World Journal

of Microbiology

& Biotechnology

10, 612-630

Review

Molecular functioning

basis of the establishment of a N2-fixing root nodule

and

J. Michiels and J. Vanderleyden* Compatible interactions between rhizobia and their leguminous host plant(s) culminate in the formation of a new plant organ, the root nodule. Within this structure, the bacteria reduce N, to NH, which is then assimilated by the plant. The formation of a N,-fixing nodule requires a continuous process of two-way signalling and cellular recognition between the prokaryote and the plant. Such a process involves the sequential activation and/or repression of host plant- and bacteria-encoded genes. Finally, functioning of a legume-nodule necessitates not only the adaptation of plant and bacterial carbon, nitrogen and oxygen metabolism to an environment allowing N,-fixation to occur, but also requires a tight co-ordination and integration of these plant and bacterial metabolic processes. Key words: Bacteroid,

Brudyrhiwbium,

legume, nitrogen

fixation, nodulation,

Biological N, fixation constitutes one of the most fundamental global processes as it produces a key nutrient for plant growth, namely fixed nitrogen. Bacterial N, fixation occurs in the free-living state, in association with plants or animals, or in symbiosis with plants. From an ecological and agricultural point of view, the most important N,-fixing systems are the symbioses. By far the best characterized symbiotic N,-fixing system is the symbiosis between legumes and rhizobia. During this interaction, rhizobia induce nodules on the roots of their leguminous host-plant, or in rare cases on stems, in which they convert N, to NH,, obviating the presence of other nitrogen sources for plant growth. In return, the host plant supplies the bacteria with carbon compounds and other nutrients necessary for the support of bacterial growth and N, fixation within the nodule. This unique system has been the object of intensive study over the past two decades. Beyond its economical and ecological relevance, analysis of the process of symbiotic N, fixation may contribute to the understanding of such complex biological activities as developmental gene regulation, signal transduction and plant organogenesis.

The authors are with the F.A. Janssens Laboratory of Genetics, University of Leuven. Willem de Croylaan 42, B-3001 Heverlee, fax: 32 16 200720. ‘Corresponding author. @ 1994 Rapid Communications

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Catholic Belgium;

Rhizobium.

This review concerns the symbiotic process from the early plant-bacterium signal exchange up to the stage of a mature nodule. Attention will be drawn to both partners during the different stages of the interaction, with an emphasis on the molecular communication and metabolic co-operation between the prokaryote and the plant.

Taxonomy

of the Rhizobia

Root nodule bacteria, collectively known as rhizobia, are currently classified into three genera: Azorhizobitlm, Bradyrhizobium and Rhizobium. The existence of a fourth genus, Sinorhiwbium, has been questioned recently (Young et al. 1993) and it is therefore not included in the present classification. So far, the genera Azorhizobium and Bradyrhizobium have only one named species each while eight species have been described in the genus Rhiwbium (summarized in Table 1). With the exception of Purusponiu, a member of the Ulmaceae family, all rhizobial host-plants belong to the family Leguminosae. This family comprises three subfamilies (Caesalpinoideae, Mimosoideae and Papilionoideae) of which most genera are nodulated by rhizobia (Young & Johnston 1989). Symbionts from very few of the 16,000 to 19,000 legume species have been studied so far. It can

Rhizobium-legume therefore be expected that in the future newly described species and genera will result as further symbioses are examined.

Early Plant-bacterium

Signal Exchange

The development of N,-fixing nodules is a multi-step process requiring the exchange and correct recognition of specific signals by both partners during the different stages of symbiosis. Failure to recognize one of such signals may cause early arrest of the symbiotic process. Closely related with this is the concept of host-specificity, i.e. a particular Rhizobitim species or strain elicits N,-fixing nodules only on a limited set of host plants. Rhizobia may have a broad or a narrow host range depending on whether they are able to successfully nodulate many or only one or a few hosts. In addition to the capacity to nodulate, host specificity may also be determined by the late stages of symbiosis. In some instances, rhizobia may induce the formation of nodules and colonize the nodule tissue without differentiating to N,-fixing bacteroids. Rhizobia are chemotactically attracted toward the root surface through the presence of attractants in plant-root exudates (Gaworzewska & Carlile 1982). Some of these root exudate components, such as amino acids, sugars, phenolic compounds and carboxylic acids, are of nutritional value while others, flavonoids and chalcones, are potent inducers or repressors of bacterial nodulation (nod) genes (Rolfe 1988, Gottfert 1993). Flavonoids (flavones, flavanones and isoflavones) are three-ringed, aromatic, phenolic compounds derived from the phenylpropanoid pathway in plants (Table 2). Prominent natural inducers of nod genes are luteolin (3’, 4’, 5, 7-tetrahydroxyflavone) and 4, 4’-dihydroxy-2’-methoxychalcone from alfalfa roots (Peters et al. 1986; Peters & Long 1988; Maxwell et al. 1989) daidzein (4’, 7-dihydroxyisoflavone) or genistein (4’, 5, 7trihydroxyisoflavone) from soybean (Kosslak et al. 1987) and DHF (a’, 7-dihydroxyflavone) from clover (Redmond et al. 1986). nod genes from broad-host-range rhizobia, such as Rhizobium sp. strain NGR234, are inducible by a wide range of compounds whereas nod genes from narrow-hostrange rhizobia, such as R. melilofi, R. legtlminosarum bv frifalii, viciae and phaseoli, respond to few flavonoids. The spectrum of flavonoids is dependent on the plant species (Rolfe 1988) and the stage of plant development (Hartwig et al. 1990; Graham 1991). Also, root exudation is not evenly distributed along the root axis. In alfalfa, the zone of maximal nod-gene-inducing capacity, the zone of emerging root hairs, also corresponds to the zone most susceptible to infection and nodulation (Redmond et al. 1986; Peters & Long 1988). In response to specific root exudates, the bacterial nod genes are induced. The nod gene products participate in the production of secreted signal molecules, the Nod factors

symbiosis

(Lerouge et al. 1990; Spaink ef al. 1991). These molecules are substituted lipo-oligosaccharides consisting of an Nacetyl glucosamine backbone with different host-specific modifications. Nod factors act in a host-specific way, causing several morphological changes in the root of the host plant such as root hair deformations (Lerouge et al. 1990) and formation of pre-infection thread structures (van Brusse1 et al. 1992) and nodule primordia (Spaink et al. 1991; Truchet et al. 1991). In addition, Nod factors induce alterations in flavonoid composition of root exudates, causing increased nod-gene-inducing activity (Ini; van Brussel et al. 1990).

