The Function Of Coding Genes Located On Linear Plasmid PBSSB1 Of Salmonella Enterica Serover Typhi

Objective:The investigation of linear plasmid has become a new hotspot in studies of prokaryote since the first bacterial linear plasmid was discovered in Streptomyces rochei. It has been reported that linear plasmid played an important role in in virulence and the synthesis of some antibiotics as the extra-chromosomal genetic elements and self replicating genetic elements. Linear plasmid p BSSB1 is the first non-bacteriophage-related linear plasmid to be reported in the Enterobacteriaceae. To know more about of p BSSB1 in S. Typhi, we constructed several strains and researched the function of two genes located on p BSSB1.Methods:1. The construction of LP008 gene over-expressing strain, heterologous expression strain, mutant strain and LP002 gene mutant strain. The identified recombinant vector p BAD-LP008 and empty p BAD/Myc-His A were cloned into the wild type train(S. Typhi GIFU10007) as LP008 gene over-expression strain(WT-LP008) and control strain(WT-p BAD), into S. Typhi Ty2 as heterologous expression strain(Ty2-LP008) and control strain(Ty2-p BAD). The mutant strains of LP008 and LP002 genes were constructed using a homologous recombination system. Homologous recombination fragments of LP008 and LP002 genes were cloned to the sucrose-sensitive suicide vector p GMB151 and the wild type strain transformed with the recombinant vector were cultured on LB plates with sucrose to filtrate correct mutant strains.2. The preparation and purification of LP008 protein. The identified recombinant vector p ET28b-LP008 was transformed into E. coli JM109(DE3) competent cells to gain the positive strain. After induction with IPTG, bacteria were collected to be splited by ultrasonic treatment. The LP008 protein with 6x His Tag in the supernatant was purified using a Ni-colum.3. The growth curves under different conditions. To compare the growth condition between different strains, the growth curves was drew with the OD600 value as Y-axis and the time bacteria had been cultured as X-axis.4. Analysis of Biofilm formation. The LP008 gene over-expressing strain and the control strain were incubated to log-phase(OD600 0.5~ OD600 0.6) in TSB medium.After that, the bacteria were inoculated into 96-well microtitre plate and incubated at 30 °C for 96 hrs to facilitate the formation of biofilm. The medium was discarded and the biofilm were stained with crystal violet. The crystal violet was removed and rinsed with water prior to extracting biofilm with acetic acid. The absorption of the eluted stain stands the quantity of biofilm.5. The motility assay. Each strain was incubated to log-phase(OD600 0.4) and 1 μl of bacterial LB were stab incubated through a motility-swim agar plate at 37 °C for 12 hours. The diameter of the circular zone was measured to evaluate the motility.6. The epithelial cells invasion assay. Each strain was incubated to log-phase and incubated with He La cells. The ability of invasion was compared between the strains to research the function of each gene.7. The macrophages intracellular survival assay. Each strain was incubated to log-phase and incubated with THP-1 cells. The ability of phagocytosis of THP-1cells and the ability of intracellular survival ability were compared between the strains to evaluate the function of these two genes.8. q RT-PCR. Total RNAs were extracted for reverse transcription. The target genes were found the mechanism of the regulation was explored by using q RT-PCR.9. Gene chip. Total RNAs of heterologous expression strain(Ty2-LP008) and control strain(Ty2-p BAD) were extracted for reverse transcription. Total c DNAs were marked with fluorescein and hybridized with the genomic microarray. The scanning signals of fluorescence were digitized to analyze the gene expression difference between the two strains.10. EMSA. The purified LP008 protein was incubated with the promoter DNAs of target genes, and then subjected to electrophoresis on 6% polyacrylamide nondenaturing gels to test the binding between LP008 protein and the promoter DNAs.Results:1. LP008 gene over-expressing strain, heterologous expression strain, LP002 gene mutant strain were constructed successfully, however, the construction of LP008 gene mutant strain was failed.2. The LP008 protein with His6 Tag was purified and acquired.3. The results of growth curves under normal condition shows that LP008 gene over-expression strain grows slower than the control strain at first and faster after the value of OD600 turn to 1.5; there is no significant difference between LP002 gene mutant strain and the wild type strain under any condition.4. The formation of biofilm in LP008 gene over-expression strain was decreased compared with the control strain.5. The motility of LP008 gene over-expression strain was decreased compared with the control strain. There is no significant difference between LP002 gene mutant strain and the wild type strain in motility assay.6. Compared with the control strain, the invasion ability to epithelial cells was decreased after over-expressing LP008 gene, while the ability of LP002 gene mutant strain was significantly increased when bacteria were incubated to early log-phase and decreased when bacteria were incubated to late log-phase.7. Unlike the result of invasion assay, LP002 gene mutant strain kept the same trend no mater what log-phase bacteria were incubated to: ability of intracellular survival in macrophages was decreased compared with the wild type strain the wild type strain.8. The result of q RT-PCR indicated that the expression of flh D, fli A, fli L, flj A, flj B,inv F, iag A, pil S and csg D decrease in LP008 gene over-expression strain.9. There is no obvious gene expression difference between heterologous expression strain and the corresponding control strain.10. The LP008 protein can bind the promoter DNAs of target genes but the binding between protein and DNA is not specific.Conclusions:The two genes, LP008 and LP002, located on p BSSB1 in S. Typhi play a role in different aspects of pathopoiesia. The LP008 protein regulates the bacterial motility,biofilm formation and invasion; while the LP002 protein affects the invasion and intracellular survival in macrophages. This study of linear plasmid p BSSB1 lays the foundation of further research to reveal more function of the 33 genes located on p BSSB1.