Due to their involvement in processes such as DNA replication, repair, and recombination, bacterial single-stranded DNA binding (SSB) proteins are essential for the survival of the bacterial cell. Whereas most bacterial SSB proteins form homotetramers in solution, dimeric SSB proteins were recently discovered in the Thermus/Deinococcus group. In this work we characterize the biophysical properties of the SSB protein from Thermus aquaticus (TaqSSB), which is structurally quite similar to the tetrameric SSB protein from Escherichia coli (EcoSSB). The binding of TaqSSB and EcoSSB to single-stranded nucleic acids was found to be very similar in affinity and kinetics. Mediated by its highly conserved C-terminal region, TaqSSB interacts with the chi-subunit of E. coli DNA polymerase III with an affinity that is similar to that of EcoSSB. Using analytical ultracentrifugation, we show that TaqSSB mutants are able to form tetramers in solution via arginine-mediated hydrogen-bond interactions that we identified in the crystal packing of wild-type TaqSSB. In EcoSSB, we identified a homologous arginine residue involved in the formation of higher aggregates and metastable highly cooperative single-stranded DNA binding under low salt conditions.