The physiological contribution of Acinetobacter PcaK, a transport system that acts upon protocatechuate, can be masked by the overlapping specificity of VanK

Abstract

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.

FIG. 1

Positive selection of mutants blocked in catabolism of aromatic or hydroaromatic compounds. Strain ADP500 contains the engineered ΔpcaBDK1 deletion and cannot grow with succinate in the presence of protocatechuate (or compounds upstream in the β-ketoadipate pathway) due to the toxic accumulation of β-carboxy-cis,cis-muconate. ADP500 derivatives in which a spontaneous mutation blocks the β-ketoadipate pathway upstream of carboxymuconate can therefore be selected. After correction of the ΔpcaBDK1 deletion to wild type, the phenotype of the remaining spontaneous mutation can be revealed by growth tests with single carbon sources.

FIG. 2

Chromosomal region containing vanA and vanB, genes for vanillate demethylase. Arrows denote direction of transcription. DNA fragments cloned in plasmids pZR139, pZR143, and pZR144 (69) were used to map vanK mutations by marker rescue.

FIG. 3

Nucleotide sequence of vanK and deduced amino acid sequence of the encoded protein. Underlined with a dashed line are 12 segments with amino acids potentially forming transmembrane α-helices (identified by the TMpred program of the ISREC-Bioinformatics Group, Geneva, Switzerland). The orientation in the membrane of these segments is predicted to alternate with the first helix oriented inside to outside. Sequenced spontaneous mutations are indicated above vanK nucleotides and above VanK amino acids; all were selected based on loss of VanK function except for the intragenic suppressors of vanK1113: the underlined vanK7610 and vanK7611 (together in ADP7610) and vanK7612 (in ADP7612). The TAG sequence duplicated as a result of IS1236 insertion in vanK7603 is underlined below a vertical arrow. Underlined below an open triangle are the G₆ and A₇ mononucleotide repeats in which 1 bp is deleted, in ΔvanK1103 and ΔvanK1108, respectively. Horizontal arrows are shown above the first and last nucleotides deleted in ΔvanK7607 and ΔvanK7602, the latter deletion including one of two repeats of the 8-bp sequence GCTGGCGT (underlined). Downstream from vanK is a gene for a putative porin (69) (Fig. 2).

FIG. 4

Relative sequence divergence in the aromatic acid permease family. The organisms in which these proteins were identified and the substrates, inferred or demonstrated, for the transporters are as follows: PcaK (Ac) in Acinetobacter sp. strain ADP1, protocatechuate and p-hydroxybenzoate (reference and this study); PcaK (Ps) in P. putida, protocatechuate and p-hydroxybenzoate (34, 56); MhtP in E. coli, 3-(3-hydroxyphenyl)propionate (20); BenK in Acinetobacter sp. strain ADP1, benzoate (9); TfdK in Ralstonia eutropha: 2,4-dichlorophenoxyacetate (48); HppK in Rhodococcus globerulus: 3-(3-hydroxyphenyl)propionate (2); VanK in Acinetobacter sp. strain ADP1, protocatechuate and p-hydroxybenzoate (this study); MucK in Acinetobacter sp. strain ADP1, cis,cis-muconate (77). The degree to which each of these proteins may facilitate transport of multiple related compounds remains unknown. A new family of carboxylic acid permeases including MucK, distinct from the aromatic acid permease family (61), may become apparent with additional sequencing (2). The sequence of Acinetobacter PcaK in GenBank has been updated (accession no. AF009672, June 1998).

FIG. 5

Predicted periplasmic localization of quinate and shikimate catabolism to protocatechuate. Genetic evidence from this study suggests that the sequential action of the QuiA membrane-bound quinate/shikimate dehydrogenase (17), the QuiB dehydroquinate dehydratase (18), and the QuiC dehydroshikimate dehydratase (18) produces protocatechuate in the periplasm. Protocatechuate thus formed could then be transported across the inner membrane (dashed horizontal lines) into the cytoplasm by the PcaK and VanK permeases. Such compartmentation would reduce competition for intermediates that are common to both the catabolic pathway and the biosynthetic pathway for generation of aromatic amino acids. The putative porin QuiX (18) may mediate entry of quinate and shikimate into the periplasm from outside the cell.

FIG. 6

Detection of extracellular protocatechuate generated by mutant strains growing in the presence of quinate. A 20-μl aliquot of an LB culture of each strain was spotted onto a plate with 10 mM succinate and 5 mM quinate and incubated overnight at 37°C. In the top and bottom rows are cells of ADP603 (Δqui1 ΔpcaBDK1) added simultaneously (top row) or 5 h after (bottom row) strains in the middle row (from left to right): ADP1 (wild type), ADP859 (ΔpcaK859), ADP7618 (ΔvanK1103), and ADP7577 (ΔpcaK859 ΔvanK1103). Deletion of quinate genes in ADP603 prevents the toxic accumulation of carboxymuconate during growth of this strain in the presence of quinate but not protocatechuate (Fig. 1). Growth of ADP603 can therefore serve as a detector of protocatechuate diffusing from adjacent cells of mutant strains impaired to various degrees in transport of protocatechuate across the inner cell membrane after its generation from quinate in the periplasm (Fig. 5). The speckling observed in the spot of ADP7577 cells is due to reversion of ΔvanK1103.

FIG. 7

Intragenic suppression of VanK1113 (Gly₇₁Asp). Transformation-facilitated PCR mutagenesis in ADP7587 (vanK1113 ΔpcaK859) generated two strains with second-site suppressors of VanK1113, a Gly₇₁Asp substitution in VanK transmembrane helix 2. ADP7610 had two suppressors in transmembrane helix 7, Phe₂₆₉Ser and Phe₂₇₁Ser, and ADP7612 had one suppressor adjacent to transmembrane helix 8, Gly₂₉₄Arg (Fig. 3).