Differential Single-stranded DNA Binding Properties of the Paralogous SsbA and SsbB Proteins from Streptococcus pneumoniae

Diane E Grove,Smaranda Willcox,Jack D Griffith,Floyd R Bryant

doi:10.1074/jbc.m414057200

Abstract

The naturally transformable Gram-positive bacterium Streptococcus pneumoniae has two single-stranded DNA-binding (SSB) proteins, designated SsbA and SsbB. The SsbA protein is similar in size to the well characterized SSB protein from Escherichia coli (SsbEc). The SsbB protein, in contrast, is a smaller protein that is specifically induced during natural transformation and has no counterpart in E. coli. In this report, the single-stranded DNA (ssDNA) binding properties of the SsbA and SsbB proteins were examined and compared with those of the SsbEc protein. The ssDNA binding characteristics of the SsbA protein were similar to those of the SsbEc protein in every ssDNA binding assay used in this study. The SsbB protein differed from the SsbA and SsbEc proteins, however, both in its binding to short homopolymeric dT(n) oligomers (as judged by polyacrylamide gel-shift assays) and in its binding to the longer naturally occurring X and M13 ssDNAs (as judged by agarose gel-shift assays and electron microscopic analysis). The results indicate that an individual SsbB protein binds to ssDNA with an affinity that is similar or higher than that of the SsbA and SsbEc proteins. However, the manner in which multiple SsbB proteins assemble onto a ssDNA molecule differs from that observed with the SsbA and SsbEc proteins. These results represent the first analysis of paralogous SSB proteins from any bacterial species and provide a foundation for further investigations into the biological roles of these proteins.

Highlights

Spection of the genome sequence reveals that S. pneumoniae has two single-stranded DNA-binding (SSB) proteins, designated SsbA and SsbB
These results indicate that the SsbA and SsbB proteins form stable tetramers in solution, and it will be presumed in the discussion below that it is the tetrameric form of these proteins that binds to singlestranded DNA (ssDNA) as well
The polyacrylamide gel shift results indicated that the SSB protein from Escherichia coli (SsbEc), SsbA, and SsbB proteins each bind to the shorter oligomer, dT50, to form a complex in which a single SSB tetramer is bound to the ssDNA

Summary

EXPERIMENTAL PROCEDURES

Materials—S. pneumoniae SsbA protein was prepared as described (5). E. coli SSB protein was from Promega. 32P-end-labeled dTn oligomers were prepared using [␥-32P]ATP and polynucleotide kinase (Amersham Biosciences). G250 BioSafe Coomassie protein gel stain was from Bio-Rad. ␾X ssDNA concentrations were determined by absorbance at 260 nm using the conversion factor 36 ␮g mlϪ1 A260Ϫ1. DTn concentrations were determined by absorbance at 260 nm using the extinction coefficient 8.4 mMϪ1 cmϪ1 (2). All ssDNA concentrations are expressed as total nucleotides. Gel-exclusion Chromatography—Gel-exclusion chromatography analysis of the SSB proteins was carried out using a Superose 12-gelexclusion column (24 ml, Amersham Biosciences) with 20 mM Tris-Cl. 2 M.

Streptococcus pneumoniae SsbA and SsbB Proteins

RESULTS

The complexes that were formed by the SsbA and SsbB

DISCUSSION