Acinetobacter species are widely distributed bacteria in the environment, and have recently gained notoriety as opportunistic nosocomial pathogens. Here we characterize a novel RNA polymerase-interacting protein named acidic transcription factor A, AtfA. It is small and highly acidic, and is widely distributed throughout the γ proteobacteria, including other significant pathogens in the genera Moraxella, Pseudomonas, Legionella and Vibrio. In the model species A. baylyi ADP1, deletion of atfA significantly affects expression of over 500 genes, resulting in a large cell phenotype, reduced cell fitness, impaired biofilm formation and twitching motility, and increased sensitivity to antibiotics. Deletion of atfA also causes dramatically enhanced sensitivity to ethanol, which is an important growth promoter and virulence factor in Acinetobacter spp. The results suggest that auxiliary factors of RNA polymerase with important biological roles remain to be discovered.