Out of the iron age: new insights into the critical role of manganese homeostasis in bacteria

Nicholas S. Jakubovics1 and Howard F. Jenkinson1

Oral Microbiology Unit, Department of Oral and Dental Science, University of Bristol, Dental School and Hospital, Lower Maudlin Street, Bristol BS1 2LY, UK1

Author for correspondence: Howard F. Jenkinson. Tel: +44 117 928 4358. Fax: +44 117 928 4313. e-mail: howard.jenkinson{at}bristol.ac.uk

Keywords: Bacillus development, virulence, oxidative stress, Streptococcus, metal ion transport


   Overview
TOP
Overview
Cellular functions of Mn
Mn2+ transport systems
Transcriptional regulation
Mn2+ homeostasis and virulence
Future perspectives
REFERENCES
 
Manganese (Mn) is required for the growth and survival of most, if not all, living organisms. Until recently, relatively little was known about how bacteria take up trace nutrients such as Mn, nickel (Ni), copper (Cu) and zinc (Zn), or about how they regulate intracellular levels of these in response to availability and demand. Over the last 5 years a large number of systems involved in these processes have been identified and characterized. This has led to new insights into transition metal ion homeostasis in Gram-positive and Gram-negative bacteria. Metal ions, including Mn, iron (Fe), cobalt (Co), Ni, Cu and Zn, are both essential and potentially toxic. Therefore, homeostatic regulation of their intracellular concentrations is critical.

In prokaryotic cells, which lack internal compartmentalization, metal ion homeostasis is maintained primarily by tight regulation of metal cation flux across the cytoplasmic membrane. Studies on transition metal ion homeostasis in bacteria have focused mainly on Fe metabolism (reviewed by Braun & Killmann, 1999 ). The acquisition of Fe poses unique problems because of the poor solubility of Fe(III) species that predominate around neutral pH in oxygenated environments. Furthermore, Fe is tightly sequestered by eukaryotic proteins, so the ability of bacteria to wrest Fe from the host is fundamental to the pathogenesis of many infectious diseases. Despite these potential difficulties, many bacteria accumulate relatively high levels of Fe. For example, laboratory-cultured Escherichia coli cells contain greater than fivefold more Fe than Mn (Posey & Gherardini, 2000 ). Fe is required as an essential cofactor for many proteins, including components of the respiratory chain (cytochromes, cytochrome oxidase), tricarboxylic acid cycle (aconitase, succinate dehydrogenase) and oxidative defence systems [catalase, peroxidase, superoxide dismutase (SOD)]. Microbial Fe acquisition has been extensively studied and shown to occur by at least three very different strategies. These include the production of small molecules (siderophores) to chelate extracellular Fe3+, the direct capture of Fe3+ from host Fe-containing proteins such as transferrin, lactoferrin or haem proteins, and the uptake of Fe2+ by the Feo transport system (Braun & Killmann, 1999 ). Within cells, unbound Fe2+ (and Cu+) ions are especially noxious because they catalyse Fenton-type reactions that lead to the production of damaging hydroxyl radicals (Pierre & Fontecave, 1999 ). By contrast, Mn(II) is highly soluble and does not catalyse hydroxyl radical formation (Cheton & Archibald, 1988 ).

Interestingly, new experimental and genomic sequence data suggest that some micro-organisms may have no requirement for Fe, having evolved metabolic and survival strategies that can be accommodated by Mn. For example, the Lyme disease pathogen Borrelia burgdorferi appears to have an obligate requirement for Mn (Posey & Gherardini, 2000 ). In the absence of Fe, the cell is limited in reactions that can be carried out; for example, aerobic respiration is impossible without cytochromes. However, the ability to forgo Fe may confer a selective advantage in natural environments in which there is intense competition for available Fe. An absolute requirement for Mn, whether in place of or in addition to Fe, would necessitate acquisition of strategies to effectively compete for Mn. Indeed, high-affinity uptake of Mn2+ was reported in E. coli over three decades ago (Silver & Lusk, 1987 ), but the candidate gene for a high-affinity transporter was identified only last year (Kehres et al., 2000 ). Also, bacterial transcriptional regulators that respond to Mn2+ have only recently been discovered, with homologous protein families shown to be present across the eubacteria (Posey et al., 1999 ; Jakubovics et al., 2000 ; Que & Helmann, 2000 ). This review will summarize some of the intracellular functions of Mn and discuss recent advances that have been made in understanding the mechanisms utilized by bacteria for Mn uptake and homeostasis.


   Cellular functions of Mn
TOP
Overview
Cellular functions of Mn
Mn2+ transport systems
Transcriptional regulation
Mn2+ homeostasis and virulence
Future perspectives
REFERENCES
 
Mn metalloenzymes have many diverse functions within bacterial cells (Christianson, 1997 ; Yocum & Pecoraro, 1999 ), some of which are summarized in Table 1. The ionic radius of Mn2+ (0·80 ) in aqueous solutions lies between that of Mg2+ (0·65 ) and Ca2+ (0·99 ), and is close to that of Fe2+ (0·76 ) and several other transition metal ions. It is therefore not surprising that Mn2+ and other cations may be interchangeable in the metal-binding sites of many proteins. Most commonly, Mn2+ and Mg2+ are interchangeable on account of the similarities between chelate structures of these ions. However, Mn is essential for certain metabolic pathways. For example, oxygenic photosynthesis in cyanobacteria requires a tetra-Mn cluster present in the reaction centre complex of photosystem II (Table 1; Yocum & Pecoraro, 1999 ), and a number of enzymes, including MnSOD, mangani-catalase and arginase, specifically require Mn2+ for activity (Christianson, 1997 ). In addition, glycolysis cannot proceed fully without 3-phosphoglycerate mutase (PGM) which, in several Gram-positive endospore-forming bacteria, is active only when associated with Mn2+ (Chander et al., 1998 ). The pH-dependent dissociation of Mn2+ from PGM represents a novel signalling mechanism for rapid enzyme inactivation (see below). It has been suggested that similar mechanisms may be responsible for the regulation of other strictly Mn2+-dependent enzymes, such as arginase (Kuhn et al., 1995 ). In addition, it appears that Mn has an important role in bacterial signal transduction. The recently identified E. coli proteins PrpA and PrpB belong to a family of Mn2+-containing serine/threonine protein phosphatases that are present widely in eukaryotes where they modulate complex signalling pathways (Barford, 1996 ). PrpA and PrpB are linked to the activity of a two-component sensor, CpxAB, and regulation of the periplasmic stress response protease HtrA/DegP (Missiakas & Raina, 1997 ). The involvement of Mn in signalling during Bacillus sporulation is discussed below.


