Metals and the rhizobial-legume symbiosis — Uptake, utilization and signalling

Metals and the Rhizobia I -Legume Symbiosis - Uptake, Ut i l izat ion and

Signall ing

Andrew W. B. Johnston, Kay H. Yeoman and Margaret Wexler

School of Biological Sciences, University of East Anglia, Norwich NR4 7T J, UK

ABSTRACT

In this review, we consider how the nitrogen-fixing root nodule bacteria, the 'rhizobia', acquire various metals, paying particular attention to the uptake of iron. We also review the literature pertaining to the roles of molybdenum and nickel in the symbiosis with legumes. We highlight some gaps in our knowledge, for example the lack of information on how rhizobia acquire molybdenum. We examine the means whereby different metals affect rhizobial physiology and the role of metals as signals for gene regulation. We describe the ways in which genetics has shown (or not) if, and how, particular metal uptake and/or metal-mediated signalling pathways are required for the symbiotic interaction with legumes.

1. I n t r o d u c t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114

2. I r on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116

2.1. The importance of iron for Rhizobium . . . . . . . . . . . . . . . . . . . . . . . . . . . 116

2.2. Siderophore production by rhizobia . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

2.3. Other iron uptake systems in rhizobia . . . . . . . . . . . . . . . . . . . . . . . . . . . 127

2.4. Iron uptake in the nodule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131

2.5. Iron and gene regulation in rhizobia . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132

3. M o l y b d e n u m . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136

3.1. Molybdenum uptake and rhizobia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136

3.2. Molybdenum uptake in other bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . 137

4. N icke l . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138

5. Other metals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141

5.1. Z i nc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142

5.2. M a n g a n e s e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142

5.3. C a l c i u m . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142

5.4. C o p p e r . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143

ADVANCES IN MICROBIAL PHYSIOLOGY VOL 45 Copyright © 2001 Academic Press ISBN 0-12-027745-X All rights of reproduction in any form reserved

114 ANDREW W. B. JOHNSTON, KAY H. YEOMAN AND MARGARET WEXLER

6. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145

1. INTRODUCTION

The symbiosis between legumes and the bacteria known collectively as the 'rhizobia' is the most important single beneficial interaction between prokaryotes and eukaryotes in agriculture. It allows many of the world's principal grain crops, such as soybeans, peas, peanuts, chickpeas and various other beans, to be grown without the need for exogenous nitrogen fertilizer; in addition, forage legumes sustain large parts of animal production. The interaction also represents a beautiful example of coupled, complex differentiation and development between two very different organisms, from two different kingdoms. Molecular genetics has, as in many spheres of biology, been piv- otal in elucidating many steps of the interaction (see reviews by Denarie et al., 1996; Hirsch and LaRue, 1997; Niner and Hirsch, 1998; Schultze and Kondorosi, 1998). Not surprisingly, the greatest advances have been in the genetic analysis of the bacterial contribution, with the identification of so many nodulation (nod) genes that the alphabet has long been saturated and the term nol has had to be recruited! In short, many of these nod and/or noi genes work in concert to manufacture a series of lipo-oligosaccharide 'Nod factors'. Remarkably, these elicit many of the early steps of the infection process even in the absence of live bacteria.

An important feature of the interaction is that it is specific. It is not our job here to survey this phenomenon, simply to say three things on this topic.

The particular host-range of a given rhizobial strain or species is determined, in part, by the particular configuration of the Nod factor made by an individual strain. However, some rhizobial strains have very wide host ranges, in part due to the wide 'action spectra' of the Nod factors that they make (Perret et al., 2000). More relevant to what follows is that the name 'rhizobia' is used as a catch-all, with little justification in real taxonomic criteria based on, for example, 16S rRNA sequences. These bacteria all belong to the t~-group of Purple-Green Bacteria but, within that heading, they have little in common, other than their ability to induce N2-fixing nodules. Thus, although the genes and processes that are used strictly for the symbiosis are similar in different rhizobial genera, the determinants of other 'house-keeping' functions can vary quite significantly. We will present several examples of such differences.

METALS AND THE RHIZOBIAL-LEGUME SYMBIOSIS 115

The last general point relating to host-range specificity is that the nodules on different legumes differ significantly with regard to their morphology, physiology and biochemistry. Again, we will allude to these differences where appropriate. Strains and species of rhizobia that we mention are in Table 1.

In this review, we examine the influence of metals in the rhizobia, an aspect of the symbiosis that has received sparse attention. This is surprising, for two reasons. First, two of the metals that we will consider, iron and molybdenum, are major components of the nitrogenase complex that reduces N 2 to NH 3 and so lie at the very heart of the symbiosis. Second, there is a large amount of information on how bacterial pathogens of animals (but not, interestingly, those of plants) acquire metals, for the important reason that competition for limited metallic resources is one of the key battlegrounds in determining whether an infection takes hold or not. Over and above Fe and Mo, of course, rhizobia need other metals for their normal metabolic processes. Although we will touch on some of these, they will feature very little in what follows

Table 1 Strains and species of rhizobia mentioned in this review.

Rhizobial strain or species Typical host legumes Comments

Bradyrhizobium japonicum Soybeans

Sinorhizobium meliloti Alfalfa

Rhizobium leguminosarum bv. viciae

R. leguminosarum bv. trifolii

Peas, vetches, lentils

Clovers

R. leguminosarum bv. phaseoli Phaseolus beans

Rhizobium tropici

Rhizobium spp.

Azorhizobium caulinodans

Phase olus beans; Leucaena (a tree)

Eg Cowpea, Siratro

Sesbania spp.

Slow-growing rhizobial species: hosts have determinant nodules

Fast growing, previously known as ' Rhizobium meliloti' ; closely related to Agrobacterium; indeterminant nodules

Fast growing rhizobial species; indeterminant nodules

Fast growing rhizobial species; indeterminant nodules

Fast growing rhizobial species; determinant nodules

Very wide host range among (mainly) tropical legume species

Induces nodules on stems as well as roots of host plant. Also, can fix N explanta and assimilate the fixed N


because, in truth, there is little to tell. We know next to nothing, for example, of how rhizobia obtain cobalt, zinc or manganese, yet these three, plus several other metals, are needed for the day-to-day metabolism of the bacteria, both in and out of the nodule. One exception to this paucity of information concerns nickel; this metal is a cofactor for the enzyme complex uptake hydrogenase, which is found in several rhizobia.

We will describe here what we know of how the rhizobia obtain their com- plement of metals, how they use them and how metals influence the metabolism of the bacteria. What we will not do, however, is survey the particular functions of individual metals in their capacities, for example, as cofactors, nor will we consider any agronomic aspects of the effects of metals on the symbiosis. We will compare the systems used by rhizobia with those in other bacteria and where appropriate, with other diazotrophs.

2. IRON

2.1. The Importance of Iron for Rhizobium

Iron is the fourth most abundant element on the Earth's crust and is an extremely important metal in biology. Due largely to its ready interconversion of redox states, it is a component of many enzymes and other proteins and is required in large amounts by virtually all organisms, with the possible exception of the lactobacilli and Borrelia burgdorfii, the causative agent of Lyme disease (see Pandey et aL, 1994; Elli et aL, 2000; Posey and Gherardini, 2000).

However, in an oxygenated world, there is a very severe problem with iron. In its oxidized, Fe 3÷ form, it is extremely insoluble, with a maximum solubil- ity of c. 10 -18 M at biological pH values. This is some 1012-fold lower than that required even for cells with no exceptional demand for the element. In the case of the Rhizobium-legume symbiosis, there is, of course, an even greater demand for iron, the nodule being a veritable magnet for the metal. The single most abundant protein that the plant host makes in the nodule is leghaemoglobin, an iron protein. In the bacteria, nitrogenase and nitrogenase reductase contain FeS clusters and the former has the cofactor FeMoCo at the active site for N 2 reduction. Further, bacteroids have a very high respiratory demand, requiring abundant cytochromes and other electron donors, each with their own Fe centres (see Delgado et al., 1998).

This high Fe demand impinges on the real world of agriculture, particularly in tropical soils, many of which are low in Fe (Vose, 1982). In forestry, too, high levels of Fe fertilization are needed for legume trees such as Acacia (Lesueur and Diem, 1997). In laboratory conditions, the lack of Fe has dramatic effects on nodule development. In lupin and peanut, nodule development


is much more susceptible to a shortage of Fe than are other parameters such as plant shoot and root weights (O'Hara et al., 1988; Tang et al., 1991a,b, 1992a,b). Note that these studies showed that the effects of Fe shortage occurred before the onset of nodules proper, mainly via the abortion of nodule initials. The plant seems to anticipate the lack of Fe to support the incipient nodules and shuts up shop ahead of time. Further, once nodules have formed, it has been found, perhaps not surprisingly, that the presence of active, N 2- fixing nodules puts further demands on the plant for iron acquisition (Terry et al., 1991). In Fe-replete conditions, the levels of Fe in the nodule are such that it is mobilized into developing seeds once N 2 fixation has ceased (Burton et al., 1998).

St Pierre et al. (1999) found that in free-living cells of R. leguminosarum, the majority of Fe was stored in the form of polynuclear iron(In) clusters that had some similarity to bacterioferritin, an Fe3+-binding protein found in several different bacterial species, but it is not known if this complex does, indeed, comprise this Fe-storage protein. To our knowledge, there are no reports of bacterioferritin or other Fe-storage protein in the rhizobia, in contrast to the situation with nickel storage (see below). However, sequences of the entire genome of Mesorh i zob ium loti (Kaneko et al. 2000 and http://www.kazusa.or.jp/rhizobase/,) and of Sinorhizobium meli lot i (see http://sequence.toulouse.inra.fr./meliloti.html) do reveal the presence of genes that are likely to encode bacterioferritin.

The importance of a more effective Fe supply to legumes may provide an attractive target for future engineering of improved Fe uptake and/or mobilization systems into legumes. Already, two genes (specifying a ferric-chelate reductase and the IRT1 iron transporter) that are important for Fe uptake in Arabidopsis thaliana have been cloned and analysed (Korshunova et al., 1999; Robinson et al., 1999).

