Identification of the pcaRKF gene cluster from Pseudomonas putida: involvement in chemotaxis, biodegradation, and transport of 4-hydroxybenzoate.
Pseudomonas putida PRS2000 is chemotactic to 4-hydroxybenzoate and other aromatic acids. This behavioral response is induced when cells are grown on 4-hydroxybenzoate or benzoate, compounds that are degraded via the beta-ketoadipate pathway. Isolation of a transposon mutant defective in 4-hydroxybenzoate chemotaxis allowed identification of a new gene cluster designated pcaRKF. DNA sequencing, mutational analysis, and complementation studies revealed that pcaR encodes a regulatory protein required for induction of at least four of the enzymes of the beta-ketoadipate pathway and that pcaF encodes beta-ketoadipyl-coenzyme A thiolase, the last enzyme in the pathway. The third gene, pcaK, encodes a transporter for 4-hydroxybenzoate, and this protein is also required for chemotaxis to aromatic acids. The predicted PcaK protein is 47 kDa in size, with a deduced amino acid sequence indicative of membership in the major facilitator superfamily of transport proteins. The protein, expressed in Escherichia coli, catalyzed 4-hydroxybenzoate transport. In addition, whole cells of P. putida pcaK mutants accumulated 4-hydroxybenzoate at reduced rates compared with that in wild-type cells. The pcaK mutation did not impair growth at the expense of 4-hydroxybenzoate under most conditions; however, mutant cells grew somewhat more slowly than the wild type on 4-hydroxybenzoate at a high pH. The finding that 4-hydroxybenzoate chemotaxis can be disrupted without an accompanying effect on metabolism indicates that this chemotactic response is receptor mediated. It remains to be determined, however, whether PcaK itself is a chemoreceptor for 4-hydroxybenzoate or whether it plays an indirect role in chemotaxis. These findings indicate that aromatic acid detection and transport are integral features of aromatic degradation pathways.
Harwood
C S
CS
Department of Microbiology, University of Iowa, Iowa City 52242.
Nichols
N N
NN
Kim
M K
MK
Ditty
J L
JL
Parales
R E
RE
D000102:Acetyl-CoA C-Acyltransferase / Q000235:genetics; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D001565:Benzoates / Q000378:metabolism; D019817:Benzoic Acid; D001673:Biodegradation, Environmental; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics; D002633:Chemotaxis; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics*; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D005810:Multigene Family / Q000235:genetics*; D009154:Mutation; D010226:Parabens / Q000378:metabolism*; D016958:Pseudomonas putida / Q000235:genetics*; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013386:Succinates / Q000378:metabolism; D019802:Succinic Acid
D001426:Bacterial Proteins; D001565:Benzoates; D002352:Carrier Proteins; D026901:Membrane Transport Proteins; D010226:Parabens; C090106:PcaK protein, Pseudomonas; D011994:Recombinant Proteins; D013386:Succinates; D019817:Benzoic Acid; D019802:Succinic Acid; D000102:Acetyl-CoA C-Acyltransferase; C443987:3-oxoadipyl-coenzyme A thiolase; C038193:4-hydroxybenzoic acid