View this table:
[in this window]
[in a new window]
 
Table 1. Selected functions of Mn within bacterial cells

 
The biological importance of Mn is not restricted to enzyme-mediated catalysis. For example, Mn2+ can detoxify a variety of reactive oxygen species (ROS), protecting cells that lack enzymic defences (discussed below). Additionally, non-enzymic Mn2+ is crucial for the proper function of a variety of bacterial products, including secreted antibiotics (see Archibald, 1986 ), and contributes to the stabilization of bacterial cell walls (Doyle, 1989 ).

Bacillus development
Early work in the 1950s demonstrated that Mn is unique amongst the transition metals in promoting both endospore formation and spore germination in Bacillus (Charney et al., 1951 ; Gould, 1969 ). Recent genetic studies have shown that Mn homeostasis is essential for efficient sporulation in Bacillus subtilis (Que & Helmann, 2000 ), and that Mn appears to be important at several stages in the developmental cycle (Fig. 1). Sporulation in B. subtilis involves a complex sequence of transcriptional changes, controlled by several different {sigma} factors, which are themselves subject to spatial and temporal regulation (Kroos et al., 1999 ). The initial decision to sporulate is triggered by starvation, and is transcriptionally evoked by {sigma}A. At this time, cells are exquisitely sensitive to ROS, so that exposure of bacteria to paraquat, which generates intracellular superoxide (), at a concentration that does not affect vegetative growth, results in sporulation arrest (Inaoka et al., 1999 ). MnSOD has been shown to play a crucial protective role since inactivation of the sodA gene markedly reduces the paraquat concentration required to inhibit sporulation, while provision of sodA on a multicopy plasmid results in increased sporulation efficiency in the presence of paraquat (Inaoka et al., 1999 ). In the switch from symmetric to asymmetric cell division, which signals commitment to sporulation, Mn2+ is directly involved as a cofactor of the SpoIIE serine phosphatase (Schroeter et al., 1999 ). This is a bifunctional protein that influences polar septum formation through interactions with FtsZ (King et al., 1999 ), and that separately activates {sigma}F in the forespore by dephosphorylation of SpoIIAA (Kroos et al., 1999 ). It is not yet clear how SpoIIE activates {sigma}F in the forespore, but not in the mother cell, since SpoIIE and {sigma}F are present in both cell types (King et al., 1999 ). It has been suggested that an inhibitor may bind to SpoIIE in the forespore, thus delaying activation until the septum is complete (Arigoni et al., 1999 ). However, since Mn2+ accumulates specifically within the developing forespore, it is possible that {sigma}F activation by SpoIIE may be Mn2+-regulated.



View larger version (22K):
[in this window]
[in a new window]
 
Fig. 1. Roles of Mn in the developmental cycle of Bacillus. Mn homeostasis is essential for efficient initiation of sporulation in the vegetative cell (1). The switch from medial to polar septum formation (2) is influenced by the Mn-requiring SpoIIE protein. Following septation, Mn accumulates in the developing forespore. SpoIIE localizes to both poles of the cell, but exclusively activates {sigma}F in the forespore. A drop in pH of around 1 unit accompanying forespore development (3) causes dissociation of Mn2+ from phosphoglycerate mutase (PGM), inactivating the enzyme (PGMi) and leading to accumulation of the storage molecule 3-phosphoglyceric acid (3-PGA) (4). Production of the inner (shaded) and outer (thick line) layers of the spore coat (5) requires superoxide dismutase (MnSOD). Germination of the mature spore (6) involves a rapid increase in pH, leading to the activation of PGM and the mobilization of 3-PGA reserves. Protease production at this stage is stimulated by Mn, and high levels of Mn2+-dependent pyrophosphatase (MnPPiase) are associated with an increase in metabolic activity. Chromosomal DNA is represented here by wavy lines.

 
Mn2+ also influences spore composition, structure and germination. Bacillus endospores contain a storage reservoir of 3-phosphoglyceric acid (3-PGA) that is metabolized following germination. Accumulation of 3-PGA within the developing forespore occurs following acidification of the forespore compartment, with concomitant dissociation of the essential Mn2+ cofactor from PGM (Kuhn et al., 1995 ; Chander et al., 1998 ) (Fig. 1). Upon initiation of spore germination, PGM is rapidly reactivated following an increase in intracellular pH, and 3-PGA is degraded (Kuhn et al., 1995 ; Chander et al., 1998 ). During the process of spore coat assembly, there is evidence that MnSOD may be involved in generating hydrogen peroxide (H2O2), which is required for the o,o-dityrosine cross-linking of the coat structural protein CotG (Henriques et al., 1998 ). During germination, Mn2+ is involved in the activation of PGM, proteases and inorganic pyrophosphatase (Gould, 1969 ; Kuhn & Ward, 1998 ). Following recent advances in understanding of Mn homeostasis in B. subtilis (Que & Helmann, 2000 ), more detailed information on the functions of Mn in sporulation is anticipated in the near future. It is notable that Mn2+ has recently been identified as an essential cation for cell cycle progression in Saccharomyces cerevisiae (Loukin & Kung, 1995 ).

Oxidative stress
One of the major challenges facing bacteria growing in oxygenated environments is to efficiently resist or repair damage caused by ROS such as , H2O2 and hydroxyl radicals (·OH). Oxidative stress may be induced endogenously, for example by the oxidation of flavoproteins during respiration, or exogenously, by phagocytic production of ROS (Miller & Britigan, 1997 ). Therefore, enzymes for protection against reactive oxygen, including SODs, catalases and peroxidases, are ubiquitous in bacteria. Fe is inextricably linked to oxidative damage, primarily through the ability of Fe2+ to reduce H2O2 (Touati, 2000 ). One strategy for minimizing oxidative damage therefore involves limiting intracellular Fe content. In fact, there is good evidence that Lactobacillus plantarum and B. burgdorferi have dispensed with Fe requirements (Archibald, 1986 ; Posey & Gherardini, 2000 ), and also that several species of streptococci can grow in the absence of Fe (Spatafora & Moore, 1998 ; Niven et al., 1999 ; Jakubovics et al., 2000 ). Where bacteria grow in the absence of Fe they appear to have an absolute requirement for Mn2+.

Bacteria have acquired elaborate defence mechanisms that co-ordinate Fe-sensing and Mn-sensing with oxidative stress responses (Bsat et al., 1998 ; Storz & Imlay, 1999 ). While Fe2+ is toxic and can promote ·OH radical formation, Mn2+-containing compounds react with , H2O2 and ·OH, without generating deleterious free radical species (Cheton & Archibald, 1988 ; Stadtman et al., 1990 ). Mn is essential for the detoxification of ROS in most bacteria, principally as a cofactor for MnSOD (SodA). However, several groups of lactic acid bacteria incorporate high levels of intracellular Mn2+ as a protectant in place of enzymic SOD (Archibald, 1986 ). Simple Mn(II) salts can also substitute for SOD activity in SOD-deficient laboratory mutants, providing increased resistance to toxic effects of anions (Inaoka et al., 1999 ). The precise mechanism by which non-enzymic Mn2+ scavenges in bacteria is not understood, but requires considerably higher intracellular Mn2+ levels than those needed for efficient MnSOD-mediated protection. In natural environments, enzymic SOD is probably dispensable only in bacteria such as L. plantarum that grow on Mn2+-rich plant material.

