title: Chimeras of Escherichia coli and Mycobacterium tuberculosis single-stranded DNA binding proteins: characterization and function in Escherichia coli.
abstract: Single stranded DNA binding proteins (SSBs) are vital for the survival of organisms. Studies on SSBs from the prototype, Escherichia coli (EcoSSB) and, an important human pathogen, Mycobacterium tuberculosis (MtuSSB) had shown that despite significant variations in their quaternary structures, the DNA binding and oligomerization properties of the two are similar. Here, we used the X-ray crystal structure data of the two SSBs to design a series of chimeric proteins (mβ1, mβ1'β2, mβ1-β5, mβ1-β6 and mβ4-β5) by transplanting β1, β1'β2, β1-β5, β1-β6 and β4-β5 regions, respectively of the N-terminal (DNA binding) domain of MtuSSB for the corresponding sequences in EcoSSB. In addition, mβ1'β2(ESWR) SSB was generated by mutating the MtuSSB specific 'PRIY' sequence in the β2 strand of mβ1'β2 SSB to EcoSSB specific 'ESWR' sequence. Biochemical characterization revealed that except for mβ1 SSB, all chimeras and a control construct lacking the C-terminal domain (ΔC SSB) bound DNA in modes corresponding to limited and unlimited modes of binding. However, the DNA on MtuSSB may follow a different path than the EcoSSB. Structural probing by protease digestion revealed that unlike other SSBs used, mβ1 SSB was also hypersensitive to chymotrypsin treatment. Further, to check for their biological activities, we developed a sensitive assay, and observed that mβ1-β6, MtuSSB, mβ1'β2 and mβ1-β5 SSBs complemented E. coli Δssb in a dose dependent manner. Complementation by the mβ1-β5 SSB was poor. In contrast, mβ1'β2(ESWR) SSB complemented E. coli as well as EcoSSB. The inefficiently functioning SSBs resulted in an elongated cell/filamentation phenotype of E. coli. Taken together, our observations suggest that specific interactions within the DNA binding domain of the homotetrameric SSBs are crucial for their biological function.
authors.affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India.
authors.forename: Sanjay Kumar
authors.initials: SK
authors.lastname: Bharti
authors.forename: Kervin
authors.initials: K
authors.lastname: Rex
authors.forename: Pujari
authors.initials: P
authors.lastname: Sreedhar
authors.forename: Neeraja
authors.initials: N
authors.lastname: Krishnan
authors.forename: Umesh
authors.initials: U
authors.lastname: Varshney
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D002918:Chymotrypsin / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000166:cytology / Q000254:growth & development / Q000378:metabolism*; D005453:Fluorescence; D005816:Genetic Complementation Test; D006801:Humans; D007700:Kinetics; D009169:Mycobacterium tuberculosis / Q000378:metabolism*; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*; D017186:Titrimetry
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D002918:Chymotrypsin