Given the demonstrated importance of iron in the relationship between rhizobia arm legumes, there has been a surprisingly small research effort into this subject compared with many other aspects of the symbiosis. Nevertheless, there are some good reviews on iron metabolism and uptake in rhizobia (Guerinot, 1991, 1994; O'Brian, 1996; Fett et al., 1998).

2.2. Siderophore Production by Rhizobia

Different bacteria have evolved various ways of getting round the problem of the insolubility of iron in the environment. One of the most widespread mechanisms involves the production, export and uptake of molecules known as siderophores. These are molecules with a high affinity for ferric iron, and which, when charged with ferric iron, can be imported by specialist transport systems. Most siderophores are of one of two chemical types: hydroxamates or


catechols. In addition to these specialized molecules, many bacteria can take up ferric iron as Fe-citrate through dedicated transporters - it may be a seman- tic point as to whether citrate is a bone fide siderophore or not. Not surprisingly, bacterial strains and species that make a given siderophore can also import the same, Fe-charged molecule. However, there are many molecular 'pirates' in which bacterial strains have all the machinery for the import of Fe-containing siderophores that are made by different microbes.

Screening for siderophore production in microorganisms is greatly aided by the chrome azurol sulfonate (CAS) plate test (Schwyn and Neilands, 1987). The CAS dye, when complexed with Fe 3+, is blue, but turns orange if the iron is removed. Therefore, it is easy to screen colonies on agar plates for orange halos, indicating the presence of extracellular siderophores. The real power of the test is its non-specificity - many different siderophores can be detected, though obviously the greater the avidity for Fe 3÷, the better the staining response. Note though, that this test is not all-embracing; some bacteria cannot grow on the plates because of sensitivity to components in the CAS staining solution.

2.2.1. Surveys of Rhizobia for Siderophore Uptake and Synthesis

It is clear from several, albeit rather limited, surveys of field isolates that many individual strains of rhizobia make at least one siderophore and, in some cases, the chemical identity, or at least the general chemical class of molecule, has been elucidated. It should be emphasized that these studies are confined to siderophores made by free-living rhizobia and do not necessarily relate to siderophore production (and utilization) by bacteroids in the nodule.

A pattern to emerge from these studies is that strains of slow-growing Bradyrhizobiumjaponicum do not make bonefide siderophores that are detected by the CAS assay (Guerinot et al., 1990; Lesueur et aL, 1993; Plessner et al., 1993; Carson et al., 2000). However, virtually all the strains of this species take up ferric citrate, pointing to the importance of citrate in the Fe nutrition of these bacteria, at least in soils. The tropical, acidic soils from which B. japonicum probably originated have higher concentrations of soluble, ferrous iron, so there may be less selection pressure for siderophore production than in the less acid soils of temperate zones. However, at least one strain of B. japonicum makes some of the apparatus involved in the uptake of hydroxamate siderophores (LeVier and Guerinot, 1996; see below), so this reasoning to explain the paucity of siderophore production by B. japonicum may need some revision.

In contrast to B. japonicum, most strains of fast-growing genera (Rhizobium and Sinorhizobium) do make siderophores in free-living culture (Fabiano et al., 1994; Carson et al., 2000) although, fike B. japonicum, they can also take up ferric citrate (Carson et al., 2000). Barran and Bromfield (1993) isolated strains of S. meliloti from two soils that differed in their available Fe. There was no apparent


difference in the frequencies of strains that do (Sid ÷) or do not (Sid-) make siderophores from the two sources, indicating that siderophore production did not confer an obvious selective advantage in response to the availability of Fe. It may be, though, that more detailed and wide-ranging ecological studies are needed to ascertain the exact role of siderophore production by free-living rhizobia.

Preliminary characterization of the structures of some Rhizobium siderophores have confirmed that, depending on the strain, they are usually either catecholates or hydroxamates (Skorpuska et al., 1988; Jadhav and Desai, 1992; Roy et al., 1994). In their more detailed chemical analyses, Carson et al. (2000) found that most strains of R. leguminosarum made a trihydroxamate that was identical to the fully characterized siderophore, vicibactin (see below). With S. meliloti, many strains made a dihydroxamate siderophore; in some cases this was indistinguishable from the well-studied molecule rhizobactin 1021 (see below), but the others were uncharacterized. Interestingly, one strain of R. tropici made a siderophore that was neither a hydroxamate nor a cate- cholate (Carson et al., 2000).

The rhizobial siderophores that have been studied in most detail are a trihydroxamate from R. leguminosarum and two rather different molecules from different strains of S. meliloti. The mechanisms and the genes involved in their synthesis, uptake and regulation, together with a consideration of the roles of these molecules, are presented in the following sections.

2.2.2. Structure, Synthesis and Uptake of Vicibactin, the 'Type ' Siderophore of R. leguminosarum

Free-living cells of R. leguminosarum bv. viciae strain WSM710, growing in low-iron conditions, make the trihydroxamate siderophore vicibactin (Carson et al., 1992a,b, 1994), which is a cyclic trihydroxamate comprising three molecules each of N2-acetyl-N5-hydroxy-D-ornithine and 3-hydroxybutyric acid (Dilworth et al., 1998; Fig. 1). Several other strains of R. leguminosarum, including the genetically well-characterized 8401 pRL 1JI, also make vicibactin (Dilworth et al., 1998; Stevens et al., 1999; Carson et al., 2000).

2.2.2.1. Genetics of Vicibactin Synthesis. Mutants defective in vicibactin synthesis were isolated by transposon mutagenesis of strain WSM710 (Worsley, 2000). The insertions were in a two-gene operon, termed vbsSO. From sequence analysis., this is thought to encode a complex, multifunctional synthase (vbsS) that catalyses the formation of the overall ring structure of the molecule and a hydroxylase (vbsO) that modifies the butyrate moieties in the nascent molecule (Worsley, 2000; R.A. Carter and K.H. Yeoman, unpublished observations). The vbs cluster also contains a gene, vbsA, which is likely to be involved in acetyla- tion of the omithine moiety of vicibactin (R.A. Carter, personal communication).


c~, o c.--c.2 -----,.f ?"

0 0%. , . / 0 " " ~ N

II X,~H 2 o , , - c - ~ . " ~ c . , CH2 / \

CH2 CH2

/ Ca-Na-C-ah CH2 / l C~O

ON--N

\ / H--CH 3

cH~cH'xo_ .~C~o ~ ' C ~ " -

O# ~ CIH ~ CH2___. CH2....~ CH 2 OH Nit

~:--o

(B)

CH~ COOH

NH

~ C=O CtXltt


CH=

C - - - O

N ~ O H

H ~ C ~ H

H ~ C ~ H H COOH

i I H - - C ...... N ~ C ~ C C ~ C ~ C

O OH O

H

H

O - - ' C

I HO--N

I H ~ C ~ H

N C - - H

I H

(c)

Figure 1 Diagrammatic representation of three siderophores synthesized by R. leguminosarum (A, vicibactin) and two strains of S. meliloti (B, rhizobactin and C, rhizobactin 1021).

2.2.2.2. Regulation ofvbs Genes. As with siderophore production in many bacteria, vicibactin synthesis by R. leguminosarum is greatly enhanced when cells are grown in low levels of available iron (Dilworth et al., 1998). This is because the vbsSO operon is expressed at high level only when Fe in the medium is at low concentration (Worsley, 2000; R.A. Carter and K.H. Yeoman, unpublished observations). In many other bacteria, siderophore biosynthetic genes are under the overall control of the 'global' transcriptional regulator fur, whose product binds to DNA sequences (fur boxes) in the vicinity of the promoters of genes that it regulates (see Crosa, 1997, Braun et al., 1998). However, in R. leguminosarum, the vbsSO operon is not regulated by the fur- like gene that had been identified in that species (see below).

We have recently found that transcription of vbsSO has an absolute requirement for a gene, termed rpoI, which is located a few kb upstream from vbsSO (Yeoman et al., 1999; K.H. Yeoman, unpublished). From its sequence, rpol appears to encode a member of the burgeoning family of extra-cytoplasmic sigma factors (ECF) of RNA polymerase, which recognize various promoters that specify proteins located near the cell surface (see Lonetto et al., 1994; Missiakis and Raina, 1998). Mutations in rpoI abolish vicibactin production, consistent with the requirement for rpoI in the transcription of the vbsSO biosynthetic genes (Yeoman et al., 1999). Further, transcription of rpoI itself


is enhanced in low-iron conditions, but, as with vbsSO expression, this is not mediated via the R. leguminosarumfur gene (M. Wexler, unpublished observations). The identity of the iron-sensing regulator of rpoI remains to be identified. It has not been established if the RpoI sigma factor acts directly on the vbsSO promoter; in Pseudomonas aeruginosa, PvdS, a homologue of RpoI (Ochsner et al., 1996), does act directly on the promoter of pvdA, which is involved in the biosynthesis of the siderophore pyoverdine (Leoni et al., 2000). However, unlike R. leguminosarum, Fe-dependent expression of the Pseudomonas pvdS gene is controlled by fur (Barton et al., 1996a).

2.2.2.3. Vicibactin Uptake. The most detailed studies on the genetics of rhizobial Fe uptake have been with R. leguminosarum. Mutants of this species were isolated, initially on the basis of their large orange halos on CAS plates. Several of these mutations were in one of two unlinked operons (fhuA) and (fhuBCD) that contained homologues of thefhu genes of E. coli and other bacteria (Stevens et al., 1999; Yeoman et al., 2000). In E. coli, these genes are in one operon, fhuABCD, which specifies the apparatus for hydroxamate uptake, the fourjhu genes respectively encoding the outer membrane receptor, the periplasmic transporter and the inner membrane ATPase proteins (see Braun et al., 1998).

In some strains of R. leguminosarum, there is a second version of thefhuA gene, downstream offhuB (Stevens et al., 1999; Yeoman et al., 2000), but it appears to be a pseudogene, with many stop codons and no promoter. Pseudogenes are rare in bacteria and most of those that have been identified appear to have arisen from genes that had encoded proteins that are located on the bacterial cell surface. The FhuA protein of E. coli not only acts as the receptor for hydroxamate siderophores, but it has been 'hijacked' by a number of colicins, bacteriophages and antibiotics that use it as their receptor (Killmann et al., 1995). Stevens et al. (1999) speculated that if the FhuA protein of R. leguminosarum also acted as a receptor for such potentially fatal agents, then there would be strong selection pressure to lose this vulnerable site. It might, therefore, be replaced by another version offhuA, which still acts as a siderophore receptor but not as a gateway for 'phages, bacteriocins or antibiotics.