Resistance to the toxic effects of superoxide anions more usually involves the concerted activities of two or more SOD enzymes. E. coli possesses three SOD activities: periplasmic Cu/ZnSOD (SodC) and two cytosolic proteins, FeSOD (SodB) and MnSOD (SodA) (Fridovich, 1995 ). The precise function of Cu/ZnSOD is unclear at present, but it has been suggested that this protein may protect against exogenous . Disruption of the sodA and sodB genes in E. coli leads to enhanced sensitivity of cells to and generates nutritional auxotrophies resulting from inactivation of dihydroxy-acid dehydratase by (Kuo et al., 1987 ). On the other hand, it appears that B. subtilis (Inaoka et al., 1999 ) and most streptococci and enterococci (e.g. Niven et al., 1999 ), may produce only MnSOD. Inactivation of the sodA gene in Streptococcus pyogenes sensitizes these cells to (Gibson & Caparon, 1996 ), and in Streptococcus pneumoniae reduces virulence in intranasal infection of mice (Yesilkaya et al., 2000 ). The cytoplasmic MnSOD in Strep. pyogenes is also secreted to the cell surface (Gerlach et al., 1998 ), where it may interact with exogenous . One of the most likely explanations for reduced virulence of bacteria that have defects in Mn2+ uptake is that they are impaired in ability to cope with oxidative stress. Another important function of enzymic Mn is in the detoxification of H2O2 by mangani-catalase. Mangani-catalases have been described in L. plantarum and Thermus thermophilus (Whittaker et al., 1999 ), and recently in a range of Gram-negative bacteria (Robbe-Saule et al., 2001 ).


   Mn2+ transport systems
TOP
Overview
Cellular functions of Mn
Mn2+ transport systems
Transcriptional regulation
Mn2+ homeostasis and virulence
Future perspectives
REFERENCES
 
Mn homeostasis in bacteria depends upon regulation of Mn2+ transport. Early work in E. coli demonstrated that Mn2+ uptake was mediated by a high-affinity import system with a Km of approximately 0·2 µM and a Vmax of 1–4 nmol min-1 per 1012 cells (reviewed by Silver & Lusk, 1987 ). This system could not be inhibited by Ca2+ or Mg2+, but was sensitive to Fe2+ or Co2+ inhibition. Uptake systems for Mn2+ were also identified in a range of Gram-positive bacteria and, in contrast to E. coli, Mn2+ transport in B. subtilis, L. plantarum and Staphylococcus aureus was competitively inhibited by Cd2+ (Silver & Lusk, 1987 ). The maximal rate of Mn2+ uptake in L. plantarum was found to be unusually high [Vmax of 24 µmol min-1 (g protein)-1], consistent with the high concentration of internal Mn2+ (30–35 mM) in this organism (Archibald, 1986 ). The mechanisms of Mn2+ uptake were unknown until Bartsevich & Pakrasi (1995) discovered the mntCAB operon encoding an ATP-dependent transporter in Synechocystis sp. PCC 6803, a cyanobacterium with a high Mn2+ requirement for photosynthesis. Subsequently, the pace of research on bacterial Mn2+ uptake has intensified, with characterization of a number of transporters for Mn2+. These transport systems are sufficiently structurally conserved to allow the location of the genes encoding them within all the currently sequenced bacterial genomes. At least three types of Mn2+ import systems have now been identified. The unusual transporter from L. plantarum is a P-type ATPase (MntA), the only one to date that has been demonstrated to have high specificity for Mn2+ (Hao et al., 1999 ). The other two types of Mn2+ uptake system conform to either the ATP-binding cassette (ABC) transporter superfamily or to the natural resistance-associated macrophage protein (Nramp) family.

ABC transporters
The mntCAB operon of Synechocystis encodes a solute-binding extracytoplasmic protein (MntC), a cytoplasmic ATP-binding component (MntA) and a transmembrane protein (MntB). Protein MntC shares 32% amino acid sequence identity with ScaA in Streptococcus gordonii, previously identified as a lipoprotein associated with an ABC transporter of unknown function (Kolenbrander et al., 1994 ). ScaA-like proteins have been found across the streptococci and enterococci (Burnette-Curley et al., 1995 ; Berry & Paton, 1996 ; Singh et al., 1998 ; Kitten et al., 2000 ). They constitute a family of proteins originally designated LraI (Jenkinson, 1994 ) and now recognized as a new cluster of solute-binding proteins for metal ion transport (Dintilhac et al., 1997 ). In Strep. gordonii (Kolenbrander et al., 1998 ) and Strep. pneumoniae (Dintilhac et al., 1997 ), the scaCBA and psaBCA operons encode the components of ABC-type uptake systems for Mn2+. The Km of the Strep. gordonii Sca transporter was estimated at 0·1–0·3 µM (Kolenbrander et al., 1998 ), somewhat lower than that of the Mnt permease in Synechocystis sp. PCC 6803 (Bartsevich & Pakrasi, 1995 ). In addition to the Psa (Mn2+) transporter, Strep. pneumoniae expresses an ABC-type Zn2+ permease, encoded by the adcCBA genes, and this is thought to transport Mn2+ also (Dintilhac et al., 1997 ). Mutations in the Strep. gordonii sca genes, or in the Strep. pneumoniae psa genes, affect Mn2+ uptake and reduce the abilities of cells to undergo DNA-mediated transformation (Dintilhac et al., 1997 ; Kolenbrander et al., 1998 ).

The form of Mn2+ that is transported by the ABC-type permeases has not been determined. Future studies will be facilitated by assays of substrate binding by purified solute-binding proteins or reconstituted transport systems. The cation specificity of the Strep. pyogenes LraI lipoprotein MtsA was investigated by ligand blotting (Janulczyk et al., 1999 ) and shown to be relatively broad, with specificity for Zn2+, Fe3+ or Cu2+, but surprisingly not for Mn2+. The X-ray crystal structure of pneumococcal PsaA has been determined to 2·0  resolution and Zn2+, rather than Mn2+, was bound to the recombinant protein (Lawrence et al., 1998 ). Thus, evidence for direct binding of Mn2+ ions to the solute-binding components of ABC transporters is not convincing. The possibility that Mn2+ is bound and transported as a chelated complex cannot be ruled out. Uptake of Mn2+ by the P-type ATPase of L. plantarum is stimulated by citrate and other tricarboxylic acids, although citrate does not seem to be imported (Archibald, 1986 ; Hao et al., 1999 ).

