Barry T.O. Lee

Molecular genetics and transport analysis of the copper-resistance determinant (pco) from Escherichia coli plasmid pRJ1004

The copper‐resistance determinant (pco) of Escherichia coli plasmid pRJ1004 was cloned and sequenced. Tn1000 transposon mutagenesis identified four complementation groups, mutations in any of which eliminated copper resistance. DNA sequence analysis showed that the four complementation groups contained six open reading frames, designated pcoABCDRS. The protein product sequences derived from the nucleotide sequence show close homology between this copper‐resistance system and the cop system of a plasmid pPT23D of Pseudomonas syringae pv. tomato. The PcoR and PcoS protein sequences show homology to the family of two‐component sensor/responder phosphokinase regulatory systems. A seventh reading frame (pcoE) was identified from DNA sequence data, and lies downstream of a copper‐regulated promoter. Transport assays with 64Cu(II) showed that the resistant cells containing the plasmid had reduced copper accumulation during the log phase of growth, while increased accumulation had previously been observed during stationary phase. Chromosomal mutants defective in cellular copper management were obtained and characterized. In two of these mutants pco resistance was rendered totally inactive, whilst in another two mutants pco complemented the defective genes. These data indicate that plasmid‐borne copper resistance in E. coli is linked with chromosomal systems for copper management.

Molecular genetics of a chromosomal locus involved in copper tolerance in Escherichia coli K-12

The cutA locus, presumably involved in copper tolerance in Escherichia coli, was characterized by a mutation leading to copper sensitivity. Copper‐accumulation measurements with radioactive 64Cu6+ showed increased uptake by cutA copper‐sensitive mutant cells, and reduced uptake when the cutA mutation was complemented in trans. The locus was mapped using complementation of the cutA mutant to partial copper tolerance with wild‐type chromosomal fragments. The 3.2 kb DNA region involved in cutA was sequenced and analysed, revealing three significant open reading frames, none of which had been previously published. The products of all three open reading frames were identified, when synthesized with the T7 phage promoter expression system, as polypeptides of about 50kDa, 24kDa, and 13kDa, consistent with the sizes predicted from the DNA sequences. The 50kDa and 24kDa polypeptides were found in the bacterial inner membrane, and the 13kDa polypeptide with the cytoplasmic fraction. In addition to being required for copper tolerance, cutA affects tolerance levels to zinC., nickel, cobalt and cadmium salts. Transcriptional fusions of cutA with the lux operon showed induction by copper, zinc, nickel, cobalt and, to a lesser extent, cadmium, manganese and silver salts.

Understanding cellular responses to toxic agents: a model for mechanism-choice in bacterial metal resistance

SummaryBacterial resistances to metals are heterogeneous in both their genetic and biochemical bases. Metal resistance may be chromosomally-, plasmid- or transposonencoded, and one or more genes may be involved; at the biochemical level at least six different mechanisms are responsible for resistance. Various types of resistance mechanisms can occur singly or in combination and for a particular metal different mechanisms of resistance can occur in the same species. To understand better the diverse responses of bacteria to metal ion challenge we have constructed a qualitative model for the selection of metal resistance in bacteria. How a bacterium becomes resistant to a particular metal depends on the number and location of cellular components sensitive to the specific metal ion. Other important selective factors include the nature of the uptake systems for the metal, the role and interactions of the metal in the normal metabolism of the cell and the availability of plasmid (or transposon) encoded resistance mechanisms. The selection model presented is based on the interaction of these factors and allows predictions to be made about the evolution of metal resistance in bacterial populations. It also allows prediction of the genetic basis and of mechanisms of resistance which are in substantial agreement with those in well-documented populations. The interaction of, and selection for resistance to, toxic substances in addition to metals, such as antibiotics and toxic analogues, involve similar principles to those concerning metals. Potentially, models for selection of resistance to any substance can be derived using this approach.

Copper resistance determinants in bacteria.

Copper is an essential trace element that is utilized in a number of oxygenases and electron transport proteins, but it is also a highly toxic heavy metal, against which all organisms must protect themselves. Known bacterial determinants of copper resistance are plasmid-encoded. The mechanisms which confer resistance must be integrated with the normal metabolism of copper. Different bacteria have adopted diverse strategies for copper resistance, and this review outlines what is known about bacterial copper resistance mechanisms and their genetic regulation.

Bacterial Response to Copper in the Environment: Copper Resistance in Escherichia coli as a Model System

In order to study the biological effects of heavy metals, it is necessary to make a distinction between two types of heavy metals (1) metals that are toxic per se and (2) metals which are essential to the growth and maintenance of living organisms but which are toxic in excess. Copper is an extremely common heavy metal in the environment. Although it is an essential element, it is toxic at high concentrations. Copper causes cell damage through, for example, modification of the active sites of cellular enzymes and the peroxidation of membranes. So, copper must not be in a free state either in the cell or at the environment/cell interface. It is involved in essential redox reactions and is a component of a number of enzymes, which means that normal copper metabolism must be closely regulated in both low and high copper environments. If any resistance to high copper concentrations is developed in response to environmental changes it must be regulated together with the normal copper utilization systems (Rouch et al. 1989a,b). In contrast, mercury salts are highly toxic to all living organisms and have no beneficial function. They are widely distributed, as a result of both geochemical and human activities and this, coupled to the toxicity, has resulted in the evolution and spread of mercury resistance in bacteria (Brown et al. 1989; Silver and Misra, 1988). Mercury resistance is the best characterized of all resistances to heavy metals and involves the sequestration of mercuric ions in the periplasmic space, their transport into the cell and their reduction to the non-toxic form, metallic mercury, Hg(0). This makes biological sense in that a highly toxic compound is sequestered in the periplasm and not released to the cellular constituents until it has been detoxified.

Chemical and Genetic Studies of Copper Resistance in E. coli

Copper is an essential trace element required for bacterial growth, but is toxic at high levels of free ions. Bacteria are thus presented with the complex problem of obtaining and storing sufficient quantities of copper for normal function of several enzymes while, on die other hand, being able to survive when confronted with concentrations of copper that exceed a toxic threshold. The molecular mechanisms of metal ion detoxification are well understood only for a few systems, most notably for mercury resistance.1,2 For recent reviews of a number of bacterial metal resistance systems, see Plasmid, Vol. 27(1), 1992 and reference 3. The discovery of plasmid-borne copper resistance in bacteria has provided accessible systems for genetic and phenomenological study of copper metabolism. Study of these extrachromosomal systems has recently provided the impetus and the methods for identification of chromosomally-encoded copper homeostasis systems in Pseudomonas syringae pv. tomato (P. syringae)4,5,6 and in Echerichia coli (E. coli) 7,8,9 The two best characterized copper resistance systems are the plasmid-encoded systems cop in P. syringae and pco in E. coli. These two determinants are remarkably similar genetically, but as will be shown they differ fundamentally in the mechanism of copper resistance. This paper will focus first on the genetics and regulation of copper resistance in the cop and pco systems, then on the chemical mechanisms of copper resistance.

Genetic and Biochemical Analysis of Copper Transport in Escherichia coli

Copper is an essential trace element yet it is also potentially toxic. Complex cellular Cu management systems should exist to ensure that supply of Cu to Cu-dependent enzymes occurs without associated toxicity. We predict that these Cu management systems should include: (i) uptake, (ii) intracellular transport and storage, (iii) repair and nullification of Cu damage, (iv) efflux, (v) regulation of cellular Cu levels. Our aim is to use a genetic approach in the microorganism Escherichia coli to investigate these systems. The findings in E. coli will provide leads for research in mammalian systems.