Just as with the vbs genes (see above) and, indeed, the siderophore uptake genes of other bacteria (Crosa, 1997), both thefhuA and thefhuBCD operons of R. leguminosarum are expressed at much higher levels when the bacteria were starved of iron (Stevens et al., 1999; Yeoman et al., 2000). As with the vbs genes, this regulation did not involve the fur gene (M, Wexler and K.H. Yeoman, unpublished). However, in contrast to the vbs genes, expression of fhuA andfhuBCD did not require the ECF gene rpoI (Stevens et aL, 1999; Yeoman et al., 2000). Although neither vbs nor fhu is regulated by fur, it is interesting to see that these two classes of genes are under different control systems with regard to the role of rpoI.


2.2.2.4. Vicibactin and a tonB-like Gene. In other bacteria, much of the energy needed to drive the uptake of the siderophore-Fe 3÷ complexes is provided by three, interacting proteins, TonB, ExbB and ExbD (see Braun et al., 1998; Higgs et al., 1998; Braun and Killmann, 1999; Postle, 1999). In R. leguminosarum, there is a gene whose product has limited homology to TonB of other bacteria (M. Wexler, unpublished). Mutations in this gene caused a large-halo phenotype on CAS plates pointing to a role for TonB in vicibactin uptake, most likely by its interaction with the Fhu uptake proteins. As described below, this gene is also involved in the uptake of haem by Rhizobium.

2.2.2.5.Vicibactin and In Planta Studies. Mutants of R. leguminosarum with insertions in either the structural vbs or the regulatory rpol genes induced nodules on peas that were capable of normal N 2 fixation (Yeoman et al., 1999; Worsley, 2000). Further, a strain of R. leguminosarum carrying vbsS fused to the reporter gene gus was used to inoculate peas. By in situ staining and assay- ing [3-glucuronidase in bacteroids of pea nodules, a surprising result was obtained (Worsley, 2000). Although the immature, non-fixing forms of rhizobia in the nodule expressed the vbs genes, the mature, N2-fixing bacteroids did not.

Strains of R. leguminosarum, in which either thefhuA or thefhuBCD operons were mutated, also induced apparently normal N2-fixing nodules. As with the vbs :system, fhuA::gus fusion strains clearly showed that fhuA was not expressed in bacteroids in pea nodules, whereas the non-fixing bacteria in the immature nodule zone expressed fhuA at high levels (Yeoman et al., 2000). Thus, in R. leguminosarum, the genes for production and for the uptake of vicibactin are switched off, just at the time when the iron requirement of the bacteria is at its highest!

Finally, strains of R. leguminosarum with mutations in the tonB-like gene (see above) also appear to be unaffected in symbiotic N 2 fixation (M. Wexler, unpublished). Therefore, any of the uptake systems that require this gene are also dispensable for normal symbiotic function.

2.2.3. S. meliloti Rhizobactin and Rhizobactin 1021

In Sinorhizobium meliloti, two very different types of siderophores have been fully characterized. One of these, rhizobactin, was obtained from Fe-starved R. meliloti strain DM4 by Smith et al. (1985). It has tx-hydroxycarboxyl- and ethylenediaminedicarboxyl- as its Fe-ligating moieties (Fig. 1), the latter being a novel natural product that is still unique amongst siderophores.

The second, structurally unrelated, S. meliloti siderophore was from strain 1021 and so was named rhizobactin 1021 (Persmark et al., 1993). It is a citrate- based dihydroxamate with structural similarity to several other bacterial


siderophores, namely schizokinen, aerobactin and arthrobactin (Fig. 1). Rhizobactin 1021 has an unusual fatty acid attachment, (E)-2-decenoic acid which, hitherto, had only been found in secretions of the occipital gland of Bactrian camels and in the royal jelly of honey bees.

2.2.3.1. Genes for the Synthesis, Uptake and Regulation of Rhizobactin 1021. Mutants that are, variously, defective in the synthesis or uptake of rhizobactin 1021 have been isolated (Gill and Neilands, 1989; Reigh and O'Connell, 1993). Lynch et al. (2001) identified a cluster of genes that are involved in these functions. A single operon, rhbABCDEF, was located on a native megaplasmid (Barloy-Hubler et al., 2000), which appeared to be sufficient for the production of rhizobactin 1021. The products of four of the rhb genes (rhbC, D, E and F) were similar to the Iuc proteins, which, in E. coli, are involved in the synthesis of the chemically similar siderophore, aerobactin (Neilands, 1992). The RhbA and RhbB proteins were similar to the Acinetobacter baumanii Dat and Dcc gene products, respectively (Lynch et al., 2001), and are both involved in the synthesis of 1,3-diaminopropane, a proposed precursor for rhizobactin 1021 biosynthesis (Lynch et al., 2001).

Next to the rhb biosynthetic operon was a gene, rhtA, with a similar sequence to iutA of E. coli, which specifies the outer membrane protein receptor for aerobactin import (Lynch et al., 2001). Mutations in rhtA abolished the uptake of rhizobactin 1021 and the production of an outer membrane protein (presumed to be the RhtA gene product) that was present in low-iron conditions in the wild-type.

As with thefhu and vbs genes ofR. leguminosarum, transcription of the rht and rhb genes of S. meliloti is enhanced when cells are grown in low concentrations of Fe. This regulation appears to be mediated by a gene, rhrA, mutations which abolish the expression of the rht and rhb genes. This regulator is closely linked to the rhb genes, and specifies a protein that is similar to the AraC family of transcriptional regulators (Lynch et al., 2001).

As with vicibactin in R. leguminosarum, it seems that rhizobactin 1021 of S. meliloti is not needed for functional nodules on alfalfa since strains defective in the rhr, rhb and rht genes all induced N2-fixing nodules on this host (Lynch et al., 2001).

2.2.4. The fegA Gene and Hydroxamate Uptake in Bradyrhizobium japonicum

Several studies on different rhizobia have revealed outer membrane proteins that are good, a priori candidates for being siderophore receptors (Reigh and O'Connell, 1993; Fabiano et al., 1994; Jadhav and Desai, 1994; Roy et al., 1994). Not surprisingly, strains of rhizobia that made a particular siderophore


normally use that siderophore, complexed to Fe 3+, as an Fe source, but, as in other bacterial groups, some rhizobia import siderophores that they do not themselves manufacture (Plessner et al., 1993; Reigh and O'Connell, 1993; Carson et al., 2000). One such 'free-loader' may have been identified by LeVier and Guerinot (1996). Strain 61A152 of B. japonicum makes an outer membrane protein, FegA, that was induced by low-Fe conditions and has a similar sequence to those of hydroxamate siderophore receptors in other bacteria (LeVier and Guerinot, 1996). FegA may, therefore, bind siderophores made by other microbes - certainly, fegA mutants are defective in the uptake of exoge- nously supplied hydroxamates (M.L. Guerinot, personal communication).

In contrast to the siderophore uptake mutants of R. leguminosarum or S. meliloti, fegA mutants of B. japonicum have dramatic effects on symbiotic N 2 fixation on soybeans - they induce nodules that have very few bacteroids, no leghaernoglobin and fail to fix any nitrogen (M.L. Guerinot, personal communication).

Why should there be such a vivid difference in the phenotypes of the siderophore-uptake-defective mutants in B. japonicum compared with those of R. leguminosarum and S. meliloti? Given that soybeans have determinant nodules whereas alfalfa and peas have indeterminant nodule structures and ontogeny, perhaps these contrasting symbiotic phenotypes are due to differences in the nature of the nodulation biochemistry and physiology in the two host plants. Certainly, the concentrations of iron within soybean nodules are substantially greater than in, for example, those on pea roots (M.J. Dilworth, personal communication). Alternatively, R. leguminosarum and S. meliloti may have back-up systems for Fe uptake (a nodule-specific siderophore?), which means that the vicibactin system is redundant in the bacteroids. Paradoxically, though, the only report for a nodule-specific siderophore is in soybeans (see below). One way of approaching this distinction would be to constructfhu or vbs mutants in R. leguminosarum bv. phaseoli, which differs from by. viciae only in the identity of a large 'Sym' plasmid that carries nod and nifgenes. Nodules of Phaseolus beans are similar to those of soybeans, so it might be instructive to examine the symbiotic phenotype of vbs and fhu mutants of R. leguminosarum by. phaseoli on Phaseolus beans. Finally, it may be that the phenotype of the fegA mutants of B. japonicum has nothing to do with Fe uptake per se. As described above, in E. coli, FhuA acts as a receptor for bacteriocins, antibiotics and bacteriophages - is it possible that the outer membrane FegA protein has a role in signalling directly to soybeans in a way that is important for nodule development in these legumes, but independent of Fe uptake? This particular point could be addressed by examining the symbiotic phenotype of anfhuB mutant of B. japonicum that would also be predicted to be deficient in the uptake of hydroxamate siderophores, but which would retain the integrity of its FegA outer membrane receptor. Recently, a tonB-like gene was identified and analysed in B. japonicum (A. Nienaber, H. Hennecke


and H.-M. Fischer, personal communication). Mutations in this gene did not affect the ability to import a heterologous siderophore, ferrichrome, so the mechanism of hydroxamate siderophore uptake appears to be significantly different in B. japonicum and R. leguminosarum (see section 2.2.2.4, above). Nonetheless, it is notable that the tonB mutant of B. japonicum was unaffected in symbiotic nitrogen fixation on soybeans (Nienaber, Hennecke and Fischer, personal communication).

2.2.5. Uncharacterized Mutants that Affect Siderophore Production

The studies described above considered genes and mutants that affect siderophore synthesis and/or uptake directly. In addition to these, there are many reports on the symbiotic properties of uncharacterized rhizobial mutants, which were identified solely on the basis of their CAS phenotypes but which were not studied further at a molecular level. We would post a serious warning concerning the interpretation of such studies. We have identified two classes of mutants that are symbiotically defective and have altered CAS phenotypes; however, the symbiotic defect has nothing to do with the failure to make siderophores per se, but to other, possibly spurious pleiotropic effects (see below).