Nramps
Mn2+ uptake in bacteria can be inhibited by uncouplers (Silver & Lusk, 1987 ), and the discovery in bacteria of proteins belonging to the Nramp transporter family (Kehres et al., 2000 ; Que & Helmann, 2000 ), accounts for this observation. The Nramp proteins form a class of pH-dependent divalent transition metal cation transporters that are required for intestinal Fe uptake and for host macrophage cell resistance to chronic bacterial infections (Gruenheid & Gros, 2000 ). The substrate specificity and cation uptake kinetics of the E. coli Nramp homologue MntH (Kehres et al., 2000 ) suggest that this transporter corresponds to the Mn2+ uptake system originally described over 30 years ago. Several other bacterial Nramps have been characterized to date, and inspection of genomic sequence data shows that they are widespread throughout many diverse groups of eubacteria. A primary role for the Nramp-like proteins in E. coli and Salmonella typhimurium may be to help protect cells against ROS, a function that is particularly important during infection of the host (Kehres et al., 2000 ; see below).

Cation export
Transition metal cations, including Co2+, Ni2+, Cu2+ and Zn2+, are actively extruded from bacterial cells (Silver, 1996 ) and regulation of export systems contributes significantly to the homeostatic control of these metal ions. In many cases, genes encoding metalloregulatory proteins are adjacent to those encoding the export apparatus. In Synechocystis sp. PCC 6803, an operon containing nine ORFs encodes the components required for the independent sensing and export of Ni2+, Co2+ and Zn2+ (García-Domínguez et al., 2000 ). It is possible that regulated export systems for Mn2+ exist, but no system for export of Mn2+ by bacteria has yet been demonstrated.


   Transcriptional regulation
TOP
Overview
Cellular functions of Mn
Mn2+ transport systems
Transcriptional regulation
Mn2+ homeostasis and virulence
Future perspectives
REFERENCES
 
The central regulator of Fe transport pathways in E. coli is Fur (Escolar et al., 1999 ). Fur is a metalloprotein that, in the presence of Fe2+ or Mn2+, binds to the operator regions of at least 20 genes involved in diverse cell functions, including siderophore production, trans-membrane Fe transport, motility, virulence factor expression and the oxidative stress and acid shock responses. In Corynebacterium diphtheriae, Fe-dependent gene expression was shown to be controlled by DtxR protein (Holmes, 2000 ), a metallorepressor with <15% amino acid identity to Fur. DtxR regulates the expression of a number of genes involved in siderophore production, haem degradation and diphtheria toxin biosynthesis. Until recently, transcriptional responses of bacteria to transition metals other than Fe were relatively poorly understood. This picture has changed dramatically over the past 5 years, with the identification of novel metalloregulators responsive to Mn2+, Co2+, Cu2+ and Zn2+. Thus, at least three distinct classes of metalloproteins control Zn2+ transport. In B. subtilis and E. coli, a high-affinity Zn2+ uptake transporter is regulated in response to Zn2+ by the Zur metalloregulator protein, which shares around 27% identity with Fur (Gaballa & Helmann, 1998 ). Cyanobacterial Zn2+ resistance is modulated by SmtB and ZiaR, both members of the ArsR metalloregulator family (Thelwell et al., 1998 ). ZntR, a Zn2+-dependent regulator of the MerR protein family, negatively controls expression of the Zn2+ efflux pump ZntA in Staph. aureus (Singh et al., 1999 ). MerR family proteins CorR of Synechocystis sp. PCC 6803 (García-Domínguez et al., 2000 ) and CueR of E. coli (Stoyanov et al., 2001 ) control the export of Co2+ and Cu2+, respectively. These responses to Co2+, Cu2+ and Zn2+ seem to be confined to cation homeostasis and there is no evidence yet that they directly affect the expression of genes involved in other cell processes.

Mn2+-sensing occurs widely in bacteria and influences both Mn2+ homeostasis and genes involved in the oxidative stress response. Several regulatory proteins related to C. diphtheriae DtxR have recently been shown to respond uniquely to Mn2+ (Posey et al., 1999 ; Jakubovics et al., 2000 ; Que & Helmann, 2000 ). These proteins provide a common mechanism for the control of Mn2+ permease production. When intracellular levels of Mn2+ rise, the metallorepressor (designated ScaR in Strep. gordonii) binds with high affinity to the transporter operon promoter, thus inhibiting transcription. Under limiting Mn2+, the apoprotein dissociates from the promoter control region such that repression is rapidly relieved, with upregulation of transcription and corresponding increase in permease levels (Jakubovics et al., 2000 ). In B. subtilis, the metallorepressor MntR is a bifunctional protein. It activates transcription of mntABCD encoding the ABC transporter in low Mn2+, but represses expression of the Nramp transporter encoded by mntH in Mn2+-replete conditions (Que & Helmann, 2000 ). Disruption of the negative regulatory control mechanisms on Mn2+ uptake may sensitize cells to Mn2+ toxicity. For example, overexpression of mntH encoding the Nramp transporter on a high-copy-number plasmid in E. coli or Sal. typhimurium decreases the growth rates of cultures in media containing >10 µM Mn2+ (Kehres et al., 2000 ). Likewise, constitutive derepression of mntH expression in B. subtilis, as a result of inactivating the metalloregulator gene mntR, greatly sensitizes the cells to Mn2+ (Que & Helmann, 2000 ). Interestingly, a mntA knockout mutant in the ABC transporter was also more sensitive to Mn2+, presumably because the MntH channel was upregulated in this strain (Que & Helmann, 2000 ). Thus, the co-ordinated expression and activities of the transporters are necessary for the cells to be able to respond to fluctuations in external Mn2+.

It is not yet known whether the Mn2+-specific DtxR-like proteins have global effects on transcription. Oxidative stress response genes are controlled in response to Mn2+ by the PerR metallorepressor, which shares about 28% identity with Fur protein. B. subtilis PerR responds to both Fe2+ and Mn2+ in controlling transcription at >=four loci, collectively known as the peroxide regulon (Bsat et al., 1998 ). PerR-repressed genes encode a variety of oxidative stress response proteins, including the vegetative cell catalase, alkyl hydroperoxide reductase, haem biosynthetic enzymes and a protective DNA-binding protein, MrgA.

Recent studies with L. plantarum have provided further evidence for an important role of Mn2+-sensing and signalling in bacteria. Expression of not only the P-type ATPase MntA, but also of two glycolytic enzymes, enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), is repressed in Mn2+-rich medium (Hao et al., 1999 ). Surface-bound enolase and GAPDH have been shown to bind human tissue proteins in a variety of organisms, and in group A streptococci they are implicated in the ability of the bacteria to modulate host cell functions (Pancholi & Fischetti, 1998 ). It is tempting to speculate, therefore, that Mn2+ concentrations might influence expression of surface proteins that are relevant for colonization or virulence.