Bacterial Nodulation Genes and Regulation of nod Gene Expression In Rhizobium species, nodulation genes, together with other symbiotic genes, are located on large plasmids (Sym plasmids). Sym plasmids vary from 50 to over 600 kb in R. leguminosarum bv frifolii (Harrison et al. 1988) to 1200 and 1500 kb in R. melilofi (Burkhardt et al. 1987). Brudyrhizobium and Azorhizobium species carry the symbiotic genes on the bacterial chromosome. The organization of N,-fixation and nodulation genes of Rhizobitrm and Brudyrhizobium species is presented in Figure 1. Nodulation genes, nod and no1 genes, are classified as regulatory, common and host-specific (Denarie et al. 1992; Gbttfert 1993). Regulation of nod genes is controlled by the nodD genes, of which all rhizobia tested so far contain one or more copies (RodriguezQuinones et al. 1987; Van Rhijn et al. 1993). NodD proteins share homology with prokaryotic, positive-regulator proteins belonging to the LysR family (Schell1993) and bind to a conserved sequence in the promoter region of inducible nod genes designated the nod box (Rostas et al. 1986). In conjunction with plant flavonoids or other phenolic compounds, NodD proteins act as transcriptional activators of inducible nod genes. Different NodD proteins respond to specific plant-signal molecules and therefore contribute to host-specificity of nodulation (Horvath et al. 1987; Spaink et al. 1987). Regulation of nodD gene expression is diverse. Most nodD genes are constitutively expressed, others are autoregulated (Innes et al. 1985; Rossen et al. 1985), inducible (Banfalvi et al. 1988) or subject to repression by NolR or NH,+ (Kondorosi et al. 1989; Dusha ef al. 1989) or to activation by SyrM (Mulligan & Long 1989). The complex regulation of nodD gene expression and the specific interaction of NodD with plant flavonoids may allow fine-tuning of nod gene expression and concomitantly of Nod-factor production, conditions that may be required for optimal nodulation of the host plant. The common nodABC genes are structurally and functionally conserved among Rhizobium, Bradyrhizobium and Azorhizobitlm strains (Martinez et al. 1990). The inactivation of these genes completely abolishes root-hair infection and

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]. Michiels and 1. Vanderleyden Table

1. Taxonomic

claaaification

of the rhisobia. Species

Genus Azorhizobium (Dreyfus et al. 1988) Bradyrhizobium (Jordan 1982) Rhizobium (Jordan 1982)

Table

2. Structures

of flavonoid

and chaicone

A. 8. ft. R. R. R. ft.

caulinodans (Dreyfus et al. 1988) japonicum (Jordan 1982) et/i (Segovia et al. 1993) fredii (Schoiia 8 Elkan 1984) galegae (Lindstrom 1989) huakuii (Chen et al. 1991) leguminosarum (Krieg 8 Hoit 1984) biovar phaseoli biovar trifolii biovar viciae R. lofi (Jarvis et al. 1982) R. meliloti (Krieg 8 Holt 1984) R. tropici (Martinez-Romero et a/. 1991)

inducers

of nod gene

Plant

host

Sesbania Glycine Phaseolus Glycine Galega Astragalus Phaseolus Jrifolium Pisum, Lathyrus, Vicia, Lens Lotus Medicago, Melilotus, Jrigonella Phaseolus, Leucaena

expression.

Flavonea Apigenin (4’, 5, 7-trihydroxyflavone) Chrysoerioi (4’, 5, 7-tetrahydroxy-3’-methoxyflavone) Kaempferol (3,4’, 5, 7-tetrahydroxyflavone) Luteolin (3’, 4’, 5, 7-tetrahydroxyflavone) Quercetin (3, 3’, 4’, 5, 7-pentahydroxyfiavone) 4’, 7-dihydroxyflavone laofiavonea Daidzein (4’, 7-dihydroxyisofiavone) Genistein (4’, 5, 7-trihydroxyisofiavone)

Flavanonea Eriodictyol Naringenin

(3’, 4’ 5, 7-tetrahydroxyflavanone) (4’, 5, 7-trihydroxyfiavanone)

Chalcone 4, 4’-dihydroxy-2’-methoxychalcone

nodule formation, regardless of the host-plant. The nodABC gene products possibly encode enzymes specifying the synthesis of the N-acylated chitin backbone of the Nod factors. This core structure may then be modified in a hostspecific way by the gene products of the host-specific nodulation genes (hsn genes). Putative functions of nodulation genes are given in Table 3. Synthesis of the N-acylated oligosaccharide may proceed in different stages. The NodC protein probably directs the synthesis of the oligosaccharide

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backbone of the Nod factor. NodC shares homology with chitin and cellulose synthases (Atkinson & Long 1992) and has been shown to be an N-acetylglucosaminyl transferase (Geremia et al. 1994). In a second step, NodB-may catalyse the deacetylation of the non-reducing N-acetylglucosamine residue of this chitooligosaccharide, thus producing a free amino group at this end (John et al. 1993). Finally, the attachment of a fatty acyl chain to this free amino group of the precursor molecule may be mediated by the NodA

Rhizobium-legume

symbiosis

R. meliloti

LJ

KNOQP

GHIS

FGHI

R. leguminosarum

F

M

NU

ABCX

K

N

viciae

biovar nif

nod

DKH

0

TNM

nif LEF

D ABCIJX

fix

B A XCBAW

B. japonicum

A

BCX

ZY

MN0

A

A

LJ NOQP

Figure 1. Organization of Rhizobium meliloti, Rhizobium leguminosarum biovar viciae and Bradyrhizobium japonicum N,-fixation and nodulation genes (not drawn to scale). The functions of some of these genes are described in the text or in Tables 3 and 4. Orientation of gene transcription is represented by arrows. Data are according to Hennecke (1990), Martinez et al. (1990), Barbour et al. (1992) and DBnarie eta/. (1992). Table

3. Possible

functions

of rhizobial

Nod proteins.

Protein

Homologies

Reference

NodC

Chitin

NodD NodE NodF

LysR family of DNA-binding P-Ketoacyl synthases Acyl carrier proteins

NodH NodL NodM NodP NodQ

Sulphotransferases Acetyltransferases D-Glucosamine ATP sulphurylase APS kinase

and cellulose

synthases

synthase

protein (Riihrig et al. 1994). The hsn genes determine the specificity of nodulation of a particular host. Mutations in these genes cause delayed nodulation or result in an alteration of the host range. These mutations cannot be complemented by homologous nod genes from other rhizobial species (Kondorosi et al. 1984). NodE and NodF proteins, exhibiting homology with Escherichia coli /Cketoacylsyn-

proteins

Atkinson & Long (1992) DebelI et al. (1992) Mulligan & Long (1989) Spaink et al. (1989) Shearman et a/. (1986) Geiger et a/. (1991) Roche et al. (1991) Downie (1989) Baev et al. (1991) Schwedock & Long (1990) Schwedock & Long (1990)

thase (condensing enzyme of fatty acid biosynthesis) and acyl carrier protein, respectively (Ttiriik et al. 1984; Geiger et al. 1991), are required for the synthesis of the unsaturated fatty acid moiety of Nod factors (Spaink et al. 1991). In R. melilofi, NodP and NodQ encode ATP sulphurylase and APS kinase (Schwedock & Long, 1990), while NodH shares similarity with steroid sulphotransferases from mam-

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J Michiels and]. Vanderleyden

H”&;~o~~~op$oH I

’

’ CH3

CHI n

R. leguminosarum

R. melilotr

R,

biovar viciae

Unsaturated BCVIS

unsaturated awls

C16:I. c162. Cl&3 orCls:I

C18:I orc,8:4

kd)-hyroxvlatcd CO(CH,),CHOH-CH,,

acvls where

x=

IS.

