Analysis Of Key Genes For The Survival Of Pantoea Agglomerans Under Nutritional Stress

Pantoea agglomerans is Gram-negative bacteria of the genus Pantoea widely distributed in nature and can cause a variety of plant diseases as pathogens.P.agglomerans CHTF15 strain was isolated from the diseased leaf tissue of walnut infected with bacterial blight,and the strain had a strong ability to infect walnut leaves.After colonizing on the leaf surface of plants,the bacteria can invade from stomata or scars and cause leaf lesions,which will cause necrosis of the leaf surface and fruit of walnut.Nutrients on plant leaf surface are relatively scarce,so the propagation of bacteria is under nutritional stress during the process of infecting the leaf surface of plants.Study the key genes of P.agglomerans under nutritional stress will help us to understand the pathogenic mechanism of P.agglomerans.It will help find the prevention and treatment of diseases caused by this pathogen.In this study,the whole genome of CHTF15 was sequenced by the second and third generation sequencing platforms,which get the complete genome map of the strain.The genome was 4 820 607 bp in size,containing 1 chromosome and 3 plasmids.The genome contained 4 336 CDS regions,22 rRNA and 75 tRNA.Compared with database,the whole genome of CHTF15 was annotated,and its pathogenicity and virulence factors were analyzed.This strain can produce a variety of toxins,such as Colibactin protein and hemolysin,The VI secretion system can inject them into host cells.Transposon insertion sequencing(Tn-seq)is a technology that combines high-throughput sequencing with transposon-constructed random mutant libraries to analyze bacterial genomes.A random mutation library of CHTF15 strain was constructed using Mariner transposon.The growth curve of CHTF15 strain in different dilution YEP liquid medium was drawn to determine the growth and reproduction of CHTF15 strain,which was limited by nutrient concentration in 0.1%YEP liquid medium,and this condition was used as the nutrient stress treatment condition.The random mutation library of CHTF15 strain under nutritional stress was processed,and used Tn-seq technology to analyze the essential genes under nutritional stress.Finally,105 essential genes were found in CHTF15 strain under nutritional stress.15 significantly different genes were screened by volcano plot(log2FC≤-1 and P-value≤0.05).The COG(Clusters of Orthologous Groups)classification of 105 genes involve in energy production and conversion,amino acid transport and metabolism,intracellular transport,secretion and vesicle transport,coenzyme transport and metabolism,transcription and posttranslational modification,protein turnover and partner,carbohydrate transport and metabolism,DNA replication recombination and repair,and other biological activities.Enrichment analysis and metabolic annotation of significantly different genes showed that protein synthesis processes such as disulfide bond formation,amino acid transport,tricarboxylic acid cycle and pentose phosphate pathway in carbon metabolism were important for strain survival under nutritional stress.At the same time,under nutritional stress,it will promote the accumulation of peroxide and cause DNA damage,which needs the dependent RecA of homologous recombination system to repair.The 15 significantly different gene annotations mainly included three classifications:amino acid or protein synthesis and metabolism,nucleic acid metabolism and recombination repair.Four genes including epmA,dksA,ribB and xerD were selected to make deletion mutant strains,epmA gene is related to protein translation,dksA is related to protein disulfide bond formation,ribB gene is the key to riboflavin anabolism,and xerD gene is related to chromosome dimer decomposition in the recombination repair phase.When the key gene deletion mutant and the wild type strain were cocultured on 0.1%YEP and walnut leaf surface,the competition indexes in 0.1%YEP and walnut leaf surface were 0.064,0.120,0.078,0.105 and 0.087,0.091,0.075,0.091 respectively.The survival ability of the mutant strain was significantly decreased compared with that of the wild strain under nutrient stress conditions,indicating that the selected genes are essential genes for P.agglomerans under nutrient stress conditions.This study laid a foundation for clarifying the mechanism of P.agglomerans survival in leaf surface and the infection process of pathogenic bacteria.