Several different siderophore-defective mutants of S. meliloti variously have no obvious symbiotic phenotype or are diminished in N2-fixation on alfalfa (Gill et aL, 1991; Fabiano et al., 1995; Barton et al., 1996b). Manjanatha et aL (1992) isolated uncharacterized mutants of R. fredii, with enhanced siderophore production as judged by the CAS test. These over-producers induced N2-fixing nodules on soybeans, but were less competitive for nodule occupancy than the wild-type. In contrast, Duhan and Dudeja (1999) found that siderophore over-producing mutants of Rhizobium isolated from pigeon pea had no effect on competition for nodulation of Cajanus cajun. These apparently contradictory sets of results simply emphasize the importance of knowing the precise molecular basis of the siderophore phenotype.

2.2.5.1. CAS Phenotypes and Pleiotropic Effects in R. leguminosarum. We have identified two classes of mutants of R. leguminosarum with altered CAS phenotypes, which are defective for nodulation, but the primary effect of the mutations does not relate to siderophore production per se.

The first concerns a mutant ofR. leguminosarum bv. viciae, which was Fix- on peas (Nadler et al., 1990). This mutant had a complex phenotype, being defective in the uptake of iron and lacking several c-type cytochromes - it was also severely affected in its ability to make vicibactin in free-living culture, as judged by CAS plates (Yeoman et al., 1997). The mutation was in an operon comprising the cycHJKL genes that are required for assembling mature


cytochrome c in the bacterial periplasm. It is known that cytochrome c is required for the respiration of bacteroids and that mutants defective in cytochrome c production are Fix- (see Delgado et al., 1995). Thus, the respiratory defect, rather than that in siderophore production, causes the Fix- phenotype of the cycHJKL mutants. At present, the biochemical basis for the siderophore defects in cyc mutants is unknown. However, the link between siderophore synthesis and cytochrome c assembly appears to be widespread, since cyc mutants of Pseudomonas and of Paracoccus have also been found to be defective in the synthesis of the respective siderophores of these bacterial genera (Gaballa et al., 1996; Pearce et al., 1998). It may be that there is a direct, oxidative, step required for the synthesis of vicibactin, or it could be due to a more indirect, physiological link.

A second 'CAS phenotype' of R. leguminosarum, which can be affected by trivial genetic changes, was demonstrated by Stevens et al. (2000). A mutant was isolated initially on the basis of its very small halo on CAS plates. The finding that it failed to nodulate peas caused an initial frisson of excitement - until it transpired that the mutation was in the purMN operon that is involved in adenine biosynthesis and that there was sufficient adenine (or a precursor thereof) to support some bacterial growth for the stabs in the CAS tests. It has long been known thatpur auxotrophs in several rhizobia are Nod- (see Stevens et al., 2000). Subsequently, we found that several different auxotrophs, if given sufficient of the required nutrient to allow some growth on CAS plates also gave no, or very small, halos. In other words, there is a real risk that almost any mutant that is 'off colour' may give a reduced CAS phenotype, and great care should be used in deducing the importance of siderophore production on nodulation ability (or, perhaps, different phenotypes in other bacteria?) based solely on CAS phenotypes.

2.3. Other Iron Uptake Systems in Rhizobia

In addition to siderophores, bacteria have several other iron acquisition systems. Here, we consider some of these 'alternative' sources in the rhizobia.

2.3.1. Haem

2.3.1.1. Haem Uptake in Rhizobia. In many bacterial pathogens of animals, haem provides a major source of iron for the bacteria during the infection and there are specific transport pathways for the import of this molecule. In Pseudomonas aeruginosa, there are two haem uptake gene systems, phu and has, which act in distinct ways (Ochsner et al., 2000). The phu genes specify an outer membrane haem receptor, PhuR, plus a typical ABC-transporter,


specified by the other phu genes that are transcribed divergently from phuR. The second, has, system involves an extracellular protein, HasA, and a cognate outer membrane receptor, HasR (Ochsner et al., 2000). The Has and Phu systems act independently; individual mutations in the has and the phu genes reduce, but do not abolish, haem uptake. As with other genes involved in Fe uptake, transcription of has and phu is enhanced in cells growing in low-iron conditions and is controlled by Fur (Ochsner et al., 2000).

Given the importance of haem in legume root nodules, it is superficially tempting to speculate that haem might be an appropriate source of Fe for N 2- fixing bacteroids. There is one report (Noya et al., 1997) that free-living strains of Rhizobium leguminosarum use haem as an Fe source. Recently, we confirmed that strain 8401pRL1JI of this species can use haem as a source of Fe and identified a gene cluster with strong homology to several genes that, in other bacterial genera, are involved in haem uptake. These genes, termed hmuPSTUV, are immediately upstream of, and transcribed divergently from, the tonB-like gene mentioned above. Mutations in hmuPSTUV did not significantly hamper growth of the ceils when haem was the sole Fe source, suggesting that R. leguminosarum, like Pseudomonas, might have more than one system for haem uptake. The hmuPSTUV operon is expressed in response to low Fe availability, but this was not under the control of either the R. leguminosarum fur or rpol genes (our unpublished observations). Interestingly, mutations in the adjacent, tonB-like gene, did interfere with haem uptake, indicating that in R. leguminosarum, as in other bacteria, TonB is also required for the import of haem (see, for example, Occhino et al., 1998). Mutations in either the R. leguminosarum tonB-like gene or the hmuPSTUV genes did not appear to affect symbiotic N 2 fixation on peas, showing that uptake of this molecule is not required as, at least, the sole Fe source for bacteroids (M. Wexler, unpublished). Similarly, tonB and hmu mutants of B. japonicum, which are defective in haem uptake are unaffected in symbiotic N 2 fixation indicating that haem is not a major source of Fe for the bacteroids in soybean nodules (Nienaber, Hennecke and Fischer, personal communication).

2.3.1.2. Haem Synthesis in Rhizobia and Leghaemoglobin - an Old Argument Resolved. It is clear from the above that R. leguminosarum, at least, can use haem as a source of Fe in the free-living state. There have also been studies on the synthesis of this molecule in free-living rhizobia and in bacteroids (see O'Brian, 1996). Had a review on metals (particularly iron) and the rhizobia been written some 10 years ago, a significant chunk of it would have been devoted to the knotty question of 'Who makes the haem for the leghaemoglobin?' At that time, there was some apparently persuasive evidence to suggest that leghaemoglobin was a molecular personification of the symbiosis, the protein being made by the host plant and the haem moiety by the bacteroids in the nodule. The most telling support for this notion was that


hemA mutants of S. meliloti that are defective in ALA synthase, and hence for haem biosynthesis, were Fix- on alfalfa (Leong et al., 1982). However, an apparent paradox was delivered by the finding that, in contrast, hemA mutants of B. japonicum induced normal, Fix + nodules on soybeans (Guerinot and Chelm, 1986). In a very nice demolition job of the inter-kingdom nature of leghaemoglobin, O'Brian (1996) explained these apparently contradictory sets of results, together with other pieces of data that pointed to a molecular division of labour in the synthesis of leghaemoglobin. In short, it is now clear that the plant makes the haem for leghaemoglobin and that the bacteria do not. However, rhizobia do make the haem that is used in, for example, the cytochromes in the bacteroids. The difference in phenotypes of hemA mutants of S. meliloti on alfalfa and B. japonicum on soybeans is simply that, in the latter, the host supplies the bacteria with the necessary ALA to allow them to grow, whereas, with the former, ALA is either not made available to S. meliloti or it is not taken up (McGinnis and O'Brian, 1995). Therefore, these latter bacteria are starved and cannot survive because of their failure to make any haem for their own respiration.

To date, there is no evidence that 'home-made' haem (or a precursor thereof, such as protoporphyrin) is actually exported and used as an iron-chelating siderophore. Indeed, we are unaware of any bacteria that make a porphyrin siderophore. Up till now, dedicated haem uptake systems have only been reported in pathogenic bacteria and it will be of real interest to know in what particular, natural niches, rhizobia come into contact with enough haem for it to be used as an effective Fe source. If it is available in the early stages in the infection of legumes, or in the bacteroids, it is clear that the hmu haem uptake system that we have identified is not the sole source of the metal for R. leguminosarum and B. Japonicum (see above).

2.3.2. Anthranilate

Rioux et al. (1986a) reported that most of the Fe in the supernatants of iron- limited R. leguminosarum was attached to anthranilate. It was established that the Fea+-anthranilate complexes were internalized by R. leguminosarum in an energy-dependent process (Rioux et al., 1986b) but no receptor or other transport proteins have been identified, nor was the nutritional importance of anthranilate-mediated uptake determined.

Barsomonian et al. (1992) isolated several tryptophan auxotrophs of S. meliloti. Mutations in trpAB, which prevent the formation of tryptophan, induce normal NE-fixing nodules on alfalfa, showing that synthesis of tryptophan per se is not needed for the symbiosis. In contrast, trpE mutants that are blocked at an early stage and fail to make anthranilate make unusual, Fix- nodules. Although not proven, it was suggested that this might be due to the role


of anthranilate in Fe sequestration by bacteroids. A striking aspect of trpE mutants is that they had a bimodal phenotype on plants, some nodules appear- ing to be normal, but others being Fix-. This suggests that the metabolic failure of these mutants triggers an 'all-or-none' response in the plant in which the host either fails to develop a normal nodule or, somehow, can locally overcome the defect. It is not known if this is due to anthranilate acting as a siderophore or, perhaps, to its role as a precursor to auxin phytohormones.

2.3.3. Citrate Uptake and Synthesis

Many rhizobia use Fe3÷-citrate, but we know essentially nothing of the molecular basis of its uptake in rhizobia, even though the details of this process in E. coli reveal some exquisite interactions among the various (Fec) transport cell-surface proteins that import Fea÷-citrate (see Braun and Killmann, 1999).