17, 19, 21.23

R,

CH,CO

R,

SO,H

H

n

I, 2 or 3

2 or 3

or H

CH,CO

Figure 2. Structures of R. meliloti and R. leguminosarom biovar viciae Nod factors (Lerouge et a/. 1990; Spaink et al. 1991; Schultze et a/. 1992; Demont et al. 1994). Nod factors vary in the numbers of glucosamine residues (n) and substitutions of the sugar backbone (R,, R,, R,).

mals. nodH and nodPQ genes have been shown to be involved in the sulphation of Nod factors (Roche et al. 1991). NodL is homologous to acetyl transferases and is required for the o-acetylation of the Nod factor (Downie 1989; Spaink et al. 1991). As a result of these modifications, Nod factors with different specificities for host plants are produced (Figure 2; DCnariC et al. 1992; Fisher & Long 1992).

Infection

Process

The attachment of bacteria to root hair constitutes one of the earliest steps in the Rhizobittm-plant interaction. Rhizobium attachment to the root-hair tip is a two-step process (Dazzo et al. 1984; Smit et al. 1989). The first step results in the attachment of single rhizobial cells to the root-hair tips and is mediated in R. leguminosarum bv viciae by the Ca2+-binding protein rhicadhesin (Smit et al. 1989). In the second step, additional bacteria adhere to the root hairbound rhizobia, leading to the formation of bacterial aggregates at the root-hair tip. This step involves plant lectins and/or bacterial cellulose fibrils or fimbriae (Vesper & Bauer 1986; Smit et al. 1987; Kijne et al. 1988). Meanwhile, being exposed to root-exudates, rhizobia secrete a set of lipo-oligosaccharides inducing root-hair curling, initiation of infection thread formation and development of nodule primordia. Entrapped in a root hair curl or in between two adjacent root hairs, rhizobia cause a localized cell-wall degradation. Whether these local lesions are due to rhizobial production of cell-wall degrading enzymes (pectinase, hemi-

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cellulase, cellulase) or to alterations in plant cell-wall synthesis or modification remains unclear (Kijne 1992). Next, the rhizobia are ingested by invagination of the plasma membrane of the hair cell. As the invaginated membrane extends towards the root-hair base, it is re-inforced by depositions of cell-wall-like material, forming a tubular structure, the infection thread. Within the growing infection thread, rhizobia are embedded in a matrix which may be of bacterial and/or host-cell origin. One matrix component is a hostcell-derived glycoprotein (Bradley ef al. 1988). A successful infection requires correct rhizobial cell-surface components (extracellular polysaccharides, capsular polysaccharides, cyclic p-glucans and lipopolysaccharides). Defects in these components may cause absence or early abortion of infection thread development (Noel 1992). Possible roles of rhizobial cell-surface components are the induction of nodule development through the release of oligosaccharin signal molecules and the avoidance of elicitation of a plant defense response (Gray & Rolfe 1990). Ahead of the growing infection thread, cells of the root cortex dedifferentiate, giving rise to a meristematic area, the nodule primordium. Upon contact with the nodule primordium, rhizobia are released from the tips of the infection thread into the plant cell cytoplasm. Within the plant cell cytoplasm, bacteria are enclosed by a plasmalemma-derived membrane, the so-called peribacteroid membrane. The bacteria divide and finally differentiate into pleiomorphic bacteroids able to reduce atmospheric N, into NH,. By their morphology, nodules are classified as determinate or indeterminate. The type of nodule is a characteristic of the host plant (Dart 1977). Determinate and indeterminate nodules have a central tissue consisting of uninfected cells and cells packed with rhizobia. The central tissue is surrounded by the nodule parenchyma, the endodermis and, outermost, by the cortex. Vascular bundles are located within the nodule parenchyma. In general, club-shaped indeterminate nodules develop on the roots of temperate legumes such as Medicago, Pisum, Trifoliwn and Vicia. In these legumes, the nodule primordium arises from cells located in the root inner cortex. Indeterminate nodules have a persistent apical meristem giving rise to successive zones of graded age in the nodule (Newcomb 1976). The youngest zones are located near the apical meristem, the oldest near the nodule base. On the other hand, tropical legumes such as Arachis, Glycine, Phaseolus and Lotus form spherical determinate nodules. In these nodules, the earliest mitotic activity occurs in the root outer cortex and meristematic activity within the nodule ceases during nodule development (Newcomb et al. 1979). Nodules on the non-leguminous plant Parasponia are indeterminate, with a central vascular system (Lancelle & Torrey 1985). In the Parasponia symbiosis, rhizobia are not released into the host-cell cytoplasm but fix N, in a modified infection thread, called the fixation thread (Price et al. 1984).

Rhizobium-legume Penetration through root hairs is the most widely studied mode of rhizobial infection. In addition, two other methods of infection have been described. In Arachis (groundnut) and Stylosanthes, a tropical forage legume, rhizobia gain entry through intercellular spaces in the epidennal and cortical cells (wound or crack infection), at zones of lateral root emergence (Chandler et al. 1982). Infection of Mimosa scabrella, a tropical tree, occurs through intact epidermises, between the epidermal cells, by localized cell-wall dissolution (de Faria et al. 1988). As with nodule morphology, the route of Rhizobium infection is determined by the host plant. The development of a legume nodule is accompanied by the expression of nodule-specific plant genes, nodulin genes. Nodulin genes that are expressed during the early stages of nodule development are named early nodulin genes. These are supposedly involved in infection thread formation and nodule organogenesis. Several early nodulin genes encode proline-rich proteins that are likely components of the cellwall or the infection-thread plasma membrane (Franssen et al. 1992). At least some of these early nodulin genes are inducible by purified Nod factors (Vijn et al. 1993). The expression of late nodulin genes starts around the onset of N, fixation. These genes are probably involved in nodule maintenance and functioning (see below).

