The physiological contribution of Acinetobacter PcaK, a transport system that acts upon protocatechuate, can be masked by the overlapping specificity of VanK.
VanK is the fourth member of the ubiquitous major facilitator superfamily of transport proteins to be identified that, together with PcaK, BenK, and MucK, contributes to aromatic catabolism in Acinetobacter sp. strain ADP1. VanK and PcaK have overlapping specificity for p-hydroxybenzoate and, most clearly, for protocatechuate: inactivation of both proteins severely impairs growth with protocatechuate, and the activity of either protein alone can mask the phenotype associated with inactivation of its homolog. Furthermore, vanK pcaK double-knockout mutants appear completely unable to grow in liquid culture with the hydroaromatic compound quinate, although such cells on plates convert quinate to protocatechuate, which then accumulates extracellularly and is readily visible as purple staining. This provides genetic evidence that quinate is converted to protocatechuate in the periplasm and is in line with the early argument that quinate catabolism should be physically separated from aromatic amino acid biosynthesis in the cytoplasm so as to avoid potential competition for intermediates common to both pathways. Previous studies of aromatic catabolism in Acinetobacter have taken advantage of the ability to select directly strains that contain a spontaneous mutation blocking the beta-ketoadipate pathway and preventing the toxic accumulation of carboxymuconate. By using this procedure, strains with a mutation in structural or regulatory genes blocking degradation of vanillate, p-hydroxybenzoate, or protocatechuate were selected. In this study, the overlapping specificity of the VanK and PcaK permeases was exploited to directly select strains with a mutation in either vanK or pcaK. Spontaneous mutations identified in vanK include a hot spot for frameshift mutation due to contraction of a G6 mononucleotide repeat as well as point mutations producing amino acid substitutions useful for analysis of VanK structure and function. Preliminary second-site suppression analysis using transformation-facilitated PCR mutagenesis in one VanK mutant gave results similar to those using LacY, the prototypic member of the major facilitator superfamily, consistent with the two proteins having a similar mechanism of action. The selection for transport mutants described here for Acinetobacter may also be applicable to Pseudomonas putida, where the PcaK permease has an additional role in chemotaxis.
D'Argenio
D A
DA
Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520-8103, USA.
Segura
A
A
Coco
W M
WM
Bünz
P V
PV
Ornston
L N
LN
D000150:Acinetobacter / Q000201:enzymology / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial / Q000235:genetics; D005816:Genetic Complementation Test; D062385:Hydroxybenzoates / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D009154:Mutation; D010802:Phylogeny; D011520:Protocatechuate-3,4-Dioxygenase / Q000235:genetics / Q000378:metabolism; D011801:Quinic Acid / Q000378:metabolism; D017385:Sequence Homology; D013379:Substrate Specificity; D013489:Suppression, Genetic; D027981:Symporters; D013696:Temperature; D014641:Vanillic Acid / Q000378:metabolism
D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D062385:Hydroxybenzoates; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; C090106:PcaK protein, Pseudomonas; D027981:Symporters; C119579:VanK protein, Bacillus subtilis; D011801:Quinic Acid; C009091:protocatechuic acid; C021499:lactose permease; D011520:Protocatechuate-3,4-Dioxygenase; D014641:Vanillic Acid