Evolution of Rhodopseudomonas palustris to degrade halogenated aromatic compounds involves changes in pathway regulation and enzyme specificity

Abstract

Halogenated aromatic compounds are used in a variety of industrial applications but can be harmful to humans and animals when released into the environment. Microorganisms that degrade halogenated aromatic compounds anaerobically have been isolated but the evolutionary path that they may have taken to acquire this ability is not well understood. A strain of the purple nonsulfur bacterium, Rhodopseudomonas palustris, RCB100, can use 3-chlorobenzoate (3-CBA) as a carbon source whereas a closely related strain, CGA009, cannot. To reconstruct the evolutionary events that enabled RCB100 to degrade 3-CBA, we selected for and isolated a CGA009 strain capable of growing on 3-CBA, although not as well as RCB100. Comparative whole-genome sequencing of the evolved strain and RCB100 revealed large deletions encompassing badM, a transcriptional repressor of genes for anaerobic benzoate degradation. It was previously shown that in strain RCB100, a single nucleotide change in an alicyclic acid coenzyme A ligase gene, named aliA, gives rise to a variant AliA enzyme that has high activity with 3-CBA. When we introduced the RCB100 aliA allele and a badM deletion into R. palustris CGA009, it grew on 3-CBA at a similar rate as RCB100. This work provides an example of pathway evolution that includes a variant of a promiscuous enzyme with enhanced substrate specificity and a regulatory mutation that leads to constitutive expression of a pathway that does not regulate the promiscuous enzyme. ImportanceBiodegradation of man-made compounds often involves the activity of promiscuous enzymes whose native substrate is structurally similar to the man-made compound. Based on the enzymes involved, it is possible to predict what microorganisms are likely involved in biodegradation of anthropogenic compounds. However, there are examples of organisms that contain the required enzyme(s) and yet cannot metabolize these compounds. We found that even when the purple nonsulfur bacterium, Rhodopseudomonas palustris, encodes all the enzymes required for degradation of a halogenated aromatic compound, it is unable to metabolize that compound. Using adaptive evolution, we found a regulatory mutation and a variant of promiscuous enzyme with increased substrate specificity were required, but the ability to metabolize a halogenated aromatic compound also resulted in reduced fitness on another aromatic compound. This work provides insight into how an environmental isolated evolved to use halogenated aromatic compounds and the potential ecological trade-offs associated with this adaptation.