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 inAcinetobacter 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 inAcinetobacter have taken advantage of the ability to select directly strains that contain a spontaneous mutation blocking the β-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 eithervanK 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.

Mapping the diversity of microbial lignin catabolism: experiences from the eLignin database

“…This may be explained by the fact that these compounds are commonly protonated at neutral pH and—due to the hydrophobic charge—can partition into the membrane and damage the structure (Kamimura et al 2017 ; Parales and Ditty 2017 ). Gram-negative bacteria with reported aromatic transporters include Acinetobacter baylyi ADP1 (Collier et al 1997 ; D’Argenio et al 1999 ), Bradyrhizobium japonicum USDA110 (Michalska et al 2012 ), Klebsiella pneumoniae M5a1 (Xu et al 2012 ), Pseudomonas putida KT2440 (Nishikawa et al 2008 ) and PRS2000 (Nichols and Harwood 1997 ), Rhodopseudomonas palustris CGA009 (Giuliani et al 2011 ; Michalska et al 2012 ), Sinorhizobium meliloti 1024 (Michalska et al 2012 ), and Sphingobium sp. SYK-6 (Mori et al 2018 ).…”

Substitution, Insertion, Deletion, Suppression, and Altered Substrate Specificity in Functional Protocatechuate 3,4-Dioxygenases

“…The product of protocatechuate oxygenase is carboxymuconate, and Acinetobacter strains blocked in its metabolism fail to grow with any substrate when they are exposed to either protocatechuate or its metabolic precursors. Thus, it is possible to select for strains in which spontaneous secondary mutations block earlier steps in either transport (6) or catabolism of aromatic compounds (Fig. 1).…”

Novel metabolic features in Acinetobacter baylyi ADP1 revealed by a multiomics approach

“…The last putative binding site is located between vanK and vanA/B (Table 1 ). On the one hand, it was suggested that vanK , whose product has overlapping specificities with PcaK, is also induced by protocatechuate in ADP1 (D’Argenio et al 1999 ). The authors also pointed out that this metabolite may also induce expression of vanA and vanB .…”