The involvement of two primary alcohol dehydrogenases, BDH and BOH, in butane utilization in Pseudomonas butanovora (ATCC 43655) was demonstrated. The genes coding for BOH and BDH were isolated and characterized. The deduced amino acid sequence of BOH suggests a 67-kDa alcohol dehydrogenase containing pyrroloquinoline quinone (PQQ) as cofactor and in the periplasm (29-residue leader sequence). The deduced amino acid sequence of BDH is consistent with a 70.9-kDa, soluble, periplasmic (37-residue leader sequence) alcohol dehydrogenase containing PQQ and heme c as cofactors. BOH and BDH mRNAs were induced whenever the cell's 1-butanol oxidation activity was induced. When induced with butane, the gene for BOH was expressed earlier than the gene for BDH. Insertional disruption of bdh or boh affected adversely, but did not eliminate, butane utilization by P. butanovora. The P. butanovora mutant with both genes boh and bdh inactivated was unable to grow on butane or 1-butanol. These cells, when grown in citrate and incubated in butane, developed butane oxidation capability and accumulated 1-butanol. The enzyme activity of BOH was characterized in cell extracts of the P. butanovora strain with bdh disrupted. Unlike BDH, BOH oxidized 2-butanol. The results support the involvement of two distinct NAD ؉ -independent, PQQ-containing alcohol dehydrogenases, BOH (a quinoprotein) and BDH (a quinohemoprotein), in the butane oxidation pathway of P. butanovora.Pseudomonas butanovora (ATCC 43655) is an aerobic gramnegative proteobacterium closely related to the genera Thauera and Azoarcus as shown by analysis of its 16S rRNA (1). This organism has been classified in the genus Pseudomonas based on its morphology, physiology, and biochemistry (39, 40). P. butanovora was isolated from activated sludge from an oil-refining company for the purpose of generating biomass from n-alkanes (39, 40). P. butanovora can derive energy for growth from C 2 to C 9 n-alkanes and any of their oxidation products as well as from a variety of other carbon sources (39, 40). Butane-grown P. butanovora can oxidize some chlorinated hydrocarbons by cometabolism through the action of a monooxygenase (18) and thus may have applications in bioremediation schemes.The pathway for the oxidation of butane in P. butanovora proceeds primarily from butane to 1-butanol, to butyraldehyde, to butyrate (2), and then probably to the ␤-oxidation pathway of fatty acid oxidation. As in other alkane utilizers (3, 27, 36), in P. butanovora the oxidation of the alkane (butane) is initiated by the action of a monooxygenase (19). Each intermediate in the pathway accumulated in the presence of appropriate inhibitors, supported cell growth, and stimulated O 2 consumption (2). The presence of a terminal butane oxidation pathway (i.e., production of 1-butanol) in P. butanovora was indicated (2). Although butane-grown cells consumed 2-butanol, 2-butanol production (indicative of a subterminal oxidation pathway) was not demonstrated, even in the presence of appropriate inhibitors of 2-b...

4-Chloroaniline has been released into the environment due to extensive use in chemical industries and intensive agriculture; hence, it becomes one of the hazardous pollutants in the priority pollutant list. In this study, three gram-negative bacteria were enriched and isolated from agricultural soil as 4-chloroaniline-degrading bacteria. They were identified as Acinetobacter baumannii CA2, Pseudomonas putida CA16 and Klebsiella sp. CA17. They were able to utilize 4-chloroaniline as a sole carbon and nitrogen source without stimulation or cocultivation with aniline or another cosubstrate. The biodegradation in these bacteria was occurred via a modified ortho-cleavage pathway of which the activity of chlorocatechol 1, 2-dioxygenase was markedly induced. They grew well on 0.2-mM 4-chloroaniline exhibiting a 60-75% degradation efficiency and equimolar liberation of chloride. The isolates were able to survive in the presence of 4-chloroaniline at higher concentrations (up to 1.2 mM). 2-Chloroaniline, 3-chloroaniline and aniline, but not 3, 4-dichloroaniline, were also growth substrates for these isolates. The results of cosubstrate supplementation illustrated the suitable conditions of each isolate to improve growth rate and 4-chloroaniline biodegradation efficiency. These results suggest that these isolates have a potential use for bioremediation of the site contaminated with 4-chloroaniline.

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