Degradation of phenol by

We have mutated Acinetobacter calcoaceticus NCIB-8250 to growth deficiency on phenol as sole carbon source and isolated genes with similarity to phenol hydroxylase and catechol 1,2-dioxygenase by complementation. Sequence analysis reveals the presence of six open reading frames (ORFs) with similarities to a Pseudomonas multicomponent phenol hydroxylase which are followed by an ORF with similarity to catA from A. calcoaceticus ADP1. Transformation of these genes to ADP1 confers the ability to grow at the expense of phenol as sole carbon source. Primer extension analysis indicates phenol-inducible transcription from an RpoN-dependent promoter sharing sequence similarity with the sigma 54 consensus promoter sequence, except that the -12 box is GG instead of GC. A catA::lacZ transcriptional fusion shows the same induction profile for beta-galactosidase expression as transcription from the sigma 54-dependent promoter. This result suggests that catA is cotranscribed in the same operon with the phenol hydroxylase-encoding genes and is consistent with the fact that no apparent additional promoter is found for catA by sequence analysis or primer extension. Catechol 1,2-dioxygenase activity is induced in NCIB8250 by benzoate, whereas beta-galactosidase expression from the catA::lacZ fusion is not. This observation leads to the hypothesis that two differentially regulated catA genes should be present in that strain.

The 7.5-kb polycistronic mop mRNA is differentially degraded in Acinetobacter calcoaceticus. The 4.9-kb 5¢ portion of the transcript contains the genes mopKLMNOP, encoding the multi-component phenol hydroxylase, and its 5¢ end decays three times faster than the 2.3-kb 3¢ portion encoding catechol 1,2-dioxygenase (catA). Larger amounts of the catA mRNA than the mopKLMNOP mRNA are present in the cells as a result of this processing. The site for endonucleolytic cleavage is located in the intercistronic region between mopP and catA, and contains a potential stem-loop structure and a putative RNase E cleavage site. Decay of the mop mRNA in Escherichia coli depends on RNase E. Thus, we propose that an RNase E-like activity is also present in A. calcoaceticus. Expression of MopN, one polypeptide of the multi-component phenol hydroxylase, interferes with growth of A. calcoaceticus. Thus, harmful expression of MopN may be reduced by rapid decay of its mRNA, indicating that mRNA processing contributes to differential gene expression in the large mop operon of A. calcoaceticus NCIB8250.