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Life depends upon multiple metals. It is estimated that approximately one-third of all gene products require a metal for folding and/or catalysis. How does the correct metal locate to the correct protein? Provision of sufficient atoms of each of the metals required by protein metal-binding sites is a challenge for cell biology. This is often especially true for iron, which is poorly soluble under aerobic conditions. Protein metal-binding sites follow universal affinity series. Under such a regime, exclusion of the wrong metals from metalloproteins is arguably an even greater challenge. High-fidelity homeostasis must match the number of some metal cations to the number of bonafide metal-binding sites. Selective protein–protein interactions also limit access of some atoms to the required subsets of proteins. Here we provide an overview of the contributions of metal sensors, metallochaperones, metal transporters and metal-storage proteins to the allocation of metals in cells. In this chapter an emphasis is placed on studies of the cell biology of metals in cyanobacteria.

Metal homeostasis relies on the ability of metalloregulatory proteins to coordinate the expression of transport and storage functions. Metalloregulatory proteins can be divided into two major groups: those that regulate the uptake of essential metals (the Fur, DtxR/MntR, and NikR families) and those that regulate metal efflux and detoxification mechanisms (the ArsR/SmtB and MerR families). Within each metalloregulator protein family, there is a tremendous diversity in metal selectivity and the corresponding biological responses. The availability of at least one protein structure from each family is beginning to provide insights into the origins of metal selectivity. Biochemical measurements of metal ion selectivity and affinity provide a window into the ambient metal ion conditions within the cytosol: metalloregulators that sense nutrient metals must be poised to bind the metal ion once the essential functional sites are saturated, but before adventitious associations begin to interfere with cellular function. Similarly, sensors of metal ion excess, whether for non-essential toxic metals or nutrient metals, must respond to metals, at levels below those that will inhibit or prevent cell growth, to activate appropriate defensive measures. Recent insights highlight the global nature of stress responses elicited by metal ion deficiency. In addition to the expected derepression of high affinity uptake systems, metal ion starvation leads to a large-scale remodeling of the proteome that includes: (i) metal-sparing, (ii) metal-substitution, and (iii) metal-mobilization responses.

Bacterial cellular responses to metal ion stress are often measured as changes in transcription of genes involved in metal ion homeostasis, during detoxification processes or during functioning of efflux systems. Although there has been evidence for other bacterial cellular responses to metal ion stress, a view of what these responses are has been difficult to obtain. Recent measurements from genome-wide transcriptional profiling in bacteria strongly suggests that the effects of metals on cells may be very wide-ranging, and the transcriptomic responses equally wide. This chapter integrates the known biological effects of metal ion stress with data from microarray and other gene regulation studies from different bacteria responding to these stresses. Metal ion stresses elicit responses in metal ion homeostasis, oxidative stress responses, membrane stress responses, amino acid synthesis, and the expression of other metal ion import systems.

Since details on metal cation transport proteins and on the allocation mechanisms for transition metals are provided elsewhere in this book, I will present aspects of transition metal homeostasis in a hopefully novel overview. We will start with a microbial look at the transition metal Periodic Table, cation speciation, and availability in the environment. This information provides rules that might govern microbial metal cation homeostasis from the outside of the cell. The fate of metal cations inside the cell is influenced by redox potentials and affinities to ligands in complex compounds. Understanding this topic requires study of interactions between metal cations and the consequences thereof. External availability and internal binding equilibria are connected by transport reactions. These lead to metal cation concentrations in cellular compartments, which are in flow equilibrium of import and export reactions. Thus, cellular cation homeostasis may be described as an interplay of transport flow backbone and competitive binding reactions. Both together provide an energy landscape for each metal cation and cellular compartment. As a recent part of the transport flow backbone in Gram-negative bacteria, efflux across the outer membrane from the periplasm to the outside has been identified. Active outer membrane efflux might indeed be taking place in Gram-negative bacteria. Thus, the periplasm is important in bacterial metal cation homeostasis.

Naturally occurring, regulated resistance mechanisms of bacteria against various heavy metals and metalloids have been used to construct whole-cell living biosensors or bioreporters. Molecular fusions of regulatory circuits with reporter genes encoding easily detectable reporter proteins enable bioreporters to sense metal targets, typically at concentrations in the nanomolar to micromolar range, although more sensitive sensors also exist. The biological components of extant bioreporter constructs and the target ranges and sensitivities of bioreporter constructs are presented. An outlook on developments using novel molecular interactions as triggers of the biological responses and strategies for the improvement of bioreporters is given. Application examples are presented that illustrate the capability of bioreporters to measure bioavailable fractions of the target species rather than total loads.

