Systematic Taxonomy Of Nitrogen-fixing Bacteria Belonging To Enterobacteriales Based On The Whole Genome Sequences

The bacterial order Enterobacteriales was recently proposed to be divided into seven families(Enterobacteriaceae,Erwiniaceae fam.nov.,Pectobacteriaceae fam.nov.,Yersiniaceae fam.nov.,Hafniaceae fam.nov.,Morganellaceae fam.nov.,and Budviciaceae fam.nov.)based on phylogenetic analyses and comparative genomic analyses.This study systematically classified the nitrogen-fixing bacteria belonging to the order Enterobacteriales using bacterial whole genome sequences and taxono-genomic methods.Nitrogenase and nif genes were searched from 14754 whole genome sequences of bacteria belonging to 53 genera of the order Enterobacteriales,which were released into the NCBI GenBank database.Two hundred and seventeen nitrogen-fixing bacterial strains belonging to four families(Enterobacteriaceae,Pectobacteriaceae,Yersiniaceae and Erwiniaceae)were found.Based on analyses of the 16S rRNA gene identities between nitrogen-fixing strains and type strains and analyses of the average nucleotide identities between genome pairs,and phylogenomic analyses on amino acid sequences of core proteins,these nitrogen-fixing bacteria were classified into the genera Klebseilla,Raoultella,Kosakonia and Mangrovibacter of the family Enterobacteriaceae,the genera Dickeya,Pectobacterium and Brenneria of the family Pectobacteriaceae,the genus Rahnella of the family Yersiniaceae,and the genus Pantoea of the family Erwiniaceae.The analyses also classified 38 strains into 13 novel species belonging to the genera Klebsiella,Koskonia,Dickeya,Pectobacterium,Rahnella and Pantoea.All the members of the species Klebsiella vaniicola,Klebsiella michiganensis,Raoultella terrigena,D.dadantii,D.dianthicola,D.paradisiaca,D.solani,P.atrosepticum and the 13 potential novel species are nitrogen-fixing bacteria.Phylogenomic analyses of the core proteins and analyses of the percentage of conserved proteins between genome pairs supported the combination of the genus Raoultella into the genus Klebsiella and the combinations of the species Enterobacter massiliensis and Escherichia vulneris into the genus Kosakonia.Phylogenetic analyses of the amino acid sequences of the NifH,NifD and NifK proteins showed that the nif genes had been transmitted through horizontal gene transfer between different families of the order Enterobacteriales,between different genera of a family,or between different species of a genus.For example,the only two nitrogen-fixing strains(At-9b and AS-PWVM4)of the genus Pantoea might obtain the nif genes through horizontal gene transfer from the nitrogen-fixing bacteria belonging to the genus Kosakonia.This study clarified the taxonomic status of the nitrogen-fixing bacteria belonging to the order Enterobacteriales.Most of the nitrogen-fixing enterobacteria belong to the genus Klebsiella and the genus Dickeya,which is pathogenic to humans and animals or plants,respectively,and thus should not be used as biofertilizers.