Nodule

Functioning

The Peribacteroid Membrane and the Symbiosome Space When the infection thread reaches nodule primordium cells, individual rhizobia are internalized into the plant cell by a process resembling endocytosis. In the endosymbiotic state, rhizobial cells, called bacteroids, are surrounded by the peribacteroid membrane, forming an organelle-like structure, the symbiosome. The term symbiosome includes the bacteroid, the symbiosome space and the peribacteroid membrane. Following endocytosis, bacteroids proliferate within the host-cell cytoplasm. Extension of the peribacteroid membrane occurs by incorporation of vesicles derived from Golgi and/or endoplasmic reticulum (Mellor & Werner 1987). Constituting the physical and metabolic barrier between both symbiotic partners, the peribacteroid membrane fulfills a pivotal role during symbiosis. Early disintegration of this membrane coincides with a pathogenic response in the host plant (Werner et al. 1985). The peribacteroid membrane allows passive diffusion of 0, and N, into the symbiosome and of H,, NH, and CO, outwards (Day et al. 1990). Several functions have been associated with the peribacteroid membrane of soybean, including a plasma-membrane type H+-ATPase (Blumwald et al. 1985), a dicarboxylate carrier (Day et al. 1990; Yang et al. IWO), a calcium-dependent protein kinase (Bassarab & Werner 1987) and a magnesium-dependent pyrophosphatase (Bassarab &

symbiosis

Werner 1989). In soybean, a number of late nodulins that are associated with the peribacteroid membrane have been identified but not yet characterized at the biochemical level. The soybean nodulin Ngm-26 shows significant homology with a bovine eye lens membrane protein and with the E. coli glycerol facilitator, a pore-type protein in the cytoplasmic membrane of E. co/i (Fortin et al. 1987). Therefore, it is likely that Ngm-26 forms an ion-channel across the peribacteroid membrane, facilitating the transport of metabolites between the host-cell cytoplasm and the bacteroid (Fortin et al. 1987; Verma 1992). The symbiosome space can be considered as a plant internal compartment with lysosomal properties (Mellor 1989). The symbiosome space contains, among other acid hydrolases, a-mannosidase, a vacuolar marker enzyme (Kinnback et al. 1987; Werner 1992). A drop in pH of the peribacteroid fluid could activate the acid hydrolases and turn the symbiosome into a lytic vesicle (Kannenberg & Brewin 1989). Nodule Metabolism During photosynthesis, the plant reduces CO, and transports the products to the nodule where they ultimately serve as energy source for the bacteroid. In return, the bacteroid fixes atmospheric N, into NH, which is translocated to the host. This metabolic co-operation between plant and bacterium requires several specific physiological adaptations: the protection of the nitrogenase enzyme against oxygen damage, the translocation of carbon compounds to the bacteroid to support the high energy demands of N, fixation, and the assimilation of the product of N, fixation, NH,, by the plant. These new physiological demands require plant adaptations for oxygen, nitrogen and carbon metabolism. Several plant proteins that are involved in these processes are synthesized in a nodulespecific or nodule-abundant form (Delauney & Verma 1988). Nodule-specific proteins that have been characterized both biochemically and genetically are the metabolic nodulins leghemoglobin, sucrose synthase, uricase and glutamine synthetase (Nap & Bisseling 1990). These nodulins are synthesized around the onset of N, fixation and are therefore referred to as late nodulins. The nodule free-O, concentration is very low (3 to 30 nM; Layzell et al. 1990), allowing optimal functioning of the nitrogenase enzyme which is extremely O,-sensitive. It is commonly assumed that such a low 0, concentration results from the presence of an 0, diffusion barrier in the nodule parenchyma and bacteroid respiration in the central tissue (Witty et al. 1986). The nodule parenchyma consists of tightly packed cells with few intercellular spaces, therefore restricting 0, diffusion (Sheehy & Web 1991). Oxygen diffuses several orders of magnitude slower in aqueous phase than in intercellular air spaces. The resistance of the OZ.-diffusion barrier can be varied within minutes (Witty et

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J. Michiels and ]. Vanderleyden al. 1986). The mechanism by which nodules can alter rapidly their resistance to 0, diffusion is yet unknown. The expression of the early nod&n ENOD2 from P&m, Glycine or Medicago is confined to the nodule parenchyma (Verma et al. 1992). This proline-rich protein is probably a cell-wall component and may contribute to the specific structure of the parenchyma (Nap & Bisseling 1990). In addition, the intercellular spaces of the parenchyma of Piwm and Glycine nodules accumulate a 95-kDa matrix glycoprotein which is also a component of the infection thread (VandenBosch et al. 1989; James et al. 1991). Since Rhizobiam is an obligate aerobe, 0, is required for energy production to fuel the bacterial nitrogenase enzyme. Diffusion of 0, to the bacteroids is facilitated through the presence of an O,-carrier protein, leghemoglobin (Wittenberg et al. 1974; Sheehy et al. 1985). This nod&n, localized in the cytoplasm of the infected cells, is the most abundant protein in a N,-fixing nodule, where it constitutes up to 25% of the plant cytoplasmic proteins and serves as 0, store and buffer in the nodule (Appleby 1984; Robertson et al. 1984). The leghemoglobin apoprotein is plant encoded whereas synthesis of the heme prosthetic group is thought to occur in the bacteroid. (Appleby 1984). The very high 0, affinity of leghemoglobin results from a fast 0, combination rate coupled to a moderately slow 0, dissociation rate (Appleby 1984). Oxygenated leghemoglobin delivers the bound 0, near the bacteroid surface, where the 0, concentration is very low. The released 0, is probably reduced by a high-O,-affinity bacteroid terminal oxidase. A good candidate of such a bacterial oxidase complex is the recently identified cytochrome-c-containing heme/copper oxidase from B. japonictlm (Preisig et al. 1993; see below). Ammonia, the product of bacterial N, fixation, enters the host cell cytosol by passive diffusion (Day et al. 1990). Within the host cell, NH, is assimilated through the combined actions of glutamine synthetase (GS or n-GS) and glutamate synthase (GOGAT; glutamine:2-oxoglutarate aminotransferase) producing glutamine and glutamate, respectively. The assimilation of NH,+ into glutamine probably occurs in the plant cytosol, but the incorporation of glutamine into glutamate is carried out in the plastids of the infected cells (Chen & Cullimore 1989; Streeter 1991; Cullimore & Bennett 1992). The compounds glutamine and glutamate are used in the synthesis of amides or ureides which are transported to the rest of the plant. Many temperate legumes, including Lapinas, Medicago, Pisum, Trifolitrm and Vicia, export the amino acids asparagine and/or glutamine whereas the tropical legumes, Glycine, Phaseolw and Vigna, transport the ureides allantoin and allantoic acid (Schubert 1986). One tropical legume, Arachis, exports both ureides and amides, including the unusual amino acid amide 4-methyleneglutamine (Winter et al. 1981). In the amide exporters, the synthesis of asparagine from glutamine and glutamate requires the activity of two additional en-