However, it has been found that the biosynthesis of citrate is important for normal nodulation, though it remains to be established if this is due to a direct effect on iron uptake or to some other, metabolic action in the cells. Pardo et al. (1994) showed that R. tropici has two copies of a gene for the synthesis of citrate, one of which, pcsA, is on a symbiotic plasmid that harbours nod and nif genes. Mutations in pcsA reduced the number of nodules on Phaseolus beans but the nodules that did form appeared to be normal for N 2 fixation. Conversely, a strain with multiple copies ofpcsA induced more nodules than the wild-type, one of the few cases where 'genetic engineering' has apparently 'improved' a strain of Rhizobium (Pardo et al., 1994; Martinez-Romero et al., 1998). Interestingly, pcsA was expressed at high levels when cells were grown in low-Fe medium, though induction was also noted when the cells were starved of Ca 2+ (Pardo et al., 1994). Subsequently, Hernandez-Lucas et al. (1995) identified and mutated the chromosomal copy of the gene for citrate synthase of R. tropici. Such chromosomal mutants were also reduced for nodulation of Phaseolus; significantly, the double mutant, lacking both pSym-borne and chromosomal copies of the gene was completely Fix-, inducing nodules that were devoid of bacteroids. Similarly, Mortimer et aL (1999) isolated a gltA mutant of S. meliloti that lacked citrate synthase activity. This mutant was defective in utilizing many compounds as sole C sources and, perhaps not surprisingly, induced Fix- nodules on alfalfa. To date, there is no evidence for a bacteroid-specific citrate synthase (Tabrett and Copeland, 2000). It would seem most likely that the symbiotic defects seen with the various mutants that lack this enzyme are due to general metabolic perturbations, rather than to iron deficiency (although Fe-mediated regulation ofpcsA is tempting!).

In contrast to mutants defective in citrate synthase, a B. japonicum acnA mutant that lacks aconitase induced apparently normal N2-fixing nodules on


soybeans, even though it was severely affected for growth in free-living cells (Thony-Meyer and Kunzler, 1996). Like citrate synthase, aconitase is involved in citrate metabolism, being responsible for isomerization of citrate and iso- citrate. Does the Fix + phenotype mean that there may be a bacteroid-specific version of aconitase in this rhizobial species and, if so, does it have anything to do with Fe nutrition?

2.4. Iron Uptake in the Nodule

We still do not know how N2-fixing bacteroids get their iron when they are in the nodule. The contrasting symbiotic phenotypes offegA mutants of B. japonicum on soybeans and thefhuA mutants of R. leguminosarum on peas or the S. meliloti rhb, rhr and rht mutants on alfalfa simply further complicate the issue. At any rate, the major, free-living siderophores made by R. leguminosarum and S. meliloti seem to be dispensable for normal nodule function.

If the free-living form of siderophores are not involved, is it possible that there are, at least in some rhizobia, bacteroid-specific molecules that perform the same role? After all, there are many genes that are switched on in the bacteroids (see Fischer, 1994). In this connection, Wittenberg et al. (1996) studied the distribution of iron in soybean nodule and reported that most of the water- soluble Fe is in the peribacteroid space (between the bacteroids and the peribacteroid membrane) and that much of it is bound to organic compounds. Significantly, the nature of these binding molecules appeared to vary accord- ing to the particular strain of B. japonicum in the inoculant, suggesting that they were bacterial in origin. On the basis solely of the absorbance maxima of the unpurified 'siderophore fraction' obtained with one strain of B. japonicum, ~qttenberg et al. (1996) suggested that the molecule might belong to the pseudobactin family, made by some strains of Pseudomonas (Vossen et al., 2000). Significantly, no free-living strain of any rhizobia, including B. japonicum, is known to make pseudobactin (Guerinot, 1991; Carson et al., 2000), although at least one strain of B. japonicum can use, though not make, this class of siderophore (Plessner et al., 1993). Therefore, it may be that the siderophore-like entity reported by Wittenberg et al. (1996) is expressed only in the bacteroids. If true, this would open up a very interesting and fruitful line of research to investigate the genes that specify the synthesis (and uptake?) of such a nodule-specific molecule, together with a more rigorous characterization of the Fe-binding molecules in the peribacteroid space.

It is possible, though, that the bacteroids do not employ any siderophores for their supply of iron. For example, Moreau et al. (1998) found that B. japonicum bacteroids in soybean nodules take up ferrous (Fe 2+) iron at higher rates than the ferric form. Fe 2+ is much more soluble than Fe 3+ and, being low in pO 2, the environment around the bacteroids is favourable for the production of


this reduced form of the metal. Further, the peribacteroid membrane contains a ferri-chelate reductase that could provide Fe 2÷ ions (LeVier et al., 1996). Ferri-reductase activity has also been demonstrated in the bacterial partner of the symbiosis, but the role of this enzyme in the symbiosis has not been established (Jadhav and Desai, 1997). Indeed, these authors surmised that, since this reductase was apparently located in the bacterial periplasm, its role was to 'strip' Fe 3÷ from the incoming siderophore. Given that there are dedicated transport systems to take the siderophore-Fe 3÷ complexes through the periplasm and across the cytoplasmic membrane, this does not seem, to us, to be a wholly convincing model.

In enteric bacteria, the feo genes are involved in Fe 2÷ uptake (Kammler et al., 1993; Tsolis et al., 1996). Importantly, feo mutants of Salmonella typhimurium were compromised in colonizing mice, pointing to the importance of this route of iron nutrition in pathogenesis (Stojilkovic et al., 1993). Despite this important observation, little work has been done on bacterialfeo genes in general. The genomes of M. loti and S. meliloti do contain ORFs whose products have some similarities to the known Feo proteins of E. coli and Helicobacter. However, the identities are low; in the absence of functional studies on these rhizobial homologues, it is uncertain that they function to import ferrous iron.

2.5. Iron and Gene Regulation in Rhizobia

Iron is not only essential for microbes, but it is toxic, catalysing several reac- tions that generate oxidative free radicals. Not surprisingly, then, genes that are involved in its uptake and subsequent intracellular movement and metabolism are strictly controlled (see Crosa, 1997).

2.5.1. fur and irr

At least in E. coli, the transcriptional regulator fur is a key regulator of genes involved in Fe uptake and metabolism. Homologues of fur have been found in R. leguminosarum (deLuca et al., 1998) and in Bradyrhizobium japonicum (Hamza et al., 1999). The fur gene ofR. leguminosarum is present as a single copy and appears to specify a genuine, specific transcriptional regulator since it represses expression of the E. colifur-regulated gene, bfd, which is preceded by a 'fur box' (M. Wexler, unpublished), deLuca et al. (1998) suggested that it was impossible to isolate a fur knockout mutant in R. leguminosarum, indicating that, as in other bacterial genera (though not E. coli),fur is essential for growth. However, using a slightly different strategy, we have now obtained insertional mutations in the R. leguminosarum fur homologue (M. Wexler,


unpublished). Far from being lethal, we have found no phenotypic defect in these mutants. They grow on low concentrations of Fe, do not differ in the levels of expression of the fhu, vbs or hmu genes (see above) and are not resistant to manganese (resistance to Mn is a feature of fur mutant strains in other bacterial genera). Further, fur mutants induce normal, N2-fixing nodules on peas (M. Wexler, unpublished).

In B..japonicum, fur is also present in single copy and its Fur protein can bind to an E. coli fur box (Hamza et al., 2000). Expression of hemA of B. japonicum, which specifies the enzyme aminolaevulinic acid synthase (the earliest step in the haem biosynthetic pathway), was increased in cells grown in high- compared with low-Fe conditions (Page et al., 1994), this regulation requiring an intact fur gene (Hamza et al., 2000). The B. japonicum Fur protein also regulates transcription of a gene termed irr, which is itself a member of the fur family (see below) (Hamza et al., 2000). This Fur-mediated regulation of irr was direct, since the Fur protein bound to sequences preceding the irr gene. Interestingly, there was no sign of a fur box sequence in the relevant DNA, showing that Fur, in addition to binding to known fur boxes, also inter- acts with different sequences. It remains to be seen exactly what these sequences are and how widespread they are in rhizobia and, indeed, other bacteria] taxa. Another phenotype ascribed to a fur mutant of B. japonicum was that it continued to import Fe even in cells that were replete with the metal, indicating that it controls some (unknown) pathway of Fe import (Hamza et al., 1999).

The B. japonicum irr gene (Hamza et al., 1998) is related to, but is distinct from, fur (e.g. 29% identical at the amino acid level to Fur of P. aeruginosa). An irr mutant accumulated protoporphyrin, a precursor of haem. This was, in turn, shown to be due to the derepressed expression of hemB, which specifies aminolaevulinic dehydratase, which catalyses the second step in haem biosynthesis. In the wild-type, expression of hemB occurs only in Fe-replete conditions, but in the irr mutant, the expression is constitutive. Thus, fur and irr regulate the genes for the first two steps in the haem biosynthetic pathway in B. japonicum differently and specifically. Transcription of irr itself is susceptible to the Fe status of the cell, irr transcription being enhanced in Fe-limited cells compared to Fe-replete ones. At least in part, this iron-mediated transcription of irr is mediated by the fur gene of B. japonicum (see above), even though the DNA 5' of irr does not contain a sequence that resembles after box. Further, the Irr protein is extremely unstable, being subject to post-translational degradation (Qi et al., 1999). In some elegant experiments, this instability of Irr was shown to be mediated via haem. The Irr protein has a motif at its N-terminus, which is similar to a haem-binding pocket. Both in vitro and in vivo, haem binds to this domain in cells grown in high-Fe conditions. This causes degradation of Irr, even in the absence of protein synthesis. This observation leads to the interesting thought that rhizobia (and perhaps


other bacteria) may, in some cases, sense the levels of available iron via the relative amounts of protoporphyrin (haem lacking Fe) and of haem itself. This possibility was raised earlier in a different context by Yeoman et al. (1997). They speculated that a possible reason for the reduction in siderophore production in cycHJKL mutants of R. leguminosarum was that they would accumulate extra haem in the periplasm, simply because the apo-cytochrome no longer acted as a 'sink' for the haem ligand. If true, the cells might be misled into 'thinking' that they were in an environment with a higher Fe concentration than was the case. If so, they might respond to this false signal by closing down their Fe acquisition apparatus, including the synthesis of siderophores.

