Structure–function relationships of two paralogous single-stranded DNA-binding proteins from Streptomyces coelicolor: implication of SsbB in chromosome segregation during sporulation

Abstract

The linear chromosome of Streptomyces coelicolor contains two paralogous ssb genes, ssbA and ssbB. Following mutational analysis, we concluded that ssbA is essential, whereas ssbB plays a key role in chromosome segregation during sporulation. In the ssbB mutant, ∼30% of spores lacked DNA. The two ssb genes were expressed differently; in minimal medium, gene expression was prolonged for both genes and significantly upregulated for ssbB. The ssbA gene is transcribed as part of a polycistronic mRNA from two initiation sites, 163 bp and 75 bp upstream of the rpsF translational start codon. The ssbB gene is transcribed as a monocistronic mRNA, from an unusual promoter region, 73 bp upstream of the AUG codon. Distinctive DNA-binding affinities of single-stranded DNA-binding proteins monitored by tryptophan fluorescent quenching and electrophoretic mobility shift were observed. The crystal structure of SsbB at 1.7 Å resolution revealed a common OB-fold, lack of the clamp-like structure conserved in SsbA and previously unpublished S-S bridges between the A/B and C/D subunits. This is the first report of the determination of paralogous single-stranded DNA-binding protein structures from the same organism. Phylogenetic analysis revealed frequent duplication of ssb genes in Actinobacteria, whereas their strong retention suggests that they are involved in important cellular functions.

Variations in amino acid composition in bacterial single stranded DNA–binding proteins correlate with GC content

“…While studying structure/function relationship of the paralogous SSBs in streptomycetes we noticed that bacteria with high GC content possess some structural elements previously reported for mycobacterial SSBs (23). Moreover, SSBs from streptomycetes (GC content over 70 %) exhibit the high content of glycine (57 %) in their Ct domain (Table 2) and also show some additional specific structural variations which contribute to structural stability of respective SSBs (18,23). Based on this, we proposed that all reported variations may be of significance for SSB functioning in a high GC content bacteria (18).…”

“…These strands form two additional clamp-like structures in the homotetrameric SSB which contribute to the overall stability of the quaternary structure of SSB (18,21). It is thought that the mode of DNA binding of actinobacterial SSBs is different from that of EcoSSB partly on account of the difference in the shape of the tetramers (22,23).…”

Characterization of single-stranded DNA-binding protein SsbB fromStaphylococcus aureus: SsbB cannot stimulate PriA helicase

“…Reaction products were visualized by autoradiography and quantified with a phosphorimager. an additional strand (b6), 18 SaSsbB does not contain b6. Additional b6 strands clamp two neighboring subunits together in a tetrameric SSB.…”

“…SsbA and SsbB are essential for genome maintenance and transformational recombination, respectively. [15][16][17][18] Signicant differences for SsbBs are found in their C-terminal sequences and DNA binding properties. In Bacillus subtilis, SsbB binds to ssDNA with lesser affinity than BsSsbA does.…”

“…17 However, Streptomyces coelicolor SsbB (ScSsbB) exhibits greater DNA-binding affinity than ScSsbA does. 18 Unlike Streptococcus pneumonia SsbB (SpSsbB), BsSsbB and ScSsbB lack the C-terminal acidic tail of SSB for protein-protein interactions. [16][17][18] Thus, SsbBs from different organisms exhibit different protein-DNA and proteinprotein interaction specicities.…”