   Mn2+ homeostasis and virulence
TOP
Overview
Cellular functions of Mn
Mn2+ transport systems
Transcriptional regulation
Mn2+ homeostasis and virulence
Future perspectives
REFERENCES
 
Clear links have been established between expression of the LraI family of putative metal-ion-binding proteins and the course of bacterial infections. Antibodies to EfaA, the metal-ion-binding protein of the Efa permease in Enterococcus faecalis, are elevated in patients with enterococcal endocarditis (Jiang et al., 1997 ). Mutants of Streptococcus parasanguinis and Strep. pneumoniae in which the LraI polypeptide genes are inactivated are avirulent in animal models of endocarditis (Burnette-Curley et al., 1995 ) and sepsis (Berry & Paton, 1996 ). Essential roles for the ABC-type Mn2+ transporters have recently been shown in virulence of E. faecalis (Singh et al., 1998 ), Streptococcus mutans (Kitten et al., 2000 ) and Yersinia pestis (Bearden & Perry, 1999 ). The solute-binding lipoprotein component of the streptococcal ABC transporter for Mn2+ is upregulated under Mn2+-limiting conditions (Jakubovics et al., 2000 ), such as those in serum where Mn2+ levels are characteristically around 20 nM (Krachler et al., 1999 ). Thus, these proteins may be essential for Mn2+ homeostasis and for survival of bacteria in the animal host.

Roles for the bacterial Nramps in virulence are less well established. These proteins might be necessary for the acquisition of divalent cations by bacteria within phagosomes during chronic infections. However, disruption of mntH in Sal. typhimurium, while delaying mortality in a mouse model, does not affect survival of bacteria within macrophages (Kehres et al., 2000 ). There is, on the other hand, a direct correlation between Mn2+ homeostasis and sensitivity of bacteria to oxidative stress. Inactivation of the Nramp transporter genes in B. subtilis, E. coli or Sal. typhimurium enhances the sensitivities of these bacteria to H2O2 compared with the corresponding wild-type strains (Kehres et al., 2000 ; Que & Helmann, 2000 ). In Strep. gordonii, the activity of MnSOD is 50% reduced in scaA or scaC mutants that lack the functional ABC-type Mn2+ transporter (N. S. Jakubovics, A. W. Smith & H. F. Jenkinson, unpublished). Taken collectively, these recent data indicate that bacterial systems for acquiring Mn2+ are crucial for protection against reactive oxygen. Since ROS play such an important role in host defence against bacterial infection (Miller & Britigan, 1997 ), it is logical to hypothesize that an essential function of Mn2+ sequestration by infecting bacteria is to activate defence systems against both externally and internally derived ROS.

A model for the role of Mn2+ homeostasis in the pathogenesis of streptococcal infections is shown in Fig. 2. The main features of this proposal centre on the mechanisms by which streptococci are able to resist the toxic effects of ROS. Exogenous is probably detoxified by cell-surface MnSOD (Gerlach et al., 1998 ). Internal ROS are produced following diffusion of molecular oxygen into bacterial cells from the bloodstream. H2O2 is generated intracellularly predominantly by the activities of pyruvate oxidase (Spx; Pericone et al., 2000 ) and other oxidases, which also generate (Fig. 2). The major enzyme capable of detoxifying in streptococci is MnSOD. Since streptococci lack catalase, most H2O2 generated by oxidases and MnSOD is released by the cells, causing haemolysis or tissue damage (Barnard & Stinson, 1996 ). Internal protection against H2O2 is afforded by peroxidases such as thiol peroxidase (Tpx), glutathione peroxidase (Gpo) or alkyl hydroperoxide reductase (AhpC) (Kolenbrander et al., 1998 ; King et al., 2000 ). Intracellular Mn2+ may directly protect against the damaging effects of H2O2 (Kehres et et al., 2000 ). For Mn2+ homeostasis to combat oxidative stress, it is thought that streptococci possess at least three Mn2+ transport systems (Fig. 2). The primary transporter under Mn2+-restricted conditions is postulated to be an ABC-type permease with high specificity for Mn2+. A second transporter, inhibitable by Zn2+ (Kolenbrander et al., 1998 ), may correspond to the Adc permease identified in Strep. pneumoniae (Dintilhac et al., 1997 ). A third transporter is likely to be an Nramp-like protein, the gene for which is present in Strep. gordonii (N. S. Jakubovics, A. W. Smith & H. F. Jenkinson, unpublished) and also present within the Strep. mutans genome (http://www.genome.ou.edu/smutans). In B. subtilis, activity of the Nramp transporter (MntH) system protects against H2O2 (Que & Helmann, 2000 ), while Nramp transporters from E. coli and Sal. typhimurium are upregulated in response to H2O2 (Kehres et al., 2000 ). Therefore, future characterization of streptococcal Nramps will allow the pathways for Mn2+ homeostasis and oxidative stress resistance in these organisms to be firmly established.



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 2. Model linking Mn2+ homeostasis and ROS-detoxification pathways in streptococci. Two ABC-type transporters, the high-affinity Sca permease (Strep. gordonii) and the Zn2+ (Mn2+) transporter Adc (Strep. pneumoniae), and an Nramp-like transporter, are hypothesized to mediate Mn2+ uptake. The high-affinity ABC-type transporter provides Mn2+ for MnSOD when environmental Mn2+ is limiting. MnSOD protects cells against external (dashed line with arrowhead) and detoxifies generated internally by the action of oxidases such as NADH oxidase (Nox), the primary O2-reducing enzyme in metabolism. Expression of the bacterial Nramp transporters may be upregulated by H2O2, formed by the action of pyruvate oxidase (Spx) or MnSOD, resulting in increased intracellular Mn2+ that may detoxify H2O2. In addition, H2O2 is degraded by peroxidases such as thiol peroxidase (Tpx), or is extruded from the cell (dashed line with arrowhead).

 

   Future perspectives
TOP
Overview
Cellular functions of Mn
Mn2+ transport systems
Transcriptional regulation
Mn2+ homeostasis and virulence
Future perspectives
REFERENCES
 
Mn2+ is an essential cofactor for many enzymes, some of which, for example MnSOD, are essential for bacterial growth and survival under oxidative stress. In Bacillus spp. it has long been recognized that Mn2+ is required for sporulation, but research into the topic has lain dormant until recently. With new insights into the mechanisms of Mn2+ transport, regulation of Mn2+-dependent gene expression and the importance of Mn2+ in oxidative stress defence, future advances in these areas are predicted. Several questions regarding the basic functions of Mn2+ homeostasis in bacteria remain unanswered. For example, how does Mn2+ protect against reactive oxygen in the absence of SOD? What is the physiological significance of this non-enzymic protection mechanism in bacteria? Are there other types of Mn2+ transporters to be discovered? Do efflux pumps fine-tune intracellular Mn2+ levels and thus contribute to Mn2+ homeostasis?

