Molecular Study On The Gene Containing Serine - Aspartate Repeats In Staphylococcus

Horizontal gene transfer (HGT) is recognized as one of the major forces for bacterial genome evolution. Many clinically important bacteria may acquire virulence factors and antibiotic resistance through HGT. The comparative genomic analysis has become an important tool for identifying HGT in emerging pathogens. In this study, the Serine-Aspartate Repeat (Sdr) family has been compared among different sources of Staphylococcus aureus (S. aureus) to discover sequence diversities within their genomes. Four sdr genes were analyzed for21different S. aureus strains and218mastitis-associated S. aureus isolates from Canada. Comparative genomic analyses revealed that S. aureus strains from bovine mastitis (RF122and mastitis isolates in this study), ovine mastitis (ED133), pig (ST398), chicken (ED98), and human methicillin-resistant S. aureus (MRSA)(TCH130, MRSA252, Mu3, Mu50, N315,04-02981, JH1and JH9) were highly associated with one another, presumably due to HGT. In addition, several types of insertion and deletion were found in sdr genes of many isolates. A new insertion sequence was found in mastitis isolates, which was presumably responsible for the HGT of sdrC gene among different strains. Moreover, the sdr genes could be used to type S. aureus. Regional difference of sdr genes distribution was also indicated among the tested S. aureus isolates. Finally, certain associations were found between sdr genes and subclinical or clinical mastitis isolates. Certain sdr gene sequences were shared in S, aureus strains and isolates from different species presumably due to HGT. Our results also suggest that the distributional assay of virulence factors should detect the full sequences or full functional regions of these factors. The traditional assay using short conserved regions may not be accurate or credible. These findings have important implications with regard to animal husbandry practices that may inadvertently enhance the contact of human and animal bacterial pathogens.Tandem repeats (either as microsatellites or minisatellites) in eukaryotic and prokaryotic organisms are mutation-prone DNA. While minisatellites in prokaryotic genomes are underrepresented, the cell surface adhesins of bacteria often contain the minisatellite SD repeat, especially in Staphylococcal strains. However, its relationship to biological functions is still elusive. In this study, therefore, effort was made to uncover the copy number variations of serine-aspartate (SD) repeats by bioformational analyses and detect the stability of SD repeats utilizing a plasmid-based assay, as a first step to understand its biological functions. The SD repeats were mainly present in the cell surface proteins. The SD repeats were genetically unstable and polymorphic in terms of copy numbers and sequence compositions. In addition, the change of its copy number was reversible, without frame shifting. A rearrangement hot spot, the ATTC/AGRT site, was found to be mainly responsible for the instability and reversibility of SD repeats. These characteristics of SD repeats can facilitate bacteria to respond quickly to environmental changes, with low cost, low risk and high efficiency. Consequently, we propose the SD repeats as contingency loci.The thermostability of the xylanase (xynR8) from uncultured Neocallimastigales rumen fungal was improved by directed evolution using error-prone PCR. Transformants expressing the variant xylanases were selected utilizing high-throughout96-well screening system. Whereas the wild type XynR8lost almost80%activities after5min at75℃, three mutants displayed higher thermostabilities than XynR8. Totally five amino acids replacement occurred in these three mutants after sequencing, which were138V, A104T, F116L, D137G and G151D, respectively. DNA shuffling and Site-directed mutagenesis was further used to identify the positive factors and negative factors for the increased thermostability of xylanases, and three positive factors were found as I38V, D137G and G151D. Based on the predicted results of RosettaDesign software, more site-directed mutagenesis was performed to identify the most thermostable amino acids on the three sites. Finally, the most thermostable XynR8mutant XynR8_VNE was obtained through DNA shuffling. It contained three mutations I38V, D137N and G151E, and showed an increase in melting temperature of8.8℃. We propose the decreased repulsive force; improved hydrophobic interaction and salt-bridge at the sites38,137and151are responsible for the improved thermostability. Three corresponding regions with frangibility and flexibility, which were N-terminus, the cord domain and the a-helix domain, were found to be instable in high temperature. This suggests to improve the stability of these three regions probably could have beneficial effect to increase the thermostability of xylanase.