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World Journal of Microbiology 6 Biotechnology, Vol 10, 1994

and asparagine synzymes, aspartate aminotransferase thetase. Aspartate aminotransferase is located in the plastids but asparagine synthetase is localized in the plant cytosol (Streeter 1991; Cullimore & Bennett 1992). In ureide-producing plants, nitrogen assimilation is compartmentalized into two cell types, the infected and the uninfected cells of the nodule central tissue. Glutamine is first channelled into purine biosynthesis in the plastids of the infected cells (Boland & Schubert 1983). The product of purine biosynthesis, probably xanthosine monophosphate or inosine monophosphate, is exported from the plastids to the cytosol (Shelp & Atkins 1983; Schubert 1986). Further purine oxidation and ureide synthesis is carried out in the uninfected cells. Xantosine monophosphate is dephosphorylated and subsequently oxidized to uric acid by the enzyme xanthine dehydrogenase. This enzyme is supposedly located in the cytosol of the uninfected cells (Nguyen ef al. 1986). Uric acid is converted to allantoin by the nodulin uricase II (or n-uricase), the second most abundant protein in soybean nodules (Legocki & Verma 1979), within the peroxisomes (Webb & Newcomb, 1987) and further oxidized to allantoic acid in the endoplasmic reticulum of the uninfected cells by the enzyme allantoinase (Hanks ef al. 1981). Allantoin and allantoic acid are exported from the nodule via the xylem. The product of photosynthesis, sucrose, is transported from the leaves to the nodule, where it constitutes the main energy source. In the nodule, sucrose is hydrolysed by a nodule-specific form of sucrose synthase (nodulin-100) and further degraded to C,-dicarboxylic acids (Romanov et al. 1985; Kouchi & Yoneyama 1986). These compounds are then transported and catabolised in the bacteroids, generating the energy for N, fixation (Rosendahl et al. 1990; Vance & Heichel 1991). The degradation of glucose classically proceeds through the glycolysis pathway, producing pyruvate. Pyruvate is then imported into the mitochondria and further oxidized in the citric acid cycle, also known as the tricarboxylic acid cycle (TCA cycle). However, due to the low 0, tension in the nodule, the functioning of mitochondria is probably seriously restricted. It is therefore unlikely that mitochondria produce dicarboxylic acids at rates sufficient to meet the requirements of the nodule metabolism (Rawsthome & LaRue 1986). Plants have evolved specific adaptations to the O,-limited environment of the nodule. Glycolysis is shunted towards the reductive synthesis of malate, which may be further converted to fumarate and succinate (Vance & Heichel 1991). This is achieved through the carboxylation of phosphoenolpyruvate by the enzyme phosphoenolpyruvate carboxylase, resulting in the production of oxalacetate (King et al. 1986; Rosendahl et al. 1990). Oxalacetate may then be reduced to malate by malate dehydrogenase or transported into the plastids to provide the carbon skeleton for the synthesis of aspartate. Thus, up to 25% of the carbon incorporated into

Rhizobium-legume malate or aspartate may originate from dark CO, fixation in the nodule-host cytosol. Photosynthate supply to the nodule is probably in excess to its needs (Vance & Heichel 1991). Part of it is therefore converted to starch and deposited in the amyloplasts of both the infected and uninfected cells. Degradation of starch may occur when carbon supply to the nodule is limiting. Another pool of carbon compounds in the nodule includes trehalose, malonate and cyclic alcohols. The roles of these metabolites are unknown. Trehalose is probably synthesized in the bacteroids and released back into the host cell (Streeter 1985). Malonate may accumulate to high concentrations in nodules (Kouchi & Yoneyama 1986; Streeter 1987) but is probably excluded from the symbiosome (Rosendahl ef al. 1990). Cyclic alcohols (cyclitols) have been identified in the nodules of Glycine (myo-inositol), Lupinw and Trifolium (pinitol) and in Pisum (oronitol) (Phillips et al. 1984; Streeter 1987). Bacteroid Carbon Mefabolism The plant supplies the bacteroid with carbon sources to sustain bacterial growth and N, fixation. Although rhizobia are capable of growing on a broad range of sugars, organic acids and aromatic compounds (Stowers, 1985), several lines of evidence indicate that C,-dicarboxylic acids are the main carbon source supplied to the bacteroids: (1) Malate, succinate and fumarate accumulate to high concentrations in the nodule (Vance & Heichel, 1991). (2) In contrast to sugars and amino acids except aspartate, dicarboxylic acids are rapidly transported across the peribacteroid membrane by an active mechanism (Day et al. 1990; Yang et al. 1990). (3) Bacteroids have a C,-dicarboxylate transport system (Dct-system). Rhizobium mutants with a defective Dctsystem form ineffective nodules (Ronson et al. 1981; Arwas el al. 1985; San Francisco & Jacobson 1985; Engelke et al. 1987). (4) A functional TCA cycle in the bacteroid is needed for N, fixation. Rhizobium mutants deficient in carbohydrate metabolism are generally not impaired in N, fixation (Ronson & Primrose 1979; Glenn ef al. 1984) whereas mutants lacking the TCA cycle enzymes a-ketoglutarate dehydrogenase or succinate dehydrogenase form ineffective symbioses (Duncan & Fraenkel 1979; Gardiol et al. 1982). (5) C,-dicarboxylic acids support high levels of nitrogenase activity in isolated bacteroids (Bergersen & Turner 1967). In R. melilofi and R. leguminosarum bv viciae free-living cells, a cluster of three genes, dcfA, dcfB and dctD, are essential for the uptake of the C,-dicarboxylic acids malate, fumarate and succinate (Ronson et al. 1984; Yarosh et al. 1989). Also, R. melilofi mutants defective in the uptake of C,-dicarboxylic acids fail to transport aspartate for use as a carbon source (Watson et al. 1988; Watson 1990). The