Measurements of trace metals as parts of homeostatic networks for essential and nonessential metals in microbial cells need sensitive high-resolution techniques. The term “metallome” denotes metals and metalloid species within cells, encompassing both the inorganic element content and their complexes with biomolecules, especially with peptides and proteins. Elucidation of the physiological roles of metals and their bioinorganic speciation requires a set of microanalytical purification, separation, and identification methods. This chapter summarizes analytical tools useful to investigate bacterial responses to metal stress.

Bacteria have evolved multiple mechanisms to cope with the extreme iron limitations in their natural environments. Fe forms insoluble hydroxy aquo complexes. The free Fe concentration lies orders of magnitude below the concentration required for microbial growth (0.1 μM). Bacteria synthesize and secrete low-molecular-weight compounds, called siderophores, which bind Fe with very high affinity and specificity, and host organisms of bacteria bind Fe to proteins that serve as iron sources for bacteria. Energy-coupled transport systems bring Fe, Fe-siderophores, and heme across the outer membrane, the periplasm, and the cytoplasmic membrane into the bacterial cytoplasm. There, iron is released from the carrier molecules and incorporated mostly into heme and iron-sulfur proteins. Intracellular iron metabolism is poorly understood. The transport systems and the biosynthesis of the siderophores are regulated by proteins, usually by Fur in Gram-negative bacteria, and DtxR and IdeR in Gram-positive bacteria. These proteins act as transcriptional repressors when loaded with Fe. Additional regulatory devices control siderophore biosynthesis and transport. The Fec-type of regulation is of particular interest because it involves a novel mechanism in which the ferric siderophore binds to the outer membrane transport protein and from there induces transcription of the transport and biosynthesis genes in the cytoplasm. Another recently detected device is the regulation of genes positively regulated by Fur via RhyB, a small regulatory RNA. RhyB facilitates degradation of positively regulated mRNAs, which does not occur when Fe-Fur represses RhyB synthesis.

Maintaining iron homeostasis is a necessity for almost all organisms. Microorganisms such as possess several systems for iron acquisition and storage. In recent years further systems have been discovered. These systems comprise the first characterized bacterial ZIP transporter, ZupT. ZupT is a transporter with broad substrate specificity and beside iron and zinc ZupT also transports cobalt or probably other divalent metal cations. Another novel bacterial iron transporter, EfeU, was recently found in and . These EfeU permeases are the first characterized bacterial members of the OFeT-family of iron transporters that are well studied in yeast and in other lower eukaryotes.

Enterobactin, the primary catecholate-type siderophore from and other bacteria, is secreted from the cell in a two-step mechanism, functionally connecting the major facilitator protein EntS and an efflux-complex comprising the outer membrane exit channel protein TolC. Our knowledge of iron-transport systems was extended by the identification and characterization of an iron-efflux transporter, FieF, from . FieF is a member of the largest subfamily of cation diffusion facilitators (CDF). CDF proteins were previously known to be involved in detoxification of divalent transition metal cations such as Zn(II) or Cd(II) but probably participate in efflux of ferrous iron as well.

Recent data have demonstrated that bacterial homologs of eukaryotic Nramp transporters as well as members of the LraI family of proteins are both highly selective Mn transporters. Mutation of these transporters in several pathogenic bacterial species causes decreased virulence in a variety of model systems. This implies that the Mn ions are required for one or more processes essential for bacterial virulence. However, Mn has few known enzymatic roles compared to other divalent cations. This review will describe what is currently known about the two classes of prokaryotic Mn transporters, how each is regulated and the virulence deficits that arise when they are mutated. Finally, possible enzymatic roles for Mn will be outlined, and their potential for a role in virulence discussed.

Copper in biological systems presents a formidable problem: it is essential for life, yet highly reactive and a potential source of cell damage. Tight control of copper is thus a cellular necessity. To meet this challenge, cells have evolved pumps for transmembranous transport, chaperones for intracellular routing, oxidases and reductases to change the oxidation state of copper, and regulators to control gene expression in response to copper. These systems are complemented by specific mechanisms for the insertion of copper into enzymes. Copper homeostasis has evolved early in evolution and some components have been conserved from bacteria to humans. This has allowed researchers to apply knowledge across phyla and even involving human copper homeostatic diseases to elucidate the fundamental mechanism of cellular copper homeostasis. After an introduction to the properties of copper and its role in biological systems, some of the best studied bacterial systems for copper homeostasis will be discussed.