SAAV2152 is a single-stranded DNA binding protein: the third SSB inStaphylococcus aureus

Abstract

Single-stranded DNA-binding proteins (SSBs) play crucial roles in DNA replication, repair, and recombination. Unlike E. coli, which contains only one type of SSB (EcSSB), some bacteria have two paralogous SSBs, namely, SsbA and SsbB. In this study, we found the third SSB-like protein in Staphylococcus aureus, SAAV2152, which was designated as SaSsbC. SaSsbC is a protein of 131 amino acids and shares 38%, 36%, and 33% sequence identity to SaSsbB, SaSsbA, and EcSSB, respectively. Gene map analysis showed that unlike the E. coli ssb gene, which is adjacent to uvrA gene, the S. aureus ssb gene SAAV2152 is flanked by the putative SceD, the putative YwpF, and fabZ genes. A homology model showed that SaSsbC consists of the classic oligonucleotide/oligosaccharide-binding fold at the N-terminus. At the C-terminus, SaSsbC did not exhibit sequence similarity to that of EcSSB. Electrophoretic mobility shift analysis showed that SaSsbC formed a single complex with ssDNA of different lengths. Mutational analysis revealed that Tyr36, Tyr47, Phe53, and Tyr81 in SaSsbC are at positions that structurally correspond to the important residues of EcSSB for binding to ssDNA and are also critical for SaSsbC to bind ssDNA. Unlike EcSSB, which can stimulate EcPriA, SaSsbC did not affect the activity of SaPriA. In addition, SaSsbA inhibitor 9-methyl-2,3,7-trihydroxy-6-fluorone (NSC5426) could inhibit the ssDNA-binding activity of SaSsbC with IC50 of 78 μM. In conclusion, this study has identified and characterized SAAV2152 as a kind of SSB, and further research can directly focus on determining its actual physiological role in S. aureus.

Plumbagin, a Natural Product with Potent Anticancer Activities, Binds to and Inhibits Dihydroorotase, a Key Enzyme in Pyrimidine Biosynthesis

“…The construction of StDHOase [61], KpDHOase [62], huDHOase [61], and ScD-HOase [61,63] expression plasmids were reported previously. The recombinant protein was purified using the protocol described previously for SSB-like proteins [64][65][66][67][68]. Briefly, E. coli BL21(DE3) cells were transformed with the expression vector, and the overexpression of the expression plasmids was induced by incubating with 1 mM isopropyl thiogalactopyranoside.…”

The glycine-rich flexible region in SSB is crucial for PriA stimulation

“…17 Recently, some SSB inhibitors as broad-spectrum antibacterial agents targeting P. aeruginosa and other pathogens have been discovered. 18,19 Thus, PaSSB structure may be needed as a basis for rational drug design to target P. aeruginosa.…”

Characterization of the Chimeric PriB-SSBc Protein

“…However, sequence comparisons and operon organization analyses indicate that PriB evolved from SSB via gene duplication with subsequent rapid sequence diversification [ 40 ]. The significant difference between PriB and SSB is the protein length; that is, PriB does not have SSBc consisting of IDL and SSB-Ct. IDL [ 56 ] and SSB-Ct [ 18 , 57 , 58 , 59 ] in SSB are required to stimulate the activity of PriA. Interestingly, PriB does not possess SSBc but can still stimulate the activity of PriA [ 37 ].…”

“…Although SSBs from eubacteria [ 12 ] to higher eukaryotes (e.g., RPA) [ 3 ] share basic mechanistic functioning, such as ssDNA binding and protection from damage during DNA replication, they are different in terms of structure and many other functions [ 56 , 59 , 63 , 70 , 71 , 72 ]. In addition, many bacteria have more than one paralogous SSB, such as SsbA [ 59 ], SsbB [ 58 , 62 ], and SsbC [ 57 ], in S. aureus . In E. coli , PriB is also identified as a kind of SSB [ 45 , 47 ].…”

“…To execute the specific mission in physiology, these OB-fold proteins may contain additional different functional domains and regions, such as SSBc in SSB. The amino acid residues in SSBc are not conserved in amino acid residues identity and the gene product length, such as for SaSsbA [ 59 ], SaSsbB [ 58 ], and SaSsbC [ 57 ]. Unlike the Gram-negative bacterial SSBs [ 18 , 56 ], these Gram-positive bacterial SSBs did not stimulate the activity of PriA.…”