symbiosis

structural gene dcfA encodes a permease (DctA) located in the cytoplasmic membrane (Ronson et al. 1987a; Jiang et al. 1989; Yarosh et al. 1989). Expression of dcfA in the freeliving state is regulated by C,-dicarboxylates, by the gene products of dctB (DctB) and dcfD (DctD) (Ronson et al. 1984; Yarosh et al. 1989) and by the alternative RNA polymerase sigma factor RpoN (@‘; NtrA) encoded by rpoN (Ronson ef al. 1987b). In addition, the dcfA gene product probably suppresses, directly or indirectly, its own synthesis in the absence of C,-dicarboxylic acids (Yarosh et al. 1989; Jording et al. 1992). DctB and DctD share homology with proteins of two-component regulatory systems (Ronson et al. 1987a; Jiang ef al. 1989; Watson 1990). It has been suggested that DctB is a transmembrane protein located in t-he cytoplasmic membrane and that it senses the presence of periplasmic C,-dicarboxylates. In response to the concentration of these compounds, DctB activates the cytoplasmic effector protein DctD, which, in turn, together with RpoN, stimulates dctA transcription. While mutations in dckA cause the formation of ineffective nodules, dcfB and dcfD Rhizobium mutants are still capable of reducing atmospheric N, during symbiosis, albeit at a reduced level (Watson et al. 1988; Yarosh et a/. 1989). Transcription of dcfA in the endosymbiotic state might therefore be controlled, in addition to dctB and dcfD, by a symbiosis-specific factor. The metabolism of C,-dicarboxylic acids in bacteroids most likely occurs via the TCA cycle (Streeter 1991). To be operative, the TCA cycle requires the input of acetyl-CoA (AcCoA). Acetyl-CoA may be supplied via the decarboxylation of pyruvate by pyruvate dehydrogenase. In R. melilofi bacteroids, pyruvate is not synthesized from oxalacetate by the combined activities of phosphoenolpyruvate carboxykinase (PCK) and pyruvate kinase since bacteroids lack PCK activity (Finan et a/. 1988, 1991). An alternative pathway involves the activity of malic enzyme catalysing the oxidative decarboxylation of malate to pyruvate. The two forms of malic enzyme, an NAD+dependent (dme) and NADP+dependent form @me), are present in bacteroids (McKay et al. 1988; Copeland et al. 1989; Driscol & Finan 1993). A R. meliloti NAD+ malic enzyme mutant has recently been characterized (Driscol & Finan 1993). This mutant failed to fix N, during symbiosis although the infection process proceded normally until the symbiosome stage. It therefore appears that the NAD+ malic enzyme fulfils a central role in R. melilofi bacteroid carbon metabolism. Bacteroid TCA cycle functioning may thus provide the energy to fuel the nitrogenase. It is a matter of controversy whether plant-derived amino acids serve as a carbon source to sustain bacteroid N,-fixation activity (Kahn et al. 1985; Bergersen & Turner 1988; Udvardi et al. 1988). Isolated B. japonicum (Salminen & Streeter 1987; Kouchi & Fukai 1988; Udvardi et al. 1988, 1990), R. meliloti (McRae et al. 1989) and R. leguminosarum

1. Michiels and ]. Vanderleyden bv phaseali (Herrada et al. 1989) bacteroids are reported to actively import C,-dicarboxylates (malate, succinate), amino acids (glutamate, aspartate) and some sugars (glucose, fructose). However, since the peribacteroid membrane is largely impermeable to amino acids and sugars (Herrada et al. 1989; McRae et al. 1989; Udvardi ef al. 1990), these compounds are unlikely to function as an energy source to the bacteroids. Exceptions to this rule are the presence of glucose- and aspartate-transport activities on the peribacteroid membranes of R. legminosartm bv phaseoli (Herrada et al. 1989) and R. mehloti (McRae et al. 1989) symbiosomes, respectively. However, in comparison with C,-dicarboxylate transport, the uptake of aspartate by R. meliloti symbiosomes is slow and probably completely inhibited in viva by the high concentrations of malate and succinate in the plant-cell cytosol (McRae et al. 1989). The biological meaning of the differences in permeability between symbiosome and bacteroid membranes is unclear. The selectivity of the peribacteroid membrane may allow direct plant control over the range of organic compounds available to the bacteroid. On the other hand, in the absence of a peribacteroid membrane or when its selective permeability is altered, for example during infection, early symbiosis or nodule senescence, plant-derived sugars and amino acids become available to the endosymbiont and may then constitute important carbon and nitrogen sources. McDermott et al. (1989) suggested that, under conditions of 0, limitation, the activity of a-ketoglutarate dehydrogenase is inhibited, resulting in a deceleration of the TCA cycle. The formation of y-aminobutyrate (GABA) from aketoglutarate and subsequent conversion to succinate may represent a shunt around the classical a-ketoglutarate-tosuccinate conversion in the TCA cycle. In addition to the TCA and malic enzyme pathways, the GABA shunt pathway is probably operative in R. meliloti and B. japonicwn bacteroids (McDermott et al. 1989; Fitzmaurice & O’Gara 1991). In the GABA shunt, glutamate is first synthesized from a-ketoglutarate. Glutamate is then decarboxylated, forming GABA, which is subsequently deaminated in a transaminase reaction and finally oxidized to succinate. Succinate in turn can re-enter the TCA cycle. A R. melilofi mutant lacking a functional GABA bypass also has reduced symbiotic N,-fixation activity (Fitzmaurice & O’Gara 1993). Bradyrhimbium japonicum bacteroids accumulate high amounts of the reserve polysaccharide poly+hydroxybutyrate (Klucas & Evans 1968). Degradation of poly-phydroxybutyrate to acetoacetate and subsequent oxidation via the TCA cycle may provide additional energy to support N,-fixation activity. Bacferoid Respiration Free 0, causes a rapid irreversible inactivation of the O,sensitive nitrogenase enzyme by the oxidation of the

metal-S-centers of the protein. In addition high 0, concentrations inhibit transcription of the N,-fixation genes. On the other hand, rhizobia are aerobic microorganisms, thus requiring 0, for the generation of ATP. This apparent paradox has been resolved by hypothesising the existence of a symbiosis-specific high-O,-affinity bacteroid respiration complex that is capable of scavenging 0, released from oxyleghemoglobin. The oxidation of reduced carbon compounds generates low potential electrons that can be channelled in the electron transport chain to generate energy. In general, low electric potential electrons are supplied by NADH and carried via the NADH-dehydrogenase complex to a small hydrophobic molecule known as ubiquinone (UQ). These electrons are subsequently passed via the ubiquinol-cytochrome-coxidoreductase complex (or cytochrome &c, complex) and cytochrome c to the cytochrome-c-oxidase complex (cytochrome au,), and finally transferred to 0,:

As an adaptation towards various environmental conditions, bacteria often possess a branched electron transport chain, containing terminal oxidases with different 0, affinities, Respiratory chains may branch off at the ubiquinol pool or at the cytochrome bc, complex. In B. japonicum, the respiratory pathway consists of at least three branches. The genes coding for the components of the first, classical, mitochondrial-like branch, the focFH operon (encoding Rieske Fe-S protein/cytochromes b and c,), the cycM gene (cytochrome c) and COXA (subunit I of cytochrome an,), have been identified and mutagenised (Bott et al. 1990, 1991; Gabel & Maier 1990; Thony-Meyer et al. 1991). Strains carrying mutations in cycM and COXA still form fully effective nodules, indicating that this branch is not essential for bacteroid respiration. Concurrently with the au,-type oxidase, aerobically grown B. japonicum cells contain a second oxidase, cytochrome o (Keister & Marsh 1990). The genes encoding a putative alternative cytochrome c oxidase (coxMNOP) were identified recently (Bott ef al. 1992). A B. japonicum COXN mutant was not affected in symbiotic N, fixation. It is not known whether this altemative cytochrome c oxidase corresponds to cytochrome o. The Fix- phenotype of B. japonicwn mutants carrying insertions within the cytochrome-bc, gene (fbcF and /WI) indicated that a third electron transport chain, which is essential during symbiosis, branches off at this point. Recently, Preisig et al. (1993) reported the identification of a B. japonicum gene cluster $rNOQP) located upstream from the symbiotic regulatory genes /ixL.] (see below). Strains containing mutations within these genes have a strongly reduced oxidase activity under microaerobic but not under aerobic conditions and are defective in bacteroid development and symbiotic N,-fixation. Analysis of the deduced amino acid sequence suggests that the fixNOQP cluster encodes a membrane-

Rhizobium-legume Table

4. Properties

and functions

of nif and fix genes.