2.5.2. feuPQ

As mentioned above, mutations in cycHJKL of R. leguminosarum severely reduced siderophore production (Yeoman et aL, 1997). Approximately 1 kb upstream of cycHJKL is a two-gene operon, feuPQ. Mutations in these genes virtually abolish Fe 3÷ uptake in free-living cells but the levels of vicibactin production are unaffected (Yeoman et al., 1997). The deduced products offeuPQ have striking similarities to the large family of bacterial 'two-component regulators' with FeuQ being the sensor and FeuP the response-regulator. It was suggested thatfeuPQ regulates gene(s) involved in Fe uptake, but the 'target' genes have not yet been identified. In the closely related Brucella suis, Dorrell et al. (1998) found two very near homologues offeuPQ ofR. leguminosarum. However, mutations in these B. suis genes did not appear to affect Fe uptake. It is not clear if thefeuPQ genes have different functions in the two genera or, perhaps, if B. suis has two copies of them.

2.5.3. ros (mucR) - Iron and a Zinc Link?

Transcriptional regulators in bacteria fall into a remarkably small number of groups or families. So, it is surprising that only Rhizobium, Sinorhizobium and the closely related Agrobacterium contain a gene that specifies such a protein, which seems to have no equivalent in any other prokaryote. That gene is 'ros', also known as mucR. The gene was identified first in Agrobacterium tumefaciens as a regulator of the virCD genes that are required for tumour-inducing ability in these bacteria (Close et al., 1985; Cooley et al., 1991) and was subsequently found to repress a gene (ipt) in the T-DNA (Chou et al., 1998). The Ros protein binds to the cis-acting 'ros-boxes' that precede genes that it regulates (Cooley et al, 1991; D'Souza-Alt et al., 1993; Bertram-Drogatz et al., 1997, 1998). Homologues of A, tumefaciens ros have been found in A.


radiobacter (Brightwell et al., 1995), S. meliloti (where it is termed mucR) (Keller et al., 1995; Barnett and Long, 1997) and R. etli (Bittinger et al., 1997). In addition to regulating the virCD genes, ros is also autoregulatory, repressing its own transcription (D'Souza-Alt et al., 1993). Further, mutations in ros (mucR) are defective in polysaccharide synthesis, indicating that it regulates exo genes that are involved in the synthesis of these macromolecules. In fact, it was shown that in ros mutants ofA. radiobacter, transcription of exoY was reduced (Tiburtius et al., 1996), though it is not known if this was due to a direct or indirect effect on the exoY promoter. The mucR gene and the rosR gene of S. meliloti and R. etli respectively are also involved in the production of polysaccharide (Keller et al., 1995; Bittinger et al., 1997); interestingly, in the latter case, the ros gene of R. etli was initially identified as affecting competitive nodulation ability on Phaseolus beans.

Kado and his colleagues noted that Ros contains a motif, Cys-X2-Cys-X 9- His-X3-His (where 'X' is any amino acid) that was reminiscent of zinc-finger domains that hitherto had only been found as transcriptional regulators in eukaryotes. It was, confirmed that the purified Ros protein contained Zn (Chou et al., 1998). However, in the absence of a structure for the Ros protein, it is not possible to say exactly how the metal is bound to the protein and if/how this structure relates to 'classical' zinc-finger regulators. A more detailed review of Ros and possible zinc-finger proteins in prokaryotes is presented by Bouhouche et al. (2000).

Although the physiological effects of Zn on the activity of the Ros regulator have not been studied, it is apparent that iron does affect its function. Hussain and Johnston (1997) found that the transcription of ros itself was greatly diminished in cells growing in low-Fe media. Further, for reasons that are not clear, certain mutants that were suppressed by the cloned ros gene were corrected phenotypically for exopolysaccharide production by adding extra Fe 3+ to the growth medium (Brightwell et al., 1995). A further, circumstantial link between iron and ros was that some mutants that were corrected phenotypically by the cloned ros gene also accumulated protoporphyrin, the immediate precursor of haem (Brightwell et al., 1995).

Though (so far) confined to Rhizobium, Agrobacterium and close relatives, we feel that the Ros/MucR protein is worthy of more detailed investigation from a structural point of view in the anticipation that it may reveal a novel mechanism of gene regulation in prokaryotes.

2.5.4. fixL

In addition to cytochromes, haem (see above) is required for one of the steps in the complex cascade that regulates the expression of the n/f genes in the bacteroids, in response to oxygen tension (see Fischer, 1994). A key player in this


pathway is FixL, a membrane-bound sensor in a two-component regulator, in which FixJ is the response regulator (dePhilip et al., 1990; Anthamatten and Hennecke, 1991). FixL is a haem-binding protein (Gilles-Gonzales et al., 1991) with similarities to haemoglobins and is a member of a growing group of haem proteins that act as molecular biosensors (for review, see Chan, 2000). The oxi- dation state of the haem affects the shape of FixL which, in turn, determines its kinase activity, and hence its ability to phosphorylate the FixJ protein (see Gilles- Gonzales et al., 1991; Perutz et al., 1999). FixJ-P can proceed to induce other genes (e.g. nifA andfixK), which in turn activate the structural genes that specify the nitrogenase and ancillary proteins required for nitrogen fixation in the bacteroids. Despite the importance of the haem moiety, to date, there is no evidence for a direct role of iron (or any other metal) in the regulation of n/fgene expression - the key environmental signal appears simply to be the oxygen tension.

3. MOLYBDENUM

Mo is present in cofactors of many enzymes (see Mendel and Schwarz, 1999). Its relevance to N 2 fixation is clear, given that the Mo in 'FeMoCo' cofactor is at the heart of the nitrogen reduction process - at least for most nitrogenases. Some time ago, Bishop and colleagues showed that some strains of Azotobacter had nitrogenases that had no Mo in their cofactor, having Va or Fe instead (see Pau, 1989). To date, there is no evidence that any rhizobia contain such 'alternative' nitrogenases (Dilworth and Loneragan, 1991). However, surveys of the types of nitrogenase in rhizobia are limited in terms of the num- bers of strains examined, the range of host legume species studied and the ecological niches from which samples were obtained. It is, therefore, possible that 'out there' lurk strains of rhizobia that do contain alternative nitrogenase(s).

3.1. Molybdenum Uptake and Rhizobia

There are reports that foliar applications of Mo to grain legumes in field conditions increase levels of N 2 fixation and nodule mass, resulting in higher overall N content and seed yield (Yanni, 1992; Vieira et al., 1998). In laboratory conditions, several different legumes that were severely starved of Mo showed more dramatic signs of deficiency (Dilworth and Loneragan, 1991). Despite the a priori importance of Mo in N 2 fixation, there has been very little work on how rhizobia obtain the metal as free-living bacteria, let alone in nodules. Maier and colleagues reported that there were differences (as much as eight-fold) in the rates of Mo uptake by bacteroids obtained from


soybean nodules containing different strains of B. japonicum (Graham and Maier, 1987). Interestingly, the strains that were particularly poor at import- ing Mo were those that responded best (in terms of acetylene reduction ability) to the addition of exogenous Mo to plant growth medium, indicating that the ability of strains to transport Mo does affect symbiotic performance. Maier et al. (1987) isolated a mutant of B. japonicum that was defective in Mo uptake which could be 'rescued' by adding cell-free supernatant of a wild-type strain. This points to a siderophore-like molecule that binds and delivers Mo. What makes that mutant more interesting is that it was severely reduced in the nitrogenase activity in free-living cells. Perhaps surprisingly, these observations were not followed up. Another report suggested that cowpea rhizobia might make a catechol-type siderophore that could sequester both Fe and Mo (Patel et al., 1988) but the nature of the molecule was not studied further.

3.2. Molybdenum Uptake in Other Bacteria

Here, we briefly review the mechanisms involved in Mo uptake in other bacteria and discuss whether these may pertain to rhizobia. It is important to note at the outset that, in contrast to ferric iron, molybdate ions are soluble in oxic, aqueous conditions. The Mo uptake system in E. coli has been studied in detail and has been shown to comprise yet another member of the classical 'ABC' transporter family (see Grunden and Shanmugam, 1997), First identified as mutants that were defective in a range of phenotypes that depended on Mo-containing enzymes (Hemschemeier et al., 1991), modABCD specifies a periplasmic transporter (ModA), an inner membrane integral protein (ModB) and an ATPase (ModC), plus ModD, a protein of unknown function (Maupinfurlow et al., 1995; Walkenhorst et al., 1995). Subsequently, analogous and homologous genes have been found in other bacterial genera including the diazotrophs Azotobacter (Luque et al., 1993) and Rhodobacter (Wang et al., 1993) and the Gram-positive Staphylococcus (Neubauer et al., 1999), showing that this transport system is widespread in bacteria.

Although there are no formal reports of the modABCD genes in any rhizobial strains, they do appear to exist in these bacteria. We searched the S. meliloti genomic sequence (June 2000) and found that it had sequences whose products were similar to those of the E. coli modA, modB and modC genes (43%, 47% and 49% identity at the amino acid level, respectively). Thus, it would appear that S. meliloti, and, presumably other rhizobia, contain the ABC transporter for Mo uptake. Whether this is the only such transporter for rhizobia and whether it operates in the free-living state and/or in bacteroids remains to be seen. With these sequences available, though, it will not be too demanding to obtain the corresponding mutants and to determine their symbiotic phenotypes.


