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Applied and Environmental Microbiology, March 1999, p. 951-960, Vol. 65, No. 3
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Molecular Characterization of the Genes pcaG and pcaH, Encoding Protocatechuate 3,4-Dioxygenase, Which Are Essential for Vanillin Catabolism in Pseudomonas sp. Strain HR199

Jörg Overhage,1 Andreas U. Kresse,2 Horst Priefert,1,* Horst Sommer,3 Gerhard Krammer,3 Jürgen Rabenhorst,3 and Alexander Steinbüchel1

Institut für Mikrobiologie der Westfälischen Wilhelms-Universität Münster, D-48149 Münster,1 G.B.F. National Research Center for Biotechnology, D-38124 Braunschweig,2 and Corporate Research, Haarmann & Reimer GmbH, D-37601 Holzminden,3 Germany

Received 6 April 1998/Accepted 14 December 1998

Pseudomonas sp. strain HR199 is able to utilize eugenol (4-allyl-2-methoxyphenol), vanillin (4-hydroxy-3-methoxybenzaldehyde), or protocatechuate as the sole carbon source for growth. Mutants of this strain which were impaired in the catabolism of vanillin but retained the ability to utilize eugenol or protocatechuate were obtained after nitrosoguanidine mutagenesis. One mutant (SK6169) was used as recipient of a Pseudomonas sp. strain HR199 genomic library in cosmid pVK100, and phenotypic complementation was achieved with a 5.8-kbp EcoRI fragment (E58). The amino acid sequences deduced from two corresponding open reading frames (ORF) identified on E58 revealed high degrees of homology to pcaG and pcaH, encoding the two subunits of protocatechuate 3,4-dioxygenase. Three additional ORF most probably encoded a 4-hydroxybenzoate 3-hydroxylase (PobA) and two putative regulatory proteins, which exhibited homology to PcaQ of Agrobacterium tumefaciens and PobR of Pseudomonas aeruginosa, respectively. Since mutant SK6169 was also complemented by a subfragment of E58 that harbored only pcaH, this mutant was most probably lacking a functional beta  subunit of the protocatechuate 3,4-dioxygenase. Since this mutant was still able to grow on protocatechuate and lacked protocatechuate 4,5-dioxygenase and protocatechuate 2,3-dioxygenase, the degradation had to be catalyzed by different enzymes. Two other mutants (SK6184 and SK6190), which were also impaired in the catabolism of vanillin, were not complemented by fragment E58. Since these mutants accumulated 3-carboxy muconolactone during cultivation on eugenol, they most probably exhibited a defect in a step of the catabolic pathway following the ortho cleavage. Moreover, in these mutants cyclization of 3-carboxymuconic acid seems to occur by a syn absolute stereochemical course, which is normally only observed for cis,cis-muconate lactonization in pseudomonads. In conclusion, vanillin is degraded through the ortho-cleavage pathway in Pseudomonas sp. strain HR199 whereas protocatechuate could also be metabolized via a different pathway in the mutants.

* Corresponding author. Mailing address: Institut für Mikrobiologie, Westfälische Wilhelms-Universität Münster, Corrensstrasse 3, D-48149 Münster, Germany. Phone: 49-251-8339829. Fax: 49-251-8338388. E-mail: priefer{at}uni-muenster.de.

Applied and Environmental Microbiology, March 1999, p. 951-960, Vol. 65, No. 3
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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