Gene

Propertles

nifH nifDK nifA nif6 nifEN nifS fixA6CX

Dinitrogenase reductase (component II, Fe protein, a, dimer) Dinitrogenase (component I, MoFe protein, u~/?~ tetramer) Regulatory protein, activator of most nif and fix genes FeMo-cofactor biosynthesis Similar to nifDK. form a a& tetramer. FeMo-cofactor biosynthesis Function unknown, involved in maturation of dinitrogenase reductase FixX is homologous to ferredoxins, Fix6 to human and rat electron transfer flavoproteins. Putative electron transport chain Regulatory protein, homologous to E. co/i Fnr and Crp proteins Regulatory proteins, homologous to two-component sensor/effecter proteins FixN is homologous to subunit I of heme/copper oxidases, Fix0 is predicted to be a monoheme cytochrome c and FixP a diheme cytochrome c. Proposed to be a membrane-bound terminal oxidase complex Contains transmembrane sequences. Fixl is related to cation ATPases. FixG to redox proteins. Proposed to participate in a transmembrane complex coupling a cation pump to redox processes

fixK fixLJ fixNOW

fixGH/S

and functions

bound, four-subunit, terminal-oxidase complex consisting of a heme b/copper-binding protein (FixN), a monoheme cytochrome c (FixO), a small polypeptide (FixQ) and a diheme cytochrome c (FixP). A fi.xNOQP cluster has also been identified in R. meliloti (Batut et al. 1989; Boistard et al. 1991). The firNOQP-encoded proteins are good candidates for the bacteroid high-O,-affinity oxidase. It is not yet known whether this complex is also capable of using 0, delivered by leghemoglobin as a substrate.

The N,-fixation

symbiosis

Process

Common Regulatory Mechanisms in Diazofrophs The conversion of atmospheric N, to NH, by the nitrogenase enzyme (N, + 8H + + 8e- 0 2NH, + H,) is an highly energy-demanding process which requires the hydrolysis of a minimum of 16 molecules of ATP. It is therefore not surprising that the process of N, fixation is tightly regulated. This is achieved by a number of mechanisms acting at both the levels of transcription of N,fixation genes and activity of their gene products. The regulatory signals that control the N,-fixation process in diazotrophs are related to their physiology and ecology. Since the nitrogenase enzyme is 0, sensitive, low 0, concentrations constitute a major trigger in all diazotrophs. Free-living N, fixers reduce N, to their own benefit, necessitating a tight coupling between N, fixation and assimilation processes. Rhizobia generally fix N, only in the endosymbiotic state, while they are provided with plant metabolites. In contrast to free-living diazotrophs, rhizobia are evolutionary designed to export NH, for the benefit of the plant. Therefore, the availability of a nitrogen source is, in addition to the 0, concentration, a major regulatory factor regulating the N,-fixation process in free-living but not in symbiotic N, fixers.

Reference Dean Dean Merrick Dean Dean Dean Arigoni

8 Jacobson (1992) 8. Jacobson (1992) (1992) &Jacobson (1992) &Jacobson (1992) &Jacobson (1992) et al. (1991)

Batut et al. (1989) David et al. (1988) Preisig et al. (1993)

Kahn

et al. (1989)

Rhizobial genes that are essential for symbiotic N, fixation are designated as nif and fix genes (Figure I and Table 4). The nif genes of Rhizobium, but not the fix genes, have a structural and functional homologue in the extensively studied free-living diazotroph, Klebsiella pneumoniae. Despite considerable taxonomic diversity, several basic elements of nif and fix gene expression are well conserved among diazotrophs (Merrick 1992). Many, but not all, nif and fix genes are preceded by a characteristic type of promoter. This promoter is characterized minimally by the presence of two conserved dinucleotides, GG and GC, located 24 and 12 base pairs (bp) upstream from the transcriptional start site, respectively, and is therefore referred to as the -24/ - 12 promoter (Beynon et al. 1983). This type of promoter is not limited to nif and fix genes but has also been found in front of genes whose products function in processes unrelated to N, fixation (Thiiny & Hennecke 1989). The - 24/ - 12 promoter is recognized by an alternative RNA polymerase sigma factor, c?, encoded by the rpoN gene (nfrA, g1nF). However, the interaction between the &“-RNA polymerase holoenzyme complex (EONS) and the -24/12 promoter does not result in initiation of transcription (Sasse-Dwight & Gralla 1988; Popham et al. 1989). The isomerization of the inactive closed complex between Eg54 and the -24/I2 promoter to an open complex is catalysed by a transcriptional activator protein, i.e. NifA or NtrC in the case of N,-fixation genes (SasseDwight & Gralla 1988; Popham et al. 1989). NifA- or NtrC-mediated activation of nif and fix promoters is dependent or stimulated by the presence of one or several upstream activator-binding sites (Buck et al. 1986). The NifA protein has been shown to bind to a DNA motif, called UAS (upstream activator sequence), with the consensus sequence 5’-TGT-N,,-ACA-3’ that is generally located between 80 and 150 bp upstream from the transcription initiation site

J Michiels and J. Vanderleyden

FixL

FixJ

(

FixJ-P El higO,

high 0,

+ O/\

0

-0lo

rp&

nif4XE > fix;gcx [ nifa

fiXNZP

fixefLs?

fdxN

Figure 3 Model for the regulation of nif and fix gene expression in Rhizobium meliloti (after Reyrat et al. 1993). Symbols indicate positive (+) or negative (-) regulation of transcription (0, 0) and activity (m, 8).

(Buck et al. 1986; Gussin et al. 1986; Morett & Buck 1988). Activation of the nif promoter by NifA is stimulated by the presence of the integration host factor (IHF). IHF is a DNA-bending protein that facilitates the contact between UAS-bound NifA protein and the binary $24/ # 12-EcY holoenzyme complex (Santero et al. 1992). In addition to this basic regulatory mechanism, pointing to a common origin, most diazotrophs have evolved additional control mechanisms of nif and fix gene expression. These mechanisms will be discussed in the case of R. meliloti and B. japonictlm, the endosymbionts of alfalfa and soybean, respectively. The fixLJ/nifA and fixLJ/fixK Regulatory Cascades in R. meliloti The activation of R. melilofi N,-fixation genes involves a regulatory cascade of which the firI. genes constitute the primary controllers (Figure 3; David et al. 1988). The FixL and FixJ proteins belong to a family of two-component sensor/effecter regulatory systems in which the sensor activates the effector protein by phosphorylation in response to a specific environmental signal (David et al. 1988). In R. meliloti, FixJ is activated by FixL when the free0, concentration is reduced to microaerobic levels. Activated FixJ in turn, in conjunction with the a“‘-RNA polymerase holoenzyme complex, enhances expression of the transcriptional regulatory genes nifA and fixK (David et al. 1988; Virts ef al. 1988; Hertig et al. 1989; de Philip et

