The Effect Of Iron Starvation On Growth Of Streptococcus Iniae And Cloning And Expression Of FtsABCD Genes

Streptococcus iniae is one of the most important fish pathogens that caused millions of dollars economic damage every year. In order to control Streptococcus iniae effectively, studies that focused on the virulence factors are needed. In this study, we find that iron starvation caused by addition of NTA in the final concentration 5mM postpones the log phase of bacterial growth almost 6 hours, when examined under microscope the gram stained bacteria showed a long chain appearance and the protein profile changed a lot compared with the positive control. Siderophore could not be detected by the universal CAS assay. Using the protein sequences of Streptococcus pyogene ferrichrome transporter FtsABCD, we searched the raw genome sequence of Streptococcus iniae 9117 provided by the Baylor College of Medicine. Then we designed 2 pairs of nested PCR primers for each gene based on the conserved domains and cloned part sequences of the ftsABCD of Streptococcus iniae. The cloned sequences matched perfectly with the genome sequences of Streptococcus iniae, in this case we designed the ORF primers based on the ORF predictions of the sequences from the database. As a result, the length of ftsA is 783 bp, ftsB is 927 bp, ftsC is 1029 bp, ftsD is 999bp. The results of signal peptide and transmembrane prediction, and combined with the results of NCBI's Conserved Domains search indicated that FtsABCD of Streptococcus iniae constitutes an ABC transporter in which FtsA is ATP binding protein, FtsB is substrate binding protein, FtsC and FtsD are permeases. Next, the prokaryote expression system of FtsB was constructed using the PCR products of ftsB mature sequence and the plasmid pET-32a. Then the recombinate FtsB protein was pured by Ni+ affinity chromatography and lyophilized and stored at -80℃.In the serum of mouse that have been infected by Streptococcus iniae we detected antibody against the recombinate FtsB protein specifically by western blot.In concluding, iron is an essential nutrient that Streptococcus iniae needs to survive. And the ftsABCD gene of Streptococcus iniae constitutes an ABC transporter, ftsB gene is translated during the infection of mouse by Streptococcus iniae. Finally, the product of ftsB has some kind of immunogenicity and could be used as the candidate of subunit vaccine.