the corresponding thresholds,therefore,J14,JCM 16163 and FSL H8-237 could be identified as a strain of P.odorifer,p.naphthalenovorans and P.barengoltzii,respectively.