Construction And Functional Analysis Of TonB2 Gene Deletion Mutant In Novosphingobium Pentaromativorans US6-1

Novosphingobium pentaromativorans US6-1,a marine bacterium isolated from muddy sediment of Ulsan Bay,Republic of Korea,is a gram-negative bacterium,which can effectively degrade polycyclic aromatic hydrocarbons that have two to five benzene rings.Strand-specific RNA sequencing show that 13 TonB system-related genes significantly upregulated under BaP stress.In order to reveal the response of TonB system to BaP stress in strain US6-1,the key gene tonB in TonB system was studied.Based on the genomic sequence analysis and real-time quantitative PCR analysis,tonB gene deletion mutant of US6-1 was constructed by homologous recombination of suicide plasmids,and the biological characters of tonB gene-deleted strain was studied.The main results are as follows:(1)Genome analysis revealed the locations of 4 hypothetical tonB genes in the Novosphingobium pentaromativorans US6-1 genome.We termed them tonBl,tonB2,tonB3 and tonB4 respectively.The tonB3(JI59_RS10120)gene is organized into operon with exbDl(JI59_RS10130)/exbD2(JI59_RS10135)/motA(JI59_RS 10125)genes.tonBl(JI59_RS03025),tonB2(JI59_RS06215)and tonB4(JI59_RS 15490)exist in the form of monocistronic copy,with no flanking exbB/exbD or its homologous genes were detected.These three tonB genes may interact directly with corresponding TonB-dependent receptors to accomplish a certain biological function.(2)The amino acid sequence analysis shows that the homology of the 4 TonB amino acids is 13.28%to 23.96%,and the multiple sequence alignment shows that 4 TonB proteins all contain a motif called "YP",and that they all contain a motif called"SSG" except TonB4.We can find by tertiary structure prediction that carboxyl terminal domains of TonB1,TonB2,TonB3 and TonB4 all contain 2 a-helixes and 3?-pleated sheets.(3)The result of quantitative PCR shows that the gene expression level of tonB1 tonB3 and tonB4 increase significantly after 4-day starvation,and the level is higher than that after 4-day in BaP treatment.The expression level of tonB2 increases greatly after 2-day or 6-day under BaP treatment,and it is notably higher than that under starvation.Thus,we predict that tonB2 is related to the response of US6-1 to the pressure of BaP,whiletonBl,tonB3 and tonB4 may be involved in the response to starvation.(5)In this study,tonB2 gene was deleted by homologous recombination of suicide plasmids,to generate the mutant US6-l?tonB2,and then the biological characters of US6-l?tonB2 were studied.The results show that inactivation of tonB2 had no significant effects on the growth of US6-l?tonB2;Biodegradation rate of Phe and BaP by US6-l?tonB2 were significantly impaired,suggesting that tonB2 plays an important role in PAHs degradation.On the other hand,tonB2 deletion in US6-1 remarkably enhanced biofilm formation.