In the immediate future, it will be interesting to identify other Mn2+ transporters that are related to the P-type ATPase of L. plantarum, which has an unusually high capacity for Mn2+ import (Hao et al., 1999 ). Also, the discovery of Nramp transporters in bacteria has underscored the similarities between prokaryotic and eukaryotic systems for essential cation uptake. Determining the relative significance of multiple Mn2+ import systems in homeostasis in individual species presents a major challenge for future research. Significantly, the observation that Mn2+ acquisition by bacteria is linked to virulence in the host suggests that Mn2+ transport is a potential new therapeutic target for control of bacterial infection.


   ACKNOWLEDGEMENTS
 
The authors would like to thank Anthony Smith, Paul Kolenbrander, Jean-Pierre Claverys and Andreas Podbielski for their helpful advice and discussions, Jean-Pierre Claverys for providing a manuscript ahead of publication, and David Dymock for critical reading of this manuscript. The support of the British Heart Foundation is gratefully acknowledged.


   REFERENCES
TOP
Overview
Cellular functions of Mn
Mn2+ transport systems
Transcriptional regulation
Mn2+ homeostasis and virulence
Future perspectives
REFERENCES
 
Abell, L. M., Schineller, J., Keck, P. J. & Villafranca, J. J. (1995). Effect of metal-ligand mutations on phosphoryl transfer reactions catalyzed by Escherichia coli glutamine synthetase. Biochemistry 34, 16695-16702.[Medline]

Archibald, F. (1986). Manganese: its acquisition by and function in the lactic acid bacteria. Crit Rev Microbiol 13, 63-109.[Medline]

Arigoni, F., Guérout-Fleury, A. M., Barák, I. & Stragier, P. (1999). The SpoIIE phosphatase, the sporulation septum and the establishment of forespore-specific transcription in Bacillus subtilis: a reassessment. Mol Microbiol 31, 1407-1415.[Medline]

Barford, D. (1996). Molecular mechanisms of the protein serine/threonine phosphatases. Trends Biochem Sci 21, 407-412.[Medline]

Barnard, J. P. & Stinson, M. W. (1996). The alpha-hemolysin of Streptococcus gordonii is hydrogen peroxide. Infect Immun 64, 3853-3857.[Abstract]

Bartsevich, V. V. & Pakrasi, H. B. (1995). Molecular identification of an ABC transporter complex for manganese: analysis of a cyanobacterial mutant strain impaired in the photosynthetic oxygen evolution process. EMBO J 14, 1845-1853.[Abstract]

Bearden, S. W. & Perry, R. D. (1999). The Yfe system of Yersinia pestis transports iron and manganese and is required for full virulence of plague. Mol Microbiol 32, 403-414.[Medline]

Berry, A. M. & Paton, J. C. (1996). Sequence heterogeneity of PsaA, a 37-kilodalton putative adhesin essential for virulence of Streptococcus pneumoniae. Infect Immun 64, 5255-5262.[Abstract]

Braun, V. & Killmann, H. (1999). Bacterial solutions to the iron-supply problem. Trends Biochem Sci 24, 104-109.[Medline]

Bsat, N., Herbig, A., Casillas-Martinez, L., Setlow, P. & Helmann, J. D. (1998). Bacillus subtilis contains multiple Fur homologues: identification of the iron uptake (Fur) and peroxide regulon (PerR) repressors. Mol Microbiol 29, 189-198.[Medline]

Burnette-Curley, D., Wells, V., Viscount, H., Munro, C. L., Fenno, J. C., Fives-Taylor, P. & Macrina, F. L. (1995). FimA, a major virulence factor associated with Streptococcus parasanguis endocarditis. Infect Immun 63, 4669-4674.[Abstract]

Chander, M., Setlow, B. & Setlow, P. (1998). The enzymatic activity of phosphoglycerate mutase from gram-positive endospore-forming bacteria requires Mn2+ and is pH sensitive. Can J Microbiol 44, 759-767.[Medline]

Chao, Y. P., Patnaik, R., Roof, W. D., Young, R. F. & Liao, J. C. (1993). Control of gluconeogenic growth by pps and pck in Escherichia coli. J Bacteriol 175, 6939-6944.[Abstract]

Charney, J., Fisher, W. P. & Hegarty, C. P. (1951). Manganese as an essential element for sporulation in the genus Bacillus. J Bacteriol 62, 145-148.[Medline]

Chen, Y. W., Dekker, E. E. & Somerville, R. L. (1995). Functional analysis of E. coli threonine dehydrogenase by means of mutant isolation and characterization. Biochim Biophys Acta 1253, 208-214.[Medline]

Cheton, P. L. & Archibald, F. S. (1988). Manganese complexes and the generation and scavenging of hydroxyl free radicals. Free Radic Biol Med 5, 325-333.[Medline]

Christianson, D. W. (1997). Structural chemistry and biology of manganese metalloenzymes. Prog Biophys Mol Biol 67, 217-252.[Medline]

Dintilhac, A., Alloing, G., Granadel, C. & Claverys, J.-P. (1997). Competence and virulence of Streptococcus pneumoniae: Adc and PsaA mutants exhibit a requirement for Zn and Mn resulting from inactivation of putative ABC metal permeases. Mol Microbiol 25, 727-739.[Medline]

Doyle, R. J. (1989). How cell walls of gram-positive bacteria interact with metal ions. In Metal Ions and Bacteria , pp. 275-293. Edited by T. J. Beveridge & R. J. Doyle. New York:Wiley.

Escolar, L., Pérez-Martín, J. & de Lorenzo, V. (1999). Opening the iron box: transcriptional metalloregulation by the Fur protein. J Bacteriol 181, 6223-6229.[Free Full Text]

Fridovich, I. (1995). Superoxide radical and superoxide dismutases. Annu Rev Biochem 64, 97-112.[Medline]

Gaballa, A. & Helmann, J. D. (1998). Identification of a zinc-specific metalloregulatory protein, Zur, controlling zinc transport operons in Bacillus subtilis. J Bacteriol 180, 5815-5821.[Abstract/Free Full Text]

García-Domínguez, M., Lopez-Maury, L., Florencio, F. J. & Reyes, J. C. (2000). A gene cluster involved in metal homeostasis in the cyanobacterium Synechocystis sp. strain PCC 6803. J Bacteriol 182, 1507-1514.[Abstract/Free Full Text]

Gerlach, D., Reichardt, W. & Vettermann, S. (1998). Extracellular superoxide dismutase from Streptococcus pyogenes type 12 strain is manganese-dependent. FEMS Microbiol Lett 160, 217-224.[Medline]

Gibson, C. M. & Caparon, M. G. (1996). Insertional inactivation of Streptococcus pyogenes sod suggests that prtF is regulated in response to a superoxide signal. J Bacteriol 178, 4688-4695.[Abstract]

Gould, G. W. (1969). Germination. In The Bacterial Spore , pp. 397-444. Edited by G. W. Gould & A. Hurst. London:Academic Press.