In E. coli, modABCD is expressed at high levels in cells grown in low-Mo conditions (Rech etal., 1995), mediated by the transcriptional regulatory repressor protein, ModE (Grunden et al., 1996). The modE gene also affects transcription of the E. coli hyc (formate hydrogenlyase) dmsABC (dimethyl-sul- foxide reductase) and the narGHJl (nitrate reductase) genes, all of whose products have Mo-based co-factors (Rosentel et al., 1995; McNicholas et al., 1998; Self et al., 1999). Homologues of ModE have been found in Azotobacter vinelandii (Mouncey et al., 1996). Interestingly, in Rhodobacter capsulatus, the mod genes are also under the control of the ntr genes, which are normally associated with the regulation of genes concerned with N assimilation (Kutsche et al., 1996). This may have some adaptive sense, given that several enzymes involved in steps in the N cycle include Mo in their cofactors. In M. loti, three contiguous genes, which appear to constitute an operon, have products that are similar to those of modABC of other bacteria. Interestingly, immediately downstream of the putative M. loti modABC operon is a gene with limited similarity to the product of the regulator modE. It will be of real interest to see what genes are regulated by this modE homologue and, indeed, what are the symbiotic phenotypes caused by mutations in the proposed modABC operon.

In addition to the ABC transporter, modABCD, other (uncharacterized) systems for Mo uptake have been reported in bacteria. Even in E. coli, if the mod system is knocked out, a low-affinity Mo uptake, mediated by the sulfate- import system, can operate (Rosentel et al., 1995).

Azotobacter, perhaps the most 'committed' of free-living N2-fixing bacterial genera, contains an unusual siderophore system that is affected by the relative concentrations of Fe and Mo. In A. vinelandii, the presence of high concentrations of Mo in the medium encourages the production of the tricate- cholate siderophore protochelin, at the expense of two other siderophores, azotochelin and aminochelin (Duhme et aL, 1998; Cornish and Page, 2000). These latter molecules bind to Mo with quite high efficiency, so it may be an adaptive response by these bacteria to make a specific siderophore (protochelin) that binds Fe and which is not sequestered by competing molybdate ions. The significance of these siderophore-like molecules in the nutrition of Azotobacter remains to be determined, but it would be of interest to know if this system has any functional or molecular similarity to that alluded to above, concerning Mo-binding siderophores in certain rhizobia.

4. NICKEL

An important feature of nitrogenase is that during N 2 reduction, protons are also reduced to form gaseous H 2 that can be lost into the environment. This ubiquitous feature of nitrogenases is wasteful of energy. However, in the late


1970s, it was found that some (but not all) rhizobia have an enzyme, uptake hydrogenase (Hup), that oxidizes H 2 and so generates ATP. Indeed, a real aim for 'rhizobial breeders' was to incorporate the so-called hup genes into elite inoculants (see Maier and Triplett, 1996, for a review of rhizobial breeding). Hup is a nickel-containing enzyme (Harker et al., 1984, Stults et al., 1984). Indeed, adding Ni to soybean growth medium greatly stimulated Hup activity (Klucas et al., 1983). Similarly, depriving peas of Ni caused reduction of Hup activity, due to the failure of bacteroids to process the Hup peptides into mature, functional forms. Thus, Ni is required both as a cofactor for the enzyme and for its correct maturation (Brito et al., 1994). Hup is one of only four known microbial enzymes with Ni at their active sites, the others being CO dehydrogenase, urease and methyl coenzyme M reductase (see Watt and Ludden, 1999a).

Bacteria possess different mechanisms to acquire Ni 2+ from the environment (see Eitinger and Mandrand-Berthelot, 2000), none of which resembles siderophore-mediated metal uptake (probably because Ni 2+ is much more soluble than Fe3+). For example, E. coli has five genes, nikABCDE, which specify a high-affinity ABC transporter (Navarro et al., 1993). This operon is regulated in response to Ni 2÷ availability by nikR, a repressor with limited sequence similarity to Fur (de Pina et al., 1999). To date, no clear homologues of these nik genes have been found in any rhizobial strain. Rather, rhizobia acquire the Ni needed for Hup activity by one of two quite different systems.

Much of our understanding of Ni uptake and processing has come from Maier's laboratory, working on B. japonicum and soybeans. Importantly, so- called Hup ÷ strains of this species express the enzyme both symbiotically and in free-living cells, thus facilitating genetic and biochemical studies. In contrast, Hup + strains of R. leguminosarum bv. viciae express hydrogenase only in bacteroids. One Hup- mutant of B. japonicum was isolated whose defect was suppressed by adding supernormal levels of Ni to the medium (Fu and Maier, 1991a). This mutant had a deletion close to the structural hup genes and three genes, hupNOP, were required to fully restore Hup + activity to the mutant when it was grown in low-Ni media (Fu et al., 1994). Cloned hupN alone restored hupNOP mutants to only c. 50% of wild-type Hup levels. Importantly, the HupN protein is very similar in sequence to the Ni2÷-uptake HoxN and NixA proteins of Ralstonia eutrophus and Helicobacter pylori (Eitinger and Friedrich, 1991; Mobley et al., 1995; Bauerfeind et al., 1996; Eitinger et al., 1997; Fulkerson et al., 1998). All three proteins are hydrophobic, being located in the cytoplasmic membrane (Eitinger and Friedrich, 1994). The fact that the cloned hoxN or nixA genes are Ni 2÷ transporters when introduced into E. coli indicates that they can act autonomously for this process (Mobley et al., 1995; Wolfram et al., 1995). However, in B. japonicum, the hupP and hupO genes were also required for full wild-type Hup activity (see above), suggesting that they have ancillary roles in Ni uptake and/or insertion of Ni into hydrogenase.


Surprisingly, given their homology to known Ni 2÷ transporters, there is evidence that the HupNOP proteins of B. japonicum may not be the major Ni transporter in vivo. In flee-living cells of this species, most of the Ni 2+ was in fact imported via a low specificity Mg 2+ transporter that also carries Co 2+, Mn 2+ and Zn 2+ (Fu and Maier, 1991b). The isolation of a mutant that was constitutive for Hup activity and which accumulated Ni 2+, plus these other ions, further points to a multi-metal, low-affinity uptake system in B. japonicum (Maier and Fu, 1994). Such mechanisms, acting primarily to import Mg 2+, occur in several bacteria (see Watt and Ludden, 1999b). Although it seems to exist in B. japonicum too, little is known of its molecular mechanism.

It seems that HupN, and, perhaps the HupO and P proteins, have more inti- mate, specialized roles in delivering Ni 2+ to hydrogenase or to ancillary proteins needed for Hup activity. This is a subject that might bear fruit from further investigation. Whatever the case, there is no question that once inside the rhizobial cell, all is not yet done in terms of moving Ni around. This metal is not only a constituent of the Hup itself, but is a co-inducer for several hup and hyp genes that are needed to produce mature hydrogenase.

A protein, HypB (termed 'nickelin' by Olson and Maier, 2000), is Nature's own 'His-tag'. In both R. leguminosarum and B. japonicum, it contains a long, histidine-rich amino terminus - so much so, that it was purified in one step using a Ni-affinity column (Rey et al., 1994; Fu et al., 1995). The B. japonicum and R. leguminosarum HypB proteins bind, respectively, 4 and 18 Ni atoms per protein. Curiously, although E. coli HypB also delivers Ni to hydrogenase, it does not have the 'His-tag' and does not itself sequester Ni (see Maier et al., 1995). In elegant experiments, Olson et al. (1997) obtained evidence that HypB of B. japonicum does act as a molecular 'sponge' for Ni 2+, perhaps analogous to the role of the ferritins in storing iron. When B. japonicum is grown in Ni-replete media, then starved of the metal, Hup activity was not impaired for some time, providing the strain had a functional hypB gene, again pointing to the role of HypB in Ni 2+ storage (Olson et al., 1997). Intriguingly, expression of hypB precedes that of the hup genes, pointing to the anticipation of the high Ni demand by the bacteria once the functional Hup is made. As pointed out by Olson et al. (1997), this seques- tering of Ni may be particularly appropriate for symbiotic bacteria, which must gain the metal in competition with the host plant. Although deleting hypB abolished Hup activity, removal of just the histidines caused only a drop in activity that was restored by adding extra Ni to the medium. Thus, the His-tag may be involved in Ni 2+ storage, rather than inserting the Ni into Hup, this function being mediated via a GTPase domain of HypB (see Maier et al., 1995). Olson and Maier (2000) separated the two domains of HypB by genetic techniques, confirming that the GTPase and the Hup-maturation domains worked autonomously.


Intriguingly, hypB mutants of B. japonicum are also defective in the expression of the hup genes (Olson et al., 1997). In rhizobia, Ni 2÷ is a co-inducer for the expression of hup genes, and Durmowicz and Maier (1997) found that in vitro binding of an unspecified protein(s) to the hupSL structural genes depended on the presence of Ni. Such binding and, indeed, expression of hupSL depended on two other genes, hoxA and hoxX, which appear from their sequences to be 'two-component' transcriptional regulators (Van Soom et al., 1993). Homologues of HoxA and HoxX regulate hydrogenase expression in other bacterial genera (see Van Soom et al., 1993). It was proposed that HoxA bound to hupSL regulatory sequences and that it was the positively acting transcriptional regulator. Interestingly, hoxA mutants, while totally defective in transcription of hup genes in free-living B. japonicum, are unaffected for Hup activity in bacteroids of soybean nodules (Durmowicz and Maier, 1997), pointing to the existence of different control systems in the symbiotic state. It may be significant that R. leguminosarum, which does not express Hup in the free- living cells, does not appear to have a homologue of hoxA. The role of HoxX may not simply be that of the 'sensor' in the HoxAX two-component pair but may be more complex and subtle. In B. japonicum, hoxX mutants are not totally Hup-, reaching maxima of 30% of wild-type values in free-living cells. However, much of the large subunit of Hup is in the immature state, pointing to a role of HoxX in its processing rather than the regulation of its transcription. A similar proposal was made by Rey et al. (1996), who identified a R. leguminosarum by. viciae homologue of HoxX, termed HypX. This protein resembles an Nl0-formyltetrahydrofolate-dependent enzyme family whose members are involved in transferring one-carbon units. The phenotypes of hypX mutants of R. leguminosarum bv. viciae on peas were similar to that of the hoxX mutants of B. japonicum in that bacteroids contained the HupL hydrogenase protein in the larger, unprocessed form - transcription of the hup genes was unaffected by the hypX mutation (Rey et al., 1996). This processing of the pre-form of Hup to the mature, functional type is Ni2÷-dependent, but the precise mechanisms involved are not known.

