SummaryThe gene cluster pbrTRABCD from Cupriavidus metallidurans CH34 is thought to encode a unique, specific resistance mechanism for lead. However, the exact functions of these genes are unknown. In this study we examine the metal specificity and functions of pbrABCD by expressing these genes in different combinations and comparing their ability to restore Pb 2+ , Zn 2+ and Cd 2+ resistance in a metal-sensitive C. metallidurans strain DN440. We show that lead resistance in C. metallidurans is achieved through the cooperation of the Zn/Cd/Pb-translocating ATPase PbrA and the undecaprenyl pyrophosphate phosphatase PbrB. While PbrA non-specifically exported Pb 2+ , Zn 2+ and Cd 2+ , a specific increase in lead resistance was observed when PbrA and PbrB were coexpressed. As a model of action for PbrA and PbrB we propose a mechanism where Pb 2+ is exported from the cytoplasm by PbrA and then sequestered as a phosphate salt with the inorganic phosphate produced by PbrB. Similar operons containing genes for heavy metal translocating ATPases and phosphatases were found in several different bacterial species, suggesting that lead detoxification through active efflux and sequestration is a common leadresistance mechanism.

Whole-cell bioreporters are living microorganisms that produce a specific, quantifiable output in response to target chemicals. Typically, whole-cell bioreporters combine a sensor element for the substance of interest and a reporter element coding for an easily detectable protein. The sensor element is responsible for recognizing the presence of an analyte. In the case of metal bioreporters, the sensor element consists of a DNA promoter region for a metal-binding transcription factor fused to a promoterless reporter gene that encodes a signal-producing protein. In this review, we provide an overview of specific whole-cell bioreporters for heavy metals. Because the sensing of metals by bioreporter microorganisms is usually based on heavy metal resistance/homeostasis mechanisms, the basis of these mechanisms will also be discussed. The goal here is not to present a comprehensive summary of individual metal-specific bioreporters that have been constructed, but rather to express views on the theory and applications of metal-specific bioreporters and identify some directions for future research and development.

We addressed how restoration of forestry-drained peatlands affects CH 4 -cycling microbes. Despite similar community compositions, the abundance of methanogens and methanotrophs was lower in restored than in natural sites and correlated with CH 4 emission. Poor establishment of methanogens may thus explain low CH 4 emissions on restored peatlands even 10 to 12 years after restoration.

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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...