engleski

Functional characterisation of paralogous SSBs in Streptomyces coelicolor

Single stranded DNA binding proteins (SSBs) are essential for DNA metabolism in all organisms and viruses. These proteins protect single stranded DNA (ssDNA) intermediates and disrupt unproductive secondary structures. Our bioinformatics analysis of sequenced bacterial genomes revealed the presence of paralogoues SSBs in many bacteria thus indicating highly dynamic evolution of SSB proteins in Eubacteria. However the role of duplicated SSB proteins is poorly studied. S. coelicolor is the model representative of Streptomyces bacteria, the largest genus of Actinobacteria. Streptomyces species exhibit a complex lifecycle involving mycelial growth, spore formation and ability to produce a plethora of secondary metabolites including antibiotics and many other useful compounds. Since S. coelicolor has two paralogus genes encoding SSBs (ssbA and ssbB) we have selected this complex bacterium to study biological role(s) of paralogous SSB proteins. SSB proteins from most prokaryotic species function as tetramers that bind to ssDNA through structurally conserved N-terminal folding motifs (OB folds), while the C-terminal domain is responsible for protein interactions. We will show here interesting variations of 3D structure of two SSBs. Next molecular analyses showed that promoter regions of ssb genes differ significantly. In concert with this, expressions of ssb genes varied throughout development indicating that SSB proteins acquired different cellular functions. Expression of ssbA is constant and high during life cycle, decreasing towards late stationary phase, while the expression of ssbB is low at all-time. In minimal medium the ssbB is significantly upregulated. Gene disruptions strongly indicated that SsbA is essential for survival while SsbB is important during the sporulation process. To get a better insight into the role of SsbB and the complex mechanism during chromosome segregation in reproductive phase of growth we have constructed double and triple mutant strains carrying mutations in ssbB gene and genes reported previously to be important for the chromosomal segregation (for example parB or smc). By fluorescence microscopy we examined the effect of these mutations on chromosome segregation and spore formation. The results of these experiments showed severe defects in nucleoid segregations during sporulation as well as unequal distribution of DNA in spore compartments.

paralogous SSB; Streptomyces coelicolor

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