It is also not clear if there is a link between the control of expression mediated by hoxA and the failure of hup structural genes to be expressed in hypB mutants (above). Olson et al. (1997) speculated that HypB might be an indicator of the Ni 2÷ status of the cell and/or that it acted as a donor of Ni to other proteins in a signal cascade. Olson et al. (1997) further surmised that such a 'downstream' protein in the signalling pathway could be HupV, a protein with a convincing Ni2÷-binding motif, which is required for expression of structural hup genes (Black et al., 1994). Although hupV and hupU mutants of B. japonicum do not express hup genes, the sequences of neither resemble that of DNA-binding regulatory proteins. Therefore, they may act indirectly. Possibly, one or both may act as a meter of the Ni status of the cells and this message is transmitted to the DNA-binding regulator (could


this, perhaps, be via HoxA?). Alternatively, HupV may act as a donor of Ni 2+ to another regulator, which, if it contains the metal, activates transcription at hup promoters.

Although there is a considerable amount known on the import and movement of Ni 2+ in B. japonicum and R. leguminosarum bv. viciae, questions remain. How is Ni imported by bacteroids? If HupN is not involved in import- ing Ni 2+ per se, how does it make Ni 2+ available for Hup to function? What is the nature of the protein-protein cross-talk among HupN, HypB and, perhaps HoxA, HoxX and HupU/V?

5. OTHER METALS

Turning to other metals that have major biological effects of how they are acquired by rhizobia, as with Mo, little is known from a molecular genetic point of view. Therefore, we simply mention those cases in which metals have a role in the symbiosis or where their involvement with rhizobia has some particular features of interest.

5.1. Zinc

In addition to the possible role of zinc in the function of the Ros/MucR transcriptional regulators, there is a description of protein engineering by Chauhan and O'Brian (1995) which relates to Zn and B. japonicum. In this bacterium, the enzyme S-aminolaevulinic acid dehydratase (the product of hemB) normally has Mg 2+ as a cofactor. In contrast, the corresponding enzyme in plants contains Zn 2÷. By site-directed mutagenesis of B. japonicum hemB, they sub- stituted the N-terminal amino acids of the B. japonicum enzyme, and showed that this caused the engineered protein to bind Zn 2÷ and not Mg 2÷. This did not affect symbiotic N 2 fixation, despite the known requirement for a functional hemB for N 2 fixation to occur (Chauhan and O'Brian, 1993). Thus, the plant can supply the extra load of Zn that would be required by this novel inoculant strain.

5.2. Manganese

In one of the earliest steps of the infection process, the binding of rhizobia to young root hairs is enhanced when R. leguminosarum is starved of Mn (Kijne et al., 1988). Whether this is because Mn affects the amounts and the type of rhizobial exopolysaccharide (Appanna and Preston, 1987) remains to be seen.


5.3. Calcium

Given the widespread importance of calcium as a signal molecule, it is not surprising that the intracellular movement of Ca within plant cells is a key feature in the infection process. Within minutes of adding Nod factor to alfalfa roots, characteristic spiking of calcium occurs within young root hairs (Ehrhardt et al., 1996). This, presumably, is a step in the reorganization of the cytoskeleton that is associated with root hair deformation and, in some cases, the formation of infection threads to deliver the bacteria into the plant. Since that report, there have been others on the role of calcium redistribution in the infection process (see Downie and Walker, 1999; Niebel et al., 1999), including the demonstra- tion that Nod factors can induce such changes even in tissue culture cells (Cardenas et al., 1999; Yokoyama et al., 2000). There is no doubt that the identification of the Nod factor receptor and the unravelling of the downstream signal transduction pathway will be a major challenge for rhizobiologists in their quest to understand the developmental pathway that culminates in the formation of the root nodule.

Over and above this fundamental role in the rhizobial-legume symbiosis, calcium makes another appearance in different circumstances. In R. leguminosarum, NodO is an exported protein, made in response to the addition of flavonoid nod gene inducers (DeMaagd et al., 1989; Economou et al., 1990). The NodO protein binds Ca 2÷ at its C-terminus, changing the shape of the protein (Dalla Serra et al., 1999) and, in that respect, is similar to haemolysin and other exported proteins to which NodO is homologous. The NodO protein is exported, along with other extracellular proteins of R. leguminosarum, by a specialized transport system (Finnie et al., 1997). Although individual mutations in nodO have little effect on nodulation, a functional nodO gene can compensate for mutation in nodE. This latter gene is involved in the formation of the lipid side-chain that is attached to the core lipo- oligosaccharide Nod factor of these bacteria (Economou et al., 1994), which is important in determining host-range specificity (see Denarie et al., 1996). NodO also affects host-range, since transfer of the cloned nodO of R. leguminosarum bv. viciae to a node mutant of bv. trifolii of the same species confers on the recipient the ability to nodulate vetches, which are normally hosts of the bv. viciae (Economou et al., 1994). Similarly, Vlassak et al. (1998) found that the transfer of nodO from a wide host-range strain of Rhizobium conferred an extension of the host-range to several recipient strains into which it was introduced. Like haemolysin and other proteins that are homologous to it, the purified NodO protein of R. leguminosarum bv. viciae can induce channels in membranes (Sutton et al., 1994). This points to a role in facilitating transport of low-molecular-weight substances through the membranes of root hairs. However, how this confers host-range specificity remains to be seen. Likewise, the means whereby nodO can partially compensate


nodE mutants, which are defective in a very different biochemical step, is unknown.

The nodO gene that was first described is located on a 'symbiotic' plasmid, pRL 1JI of R. leguminosarum bv. viciae. This plasmid was originally identified on the basis of its specification of a bacteriocin (Hirsch, 1979); some 20 years later, it is now known that this bacteriocin is a protein that, like NodO, also has repeat sequences that characterize Ca2÷-binding domains. Indeed, the biological activity of this bacteriocin depends on calcium in the medium (Oresnik et al., 1999).

5.4. Copper

Apart from its role in respiratory proteins that are required for N 2 fixation in rhizobia (see Delgado et al., 1998), copper also plays a role in a protein that is expressed co-ordinately with the nifgenes and may affect the efficacy of bacteroid function. Several rhizobial strains, particularly R. leguminosarum bv. phaseoli, make the pigment melanin. In this biovar, the genes for melanin production are on the same large Sym plasmid as the nod and nifgenes (Johnston et al., 1978). The meIA gene, specifying the copper-containing enzyme tyrosinase is expressed at high levels in bacteroids, this being under the control of the regulatory R. leguminosarum nifA gene (Hawkins and Johnston, 1988). In other rhizobial species, however, a different mode of control operates, which is independent of nifA (Mercado-Blanco et al., 1993). The role of the tyrosinase may be to degrade potentially harmful phenolic compounds once the nodules have begun to senesce.

The ability of rhizobia to tolerate acid conditions may be an important trait for their efficacy as inoculants in many parts of the world. Tiwari et al. (1996) characterized the S. rneliloti gene, actA, mutations in which confer hypersen- sitivity to low pH conditions. The ActA protein is a homologue of CutA, an E. coli protein that is important in conferring resistance to copper; S. meliloti actA mutants were also extra-sensitive to Cu 2÷ in the growth medium. The role of actA in the Cu nutritional status of rhizobia remains to be seen - clearly it is not required for N 2 fixation, since actA mutants can fix N 2 in alfalfa nodules.

Lastly, there is increasing interest in the phenomenon whereby bacteria enter a state that is 'viable but non-culturable'. There is a recent report that shows that, for reasons that are not clear, adding Cu to Agrobacterium or R. leguminosarum cells sends them to this state (Alexander et al., 1999).

METALS AND THE RHIZOBIAL-LEGUME SYMBIOSIS

6. CONCLUDING REMARKS

145

It is not clear why we still know so little about the uptake of iron, possibly the metallic totem of the symbiosis. There is an irony that, thanks largely to the work of one laboratory (Rob Maier's), we know more about the uptake and the signalling properties of Ni, despite the fact that most rhizobial strains do not have the Hup system.

Perhaps this simply reflects the fashions and vicissitudes of scientific research, though, if so, this reluctance also seems to extend to the study (or lack of it) of the role of metals in plant diseases that are caused by bacteria. This is in marked contrast to the situation with bacterial diseases of animals. Many questions remain to be answered, over and above the identification of the genes, proteins and processes that are involved in metal import and metal- mediated signalling in the rhizobia and the legume root nodule.

One of the most important of these questions concerns the means by which legumes sense the absence of Fe and respond by shutting down the nodulation process. Dilworth pointed out 10 years ago that it was surprising that this question had not really been addressed then. Now, that plea is even more jus- tified - we do not even know if the signals are mediated via the bacteria, the host, or both. The apparently contradictory findings on the role of siderophore receptors in R. leguminosarum on peas and B. japonicum on soybeans may reflect another important difference in the physiology and biochemistry of determinant and indeterminant legume nodules - another canvas for further research.

In this topic, as in many others in biology, the availability of genome sequences of rhizobial strains will be of great value. With that information, the identification of the full panoply of genes involved in metal uptake, for example, plus their various promoters with their own trademark regulatory 'boxes' will be greatly facilitated. For example, one group of potentially very important genes that has received little attention is those involved in citrate uptake. The most rapid way forward may be to identify homologues from genomic sequences, then mutate the variousfec homologues that may emerge.

It may be that, armed with a detailed audit of how rhizobia get and respond to metals, we may learn more of the general importance of these processes in the multifarious interactions between bacteria and higher eukaryotes. The study of iron uptake and signalling, in particular, has received much attention as an important determinant of many diseases of animals. Will bacterial pathologists have anything to learn in the future from that potential bacterial pathogen that has 'seen the light' and adopted a symbiotic lifestyle instead?


ACKNOWLEDGEMENTS

Thanks are gladly given to Mike Dilworth, Kerry Carson, Penny Worsley, Mary-Lou Guerinot, Mick O'Connell and Mark O'Brian for their generosity in sending unpublished results. We are also grateful to Rob Carter for helpful dis- cussions and his supply of unpublished data and to Neil Halliday for his constant help in the preparation of the manuscript. KHY and MW are sup- ported by the Biotechnology and Biological Research Council of the UK.

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Metals and the rhizobial-legume symbiosis — Uptake, utilization and signalling

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