Isolation, characterization, genomic sequencing, and GFP-marked insertional mutagenesis of a high-performance nitrogen-fixing bacterium, Kosakonia radicincitans GXGL-4A and visualization of bacterial colonization on cucumber roots

• Published in: Folia microbiologica 2018-11, Vol.63 (6), p.789-802
• Main Authors: Sun, Shuaixin, Chen, Yunpeng, Cheng, Jiejie, Li, Qiongjie, Zhang, Zhenchuan, Lan, Zhengliang
• Format: Article
• Language: English
• Subjects: Applied Microbiology; Biomedical and Life Sciences; Cell culture; Colonization; Deoxyribonucleic acid; DNA; Elongation; Environmental Engineering/Biotechnology; Fluorescence; Fluorescence microscopy; Gene sequencing; Genetic modification; Genomes; Immunology; Inoculation; Insertional mutagenesis; Life Sciences; Microbiology; Mutagenesis; Nitrogen; Nitrogen fixation; Nucleotide sequence; Original Article; Performance indices; Phylogeny; Physiological effects; Proteins; Ribosomal DNA; Roots; Seedlings; Species classification; Sustainable agriculture; Technology assessment; Transposon Tn5
• ISSN: 0015-5632; 1874-9356
• DOI: 10.1007/s12223-018-0608-1

A gram-negative bacterium GXGL-4A was originally isolated from maize roots. It displayed nitrogen-fixing (NF) ability under nitrogen-free culture condition, and had a significant promotion effect on cucumber growth in the pot inoculation test. The preliminary physiological and biochemical traits of GXGL-4A were characterized. Furthermore, a phylogenetic tree was constructed based on 16S ribosomal DNA (rDNA) sequences of genetically related species. To determine the taxonomic status of GXGL-4A and further utilize its nitrogen-fixing potential, genome sequence was obtained using PacBio RS II technology. The analyses of average nucleotide identity based on BLAST+ (ANIb) and correlation indexes of tetra-nucleotide signatures (Tetra) showed that the NF isolate GXGL-4A is closely related to the Kosakonia radicincitans type strain DSM 16656. Therefore, the isolate GXGL-4A was eventually classified into the species of Kosakonia radicincitans and designated K . radicincitans GXGL-4A. A high consistency in composition and gene arrangement of nitrogen-fixing gene cluster I ( nif cluster I) was found between K . radicincitans GXGL-4A and other Kosakonia NF strains. The mutants tagged with green fluorescence protein (GFP) were obtained by transposon Tn5 mutagenesis, and then, the colonization of gfp -marked K . radicincitans GXGL-4A cells on cucumber seedling root were observed under fluorescence microscopy. The preferential sites of the labeled GXGL-4A cell population were the lateral root junctions, the differentiation zone, and the elongation zone. All these results should benefit for the deep exploration of nitrogen fixation mechanism of K . radicincitans GXGL-4A and will definitely facilitate the genetic modification process of this NF bacterium in sustainable agriculture.