Gruenheid, S. & Gros, P. (2000). Genetic susceptibility to intracellular infections: Nramp1, macrophage function and divalent cations transport. Curr Opin Microbiol 3, 43-48.[Medline]

Hao, Z., Chen, S. & Wilson, D. B. (1999). Cloning, expression and characterization of cadmium and manganese uptake genes from Lactobacillus plantarum. Appl Environ Microbiol 65, 4746-4752.[Abstract/Free Full Text]

Henriques, A. O., Melsen, L. R. & Moran, C. P. J. (1998). Involvement of superoxide dismutase in spore coat assembly in Bacillus subtilis. J Bacteriol 180, 2285-2291.[Abstract/Free Full Text]

Holmes, R. K. (2000). Biology and molecular epidemiology of diphtheria toxin and the tox gene. J Infect Dis 181, S156-S167.[Medline]

Hosfield, D. J., Guan, Y., Haas, B. J., Cunningham, R. P. & Tainer, J. A. (1999). Structure of the DNA repair enzyme endonuclease IV and its DNA complex: double-nucleotide flipping at abasic sites and three-metal-ion catalysis. Cell 98, 397-408.[Medline]

Inaoka, T., Matsumura, Y. & Tsuchido, T. (1999). SodA and manganese are essential for resistance to oxidative stress in growing and sporulating cells of Bacillus subtilis. J Bacteriol 181, 1939-1943.[Abstract/Free Full Text]

Jakubovics, N. S., Smith, A. W. & Jenkinson, H. F. (2000). Expression of the virulence-related Sca (Mn2+) permease in Streptococcus gordonii is regulated by a diphtheria toxin metallorepressor-like protein ScaR. Mol Microbiol 38, 140-153.[Medline]

Janulczyk, R., Pallon, J. & Björk, L. (1999). Identification and characterization of a Streptococcus pyogenes ABC transporter with multiple specificity for metal cations. Mol Microbiol 34, 596-606.[Medline]

Jenkinson, H. F. (1994). Cell surface protein receptors in oral streptococci. FEMS Microbiol Lett 121, 133-140.[Medline]

Jiang, Y. X., Murray, B. E. & Weinstock, G. M. (1997). Enterococcus faecalis antigens in human infections. Infect Immun 65, 4207-4215.[Abstract]

Kehres, D. G., Zaharik, M. L., Finlay, B. B. & Maguire, M. E. (2000). The NRAMP proteins of Salmonella typhimurium and Escherichia coli are selective manganese transporters involved in the response to reactive oxygen. Mol Microbiol 36, 1085-1100.[Medline]

King, K. Y., Horenstein, J. A. & Caparon, M. G. (2000). Aerotolerance and peroxide resistance in peroxidase and PerR mutants of Streptococcus pyogenes. J Bacteriol 182, 5290-5299.[Abstract/Free Full Text]

King, N., Dreesen, O., Stragier, P., Pogliano, K. & Losick, R. (1999). Septation, dephosphorylation, and the activation of {sigma}F during sporulation in Bacillus subtilis. Genes Dev 13, 1156-1167.[Abstract/Free Full Text]

Kitten, T., Munro, C. L., Michalek, S. M. & Macrina, F. L. (2000). Genetic characterization of a Streptococcus mutans LraI family operon and role in virulence. Infect Immun 68, 4441-4451.[Abstract/Free Full Text]

Kolenbrander, P. E., Andersen, R. N. & Ganeshkumar, N. (1994). Nucleotide sequence of the Streptococcus gordonii PK488 coaggregation adhesin gene, scaA, and ATP-binding cassette. Infect Immun 62, 4469-4480.[Abstract]

Kolenbrander, P. E., Andersen, R. N., Baker, R. A. & Jenkinson, H. F. (1998). The adhesion-associated sca operon in Streptococcus gordonii encodes an inducible high-affinity ABC transporter for Mn2+ uptake. J Bacteriol 180, 290-295.[Abstract/Free Full Text]

Krachler, M., Rossipal, E. & Micetic-Turk, D. (1999). Concentrations of trace elements in sera of newborns, young infants, and adults. Biol Trace Elem Res 68, 121-135.[Medline]

Kroos, L., Zhang, B., Ichikawa, H. & Yu, Y. T. (1999). Control of {sigma} factor activity during Bacillus subtilis sporulation. Mol Microbiol 31, 1285-1294.[Medline]

Kuhn, N. J. & Ward, S. (1998). Purification, properties, and multiple forms of a manganese-activated inorganic pyrophosphatase from Bacillus subtilis. Arch Biochem Biophys 354, 47-56.[Medline]

Kuhn, N. J., Setlow, B., Setlow, P., Cammack, R. & Williams, R. (1995). Cooperative manganese (II) activation of 3-phosphoglycerate mutase of Bacillus megaterium: a biological pH-sensing mechanism in bacterial spore formation and germination. Arch Biochem Biophys 320, 35-42.[Medline]

Kuo, C. F., Mashino, T. & Fridovich, I. (1987). {alpha},ß-Dihydroxyisovalerate dehydratase. A superoxide-sensitive enzyme. J Biol Chem 262, 4724-4727.[Abstract/Free Full Text]

Lawrence, M. C., Pilling, P. A., Epa, V. C., Berry, A. M., Ogunniyi, A. D. & Paton, J. C. (1998). The crystal structure of the pneumococcal surface antigen PsaA reveals a metal binding site and a novel structure for a putative ABC-type binding protein. Structure 6, 1553-1561.[Medline]

Loukin, S. & Kung, C. (1995). Manganese effectively supports yeast cell-cycle progression in place of calcium. J Cell Biol 131, 1025-1037.[Abstract]

Miller, R. A. & Britigan, B. E. (1997). Role of oxidants in microbial pathophysiology. Clin Microbiol Rev 10, 1-18.[Abstract]

Missiakas, D. & Raina, S. (1997). Signal transduction pathways in response to protein misfolding in the extracytoplasmic compartments of E. coli: role of two new phosphoprotein phosphatases PrpA and PrpB. EMBO J 16, 1670-1685.[Abstract/Free Full Text]

Morgan, T. R., Shand, J. A., Clarke, S. M. & Eaton-Rye, J. J. (1998). Specific requirements for cytochrome c-550 and the manganese-stabilizing protein in photoautotrophic strains of Synechocystis sp. PCC 6803 with mutations in the domain Gly-351 to Thr-436 of the chlorophyll-binding protein CP47. Biochemistry 37, 14437-14449.[Medline]