622

World ]oumal

of Microbiology

& Bmtechnology, Vol IO. 1994

al. 1992). FixL is a membrane-bound protein containing a heme-binding domain and a carboxy-terminal kinase domain (Gilles-Gonzalez et al. 1991). The cytoplasmic FixJ protein contains a transcription-activation function in its carboxy-terminal domain which is negatively regulated by the phosphate-acceptor amino-terminal domain (Kahn & Ditta 1991). The free-O, concentration is sensed through the heme moiety of FixL located in its central domain (de Phillip et al. 1992; Monson et al. 1992). Under anaerobiosis, the FixL protein autophosphorylates and subsequently transfers its phosphate group to the FixJ protein (Monson et al. 1992; Reyrat et al. 1993). In addition to its kinase activity, FixL also possesses a phosphorylated FixJ phosphatase activity (Lois et al. 1993). Under anaerobiosis, autophosphorylation of FixL is enhanced whereas the phosphatase activity is repressed (Lois et al. 1993). The presence of both the positive and negative regulatory activities in the FixL protein may allow the cell to control the level of activated FixJ protein. Recently, the complete signal transduction pathway from the oxygen signal to the transcriptional activation of fixK in the presence of FixL and FixJ has been reconstituted in vitro (Agron et al, 1993; Reyrat et al. 1993). NifA and FixK are transcriptional regulatory proteins, each of them controlling the expression of a different set of nif and fix genes (David et al. 1987; Batut et al. 1989). Expression of the nifA gene is subject to positive and negative regulation. In addition to the FixLJ-dependent activation, nifA transcription is positively autoregulated under microaerobiosis (Kim et al. 1986). In R. melilofi, at least 50% of the nifA transcription is attributable to the transcripts originating at the firABCX promoter located approximately 4 kb upstream from nifA (Kim et al. 1986). nifA transcription is negatively regulated by FixK (Batut et al. 1989). NifA activity is also controlled post-translationally by the cellular oxygen status since the R. meliloti NifA protein is 0, sensitive (Beynon et al. 1988). The R. meliloti FixK protein acts both as a positive and negative regulator of nif and fir gene expression. FixK represses AJifA transcription, as well as its own expression, while activating transcription of the firNOQP operon which codes for a putative respiratory complex and possibly of the fixGH1.S operon which codes for a putative cation pump (Batut et al. 1989; Kahn et al. 1989). Unlike NifA, the FixK protein is not 0, sensitive (de Philip et al. 1992). Rhizobium meliloti FixK is homologous to the E. coli Fnr protein, involved in the regulation of anaerobic respiratory metabolism, and to the E. coli CAMP receptor protein (Crp) (Batut et al. 1989). The strong amino acid conservation in the helix-turn-helix motifs of the Fnr and FixK proteins led Batut ef al. (1989) to suggest that both proteins might recognize similar DNA sequences. A DNA motif resembling the Fnr-binding consensus (TTGAT-N,-ATCAA) is found in the promoters of the f;xAJOQP and firGH1S transcriptional units (Batut et al. 1989; Kahn ef al. 1989). In addition,

Rhizobium-legume unknown act1vatw

Figure 4. Model for the regulation of genes involved in symbiotic N, fixation in Bradyrhizobium japonicum (after Fischer et al. 1993). Symbols indicate positive (+) or negative (-) regulation of transcription (0, 0) and activity (B, 8). UAS-Upstream activator sequence; P-promoter.

an Fnr-like binding region is involved al. 1992).

sequence in the R. melilofi fixK promoter in negative autoregulation (Waelkens et

Tke fixLJ/fixK and nifA Regulafo y Cascades in B. japonicum Contrary to R. melilofi, B. japonicum possesses two O,responsive but largely independent regulatory cascades, the fixL]/fixK and the nifA cascade (Figure 4). While nifA regulates the expression of many N,-fixation genes, the fixL]/fi& regulatory system is more specialized in the control of anaerobic respiratory processes. Both cascades are linked by the fixL]/fixK-regulated rpoN, gene encoding a-. The B. japonicum FixL and FixJ proteins are highly homologous to their R. melilofi counterparts but, in contrast to R. melilofi, the B. japonicum FixL protein does not contain the amino-terminal hydrophobic, membrane-bound domain (Anthamatten & Hennecke 1991). Bradyrkizobium japonictrm firLj mutants have a 90% reduction of symbiotic N,-fixation activity and are unable to use nitrate as terminal electron acceptor during anaerobic growth. In response to microaerobiosis, fi.xL] activates the expression of fix& (Anthamatten et al. 1992), rpoN, (one of the two genes encoding 8*) (Kullik et al. 1991) and the firNOQP transcriptional unit. Although a constitutively expressed fix& gene can restore anaerobic nitrate respiration of a B. japonicum fixL] mutant, the inactivation of fix& does not lead to the phenotypical defects of a fixLJ mutation (Anthamatten et al. 1992). This led Fischer et al. (1993) to propose the existence of a second fixL]-regulated functional copy of fi.& (firK,). Unlike R. melilofi FixK, the B. japonicum FixK, protein is O,-sensitive (Anthamatten ef al. 1992). In the case of the fix&homologous Fnr protein from E. coli, it has been suggested that the presence of a cysteine-rich motif within fhe amino-terminal domain of the protein may be responsible for its O,-sensitivity (Green et al. 1991). A similar cysteine-rich pattern is also present in the B. japonicum

symbiosis

FixK, protein but is absent in R. melilofi FixK. fixL] dependent regulation of rpoN, and fixNOQP is possibly mediated by firK, since their promoters confain a DNA sequence resembling the Fnr-binding consensus (TTGAT-N,ATCAA). Additional putative FixK target genes are the oxygen-regulated genes hemA, encoding 5-aminolevulinic acid synthase (McClung et al. 1987) and cyrB, encoding the c-type cytochrome cSS2 (Rossbach et al. 1991). Both genes contain an Fnr-like binding sequence. Bradyrkizobium japoniczm contains two functional fY4homologous genes, rpoN, and rpoN, (Kullik et al. 1991). An rpoN,,, d ou bl e mutant induces nodules that lack N,fixation activity. rpaN, but not rpoN, is regulated by the fixLl/fi*K cascade in response to microaerobiosis. Expression of rpoN, is independent of the 0, concentration and negatively autoregulated. The mechanism underlying the negative autoregulation of rpoN, transcription is yet unclear. However, since no obvious -24/ - 12 promoter structure was detected on either strand of the rpoN, promoter, negative autoregulation by direct interaction of the rpoN, gene product to its own promoter is unlikely. Bradyrkizobitrm japonicum nifA is the promoter-distal gene of the fixRnifA operon (ThGny et al, 1