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.
Department of Microbiology, University of Iowa, Iowa City 52242.
C S
CS
Harwood
N N
NN
Nichols
M K
MK
Kim
J L
JL
Ditty
R E
RE
Parales
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