Over-initiation of DNA replication in cells containing the cold-sensitive dnaA(cos) allele has been shown to lead to extensive DNA damage, potentially due to head-to-tail replication fork collisions that ultimately lead to replication fork collapse, growth stasis and/or cell death. Based on the assumption that suppressors of the cold-sensitive phenotype of the cos mutant should include mutations that affect the efficiency and/or regulation of DNA replication, we subjected a dnaA(cos) mutant strain to transposon mutagenesis and selected mutant derivatives that could form colonies at 30 degrees C. Four suppressors of the dnaA(cos)-mediated cold sensitivity were identified and further characterized. Based on origin to terminus ratios, chromosome content per cell, measured by flow cytometry, and sensitivity to the replication fork inhibitor hydroxyurea, the suppressors fell into two distinct categories: those that directly inhibit over-initiation of DNA replication and those that act independently of initiation. Mutations that decrease the cellular level of HolC, the chi subunit of DNA polymerase, or loss of ndk (nucleoside diphosphate kinase) function fall into the latter category. We propose that these novel suppressor mutations function by decreasing the efficiency of replication fork movement in vivo, either by decreasing the dynamic exchange of DNA polymerase subunits in the case of HolC, or by altering the balance between DNA replication and deoxynucleoside triphosphate synthesis in the case of ndk. Additionally, our results indicate a direct correlation between over-initiation and sensitivity to replication fork inhibition by hydroxyurea, supporting a model of increased head-to-tail replication fork collisions due to over-initiation.