Mukhopadhyay, B., Stoddard, S. F. & Wolfe, R. S. (1998). Purification, regulation, and molecular and biochemical characterization of pyruvate carboxylase from Methanobacterium thermoautotrophicum strain {Delta}H. J Biol Chem 273, 5155-5166.[Abstract/Free Full Text]

Neidhart, D. J., Kenyon, G. L., Gerlt, J. A. & Petsko, G. A. (1990). Mandelate racemase and muconate lactonizing enzyme are mechanistically distinct and structurally homologous. Nature 347, 692-694.[Medline]

Niven, D. F., Ekins, A. & Al-Sumaurai, A. A.-W. (1999). Effects of iron and manganese availability on growth and production of superoxide dismutase by Streptococcus suis. Can J Microbiol 45, 1027-1032.[Medline]

Ohtani, N., Haruki, M., Muroya, A., Morikawa, M. & Kanaya, S. (2000). Characterization of ribonuclease HII from Escherichia coli overproduced in a soluble form. J Biochem 127, 895-899.[Abstract]

Pancholi, V. & Fischetti, V. A. (1998). {alpha}-Enolase, a novel strong plasmin(ogen) binding protein on the surface of pathogenic streptococci. J Biol Chem 273, 14503-14515.[Abstract/Free Full Text]

Pericone, C. D., Overweg, K., Hermans, P. W. & Weiser, J. N. (2000). Inhibitory and bactericidal effects of hydrogen peroxide production by Streptococcus pneumoniae on other inhabitants of the upper respiratory tract. Infect Immun 68, 3990-3997.[Abstract/Free Full Text]

Pierre, J. L. & Fontecave, M. (1999). Iron and activated oxygen species in biology: the basic chemistry. Biometals 12, 195-199.[Medline]

Posey, J. E. & Gherardini, F. C. (2000). Lack of a role for iron in the Lyme disease pathogen. Science 288, 1651-1653.[Abstract/Free Full Text]

Posey, J. E., Hardham, J. M., Norris, S. J. & Gherardini, F. C. (1999). Characterization of a manganese-dependent regulatory protein, TroR, from Treponema pallidum. Proc Natl Acad Sci USA 96, 10887-10892.[Abstract/Free Full Text]

Que, Q. & Helmann, J. D. (2000). Manganese homeostasis in Bacillus subtilis is regulated by MntR, a bifunctional regulator related to the diphtheria toxin repressor family of proteins. Mol Microbiol 35, 1454-1468.[Medline]

Rao, N. N., Liu, S. & Kornberg, A. (1998). Inorganic polyphosphate in Escherichia coli: the phosphate regulon and the stringent response. J Bacteriol 180, 2186-2193.[Abstract/Free Full Text]

Robbe-Saule, V., Coynault, C., Ibanez-Ruiz, M., Hermant, D. & Norel, F. (2001). Identification of a non-haem catalase in Salmonella and its regulation by RpoS ({sigma}S). Mol Microbiol 39, 1533-1545.[Medline]

Schroeter, R., Schlisio, S., Lucet, I., Yudkin, M. & Borriss, R. (1999). The Bacillus subtilis regulator protein SpoIIE shares functional and structural similarities with eukaryotic protein phosphatases 2C. FEMS Microbiol Lett 174, 117-123.[Medline]

Seemann, J. E. & Schulz, G. E. (1997). Structure and mechanism of L-fucose isomerase from Escherichia coli. J Mol Biol 273, 256-268.[Medline]

Sekowska, A., Danchin, A. & Risler, J. L. (2000). Phylogeny of related functions: the case of polyamine biosynthetic enzymes. Microbiology 146, 1815-1828.[Abstract/Free Full Text]

Silver, S. (1996). Bacterial resistances to toxic metal ions – a review. Gene 179, 9-19.[Medline]

Silver, S. & Lusk, J. E. (1987). Bacterial magnesium, manganese and zinc transport. In Ion Transport in Prokaryotes , pp. 165-180. Edited by B. P. Rosen & S. Silver. London:Academic Press.

Singh, K. V., Coque, T. M., Weinstock, G. M. & Murray, B. E. (1998). In vivo testing of an Enterococcus faecalis efaA mutant and use of efaA homologs for species identification. FEMS Immunol Med Microbiol 21, 323-331.[Medline]

Singh, V. K., Xiong, A., Usgaard, T. R., Chakrabarti, S., Deora, R., Misra, T. K. & Jayaswal, R. K. (1999). ZntR is an autoregulatory protein and negatively regulates the chromosomal zinc resistance operon znt of Staphylococcus aureus. Mol Microbiol 33, 200-207.[Medline]

Spatafora, G. & Moore, M. (1998). Growth of Streptococcus mutans in an iron-limiting medium. Methods Cell Sci 20, 217-221.

Stadtman, E. R., Berlett, B. S. & Chock, P. B. (1990). Manganese-dependent disproportionation of hydrogen peroxide in bicarbonate buffer. Proc Natl Acad Sci USA 87, 384-388.[Abstract]

Storz, G. & Imlay, J. A. (1999). Oxidative stress. Curr Opin Microbiol 2, 188-194.[Medline]

Stoyanov, J. V., Hobman, J. L. & Brown, N. L. (2001). CueR (Ybbl) of Escherichia coli is a MerR family regulator controlling expression of the copper exporter CopA. Mol Microbiol 39, 502-511.[Medline]

Thelwell, C., Robinson, N. J. & Turner-Cavet, J. S. (1998). An SmtB-like repressor from Synechocystis PCC 6803 regulates a zinc exporter. Proc Natl Acad Sci USA 95, 10728-10733.[Abstract/Free Full Text]

Thompson, J., Ruvinov, S. B., Freedberg, D. I. & Hall, B. G. (1999). Cellobiose-6-phosphate hydrolase (CelF) of Escherichia coli: characterization and assignment to the unusual family 4 of glycosylhydrolases. J Bacteriol 181, 7339-7345.[Abstract/Free Full Text]

Touati, D. (2000). Iron and oxidative stress in bacteria. Arch Biochem Biophys 373, 1-6.[Medline]

Whittaker, M. M., Barynin, V. V., Antonyuk, S. V. & Whittaker, J. W. (1999). The oxidized (3,3) state of manganese catalase. Comparison of enzymes from Thermus thermophilus and Lactobacillus plantarum. Biochemistry 38, 9126-9136.[Medline]

Yesilkaya, H., Kadioglu, A., Gingles, N., Alexander, J. E., Mitchell, T. J. & Andrew, P. W. (2000). Role of manganese-containing superoxide dismutase in oxidative stress and virulence of Streptococcus pneumoniae. Infect Immun 68, 2819-2826.[Abstract/Free Full Text]

Yocum, C. F. & Pecoraro, V. L. (1999). Recent advances in the understanding of the biological chemistry of manganese. Curr Opin Chem Biol 3, 182-187.[Medline]