Pmr, a nucleoid-associated protein encoded on the catabolic plasmid pCAR1, functions coordinately with TurA and TurB encoded on the chromosome of Pseudomonas putida KT2440. These MvaT homologs bind to DNA, form hetero-oligomers, and regulate transcription. Here, we assessed the DNA-binding affinity of the C-terminal DNA-binding domains: Pmr_ct, TurA_ct, and TurB_ct. The affinity for DNA duplexes containing T-A steps, A-tracts, or GC-rich sequences was evaluated using surface plasmon resonance. All proteins exhibited the highest affinity for DNA duplexes containing T-A steps. Although MvaT homologs preferentially bind to AT-rich sequences, Pmr_ct, TurA_ct, and TurB_ct exhibited higher affinity for DNA duplexes containing GC-rich sequences than A-tracts. TurB_ct exhibited the highest affinity for all DNA duplexes tested, whereas the DNA-binding affinity of TurA_ct was higher than Pmr_ct. Among the six lysine residues interacting with DNA in MvaT from Pseudomonas aeruginosa, one was substituted with alanine in Pmr, and two with valine and arginine in TurB. TurA_ct variants mimicking the amino acid substitutions in Pmr and TurB partially explained the decreased and increased DNA-binding affinity of Pmr_ct and TurB_ct, respectively, compared to TurA_ct. These results provide essential insights into how chromosomally encoded MvaT homologs function coordinately after the acquisition of an additional plasmid-encoded MvaT homolog.