Use of Microcalorimetry To Determine the Costs and Benefits to Pseudomonas putida Strain KT2440 of Harboring Cadmium Efflux Genes

Abstract

A novel microcalorimetric approach was used to analyze the responses of a metal-tolerant soil bacterium (Pseudomonas putida strain KT2440) to metal resistance gene deletions in cadmium-amended media. As hypothesized, under cadmium stress, the wild-type strain benefited from the resistance genes by entering the exponential growth phase earlier than two knockout strains. In the absence of cadmium, strain KT1, carrying a deletion in the main component (czcA1) of a Cd/Zn chemiosmotic efflux transporter (CzcCBA1), grew more efficiently than the wild type and released ϳ700 kJ (per mole of biomass carbon) less heat than the wild-type strain, showing the energetic cost of maintaining CzcCBA1 in the absence of cadmium. A second mutant strain (KT4) carrying a different gene deletion, ⌬cadA2, which encodes the main Cd/Pb efflux transporter (a P-type ATPase), did not survive beyond moderate cadmium concentrations and exhibited a decreased growth yield in the absence of cadmium. Therefore, CadA2 plays an essential role in cadmium resistance and perhaps serves an additional function. The results of this study provide direct evidence that heavy metal cation efflux mechanisms facilitate shorter lag phases in the presence of metals and that the maintenance and expression of tolerance genes carry quantifiable energetic costs and benefits.Human activities have contaminated ecosystems around the world with persistent heavy metals (30). Metal-associated stress alters microbial community structure and metabolism (14,15,40) and therefore affects higher trophic interactions. Metal efflux pumps exploit the cellular pool of energy (ATP and chemiosmotic gradients) to pump metal ions out of a cell. Consequently, they should encumber the host organism with the energetic cost of maintaining and expressing the genes coding for them (10,32,42). The overall cost of encoding these traits, while necessary for survival in a contaminated environment, should decrease fitness in environments with low concentrations of metals (43). The energetic costs of maintaining metal resistance genes in the absence of bioavailable heavy metals in bacteria are generally assumed but apparently have never been directly quantified.The main toxic effect of cadmium on bacterial cells is to bind to and denature protein (33, 34). Cadmium-protein binding induces a cascade of other putative changes in the cell, such as the release of redox-active metals bound to proteins and the general disruption of many protein-mediated processes (20,44). Efflux pumps are a way of directly and efficiently dealing with cadmium, by transporting it out of the cell. Other cellular responses to cadmium involve the large-scale synthesis of molecules, such as glutathione, polysaccharides, and chaperone proteins, to bind cadmium and cope with cadmium-induced protein denaturation (13,16,20,33).Most bacteria nonspecifically import cadmium into the cell along with other divalent metal ions (31). The result can be seen in the evolution of multiple cadmium resistance mechanisms in bacteria (44). Mode...

Cadmium resistance mechanism in Escherichia coli P4 and its potential use to bioremediate environmental cadmium

“…Hyperaccumulation of cadmium ions may result in the disruption of bacterial respiratory proteins and generation of reactive oxygen species (Schirawski et al 2002;Gibbons et al 2011;Sabdono 2011). Cadmium has also been reported to disturb the physiological functioning of cell (Zeng et al 2012).…”

Heavy metal tolerance genes alter cellular thermodynamics inPseudomonas putidaand riverPseudomonas spp. and influence amebal predation

“…Because both strains tolerate at least 1.5 mM cadmium, a divergence in performance at only the highest cadmium level was expected. These results correspond to our previous finding with P. putida KT2440, in which the strains that were more susceptible to Cd toxicity, KT1 and KT4 (Leedjarv et al ., ), performed poorly when exposed to cadmium relative to the tolerant wild‐type strain (Gibbons et al ., ). The performance between the environmental strains remained unchanged from 0.00 mM to 0.10 mM cadmium.…”

“…In contrast, the organisms without enhanced metal resistance produced more biomass, but are unable to process available glucose as soon as their metal‐tolerant counterpart strains. These trends are supported by the energetics of growth of both the P. putida KT2440 wild type and mutants (KT1 and KT4) (Gibbons et al ., ) and the Montana isolates. Pseudomonas sp .…”

“…Yet, the stressor suppresses cell functions such as respiration and metabolism (Kandeler et al, 2000;Ramsey et al, 2005;Stanaway et al, 2012) and the flow of energy to higher trophic levels (Pratt et al, 1997;Iwasaki et al, 2009). A possible explanation for these observations is that over time, genes for tolerance mechanisms become widespread and abundant (Coombs & Barkay, 2004;Martinez et al, 2006;Sobecky & Coombs, 2009;Nongkhlaw et al, 2012), but impose a continual energetic cost (Gibbons et al, 2011). The genetic mechanisms of heavy metal resistance in bacteria are well documented (Silver & Phung, 2005;Monsieurs et al, 2011;Ray et al, 2013), yet there is little knowledge of how these genes influence energy flow and trophic interactions.…”

“…To tolerate chronic heavy metal stress, bacteria express genes to expel, bind, or reduce the metal (Silver & Phung, 1996;Nies, 1999Nies, , 2003. Microcalorimetry was previously used to quantify the energetic cost of heavy metal tolerance genes on growth yield and metabolism (Gibbons et al, 2011). Cellular thermodynamics were compared between isogenic strains of Pseudomonas putida KT2440, each with different single tolerance gene deletions.…”