Acinetobacter lwoffii K24 known as an aniline degrading bacterium has also been found to utilize p-hydroxybenzoate as a sole carbon source. In this study, 2-DE using Q-Sepharose column separation was attempted for fast screening of protocatechuate 3.4-dioxygenase for catabolism of p-hydroxybenzoate in A. lwoffii K24. Two protocatechuate 3.4-dioxygenase subunits, pcaG and pcaH were detected and identified with N-terminal and internal sequencing, suggesting proteomics using a column separation may be helpful for the identification of specific protein spots and maximizing the detectable protein spots on the 2-DE gel. The PCR process using degenerate primers for protocatechuate 3.4-dioxygenase and sequence analyses of the PCR products revealed the existence of pcaH and peaG in A. lwoffii K24. These two subunits were found to be closely located and share extensive homology with peaH and peaG of Pseudomonas marginata or Pseudomonas cepacia. providing the evidence that A. lwoffi K24 has the protocatechuate branches as well as catechol branches of beta-ketoadipate pathway. (C) 2002 Elsevier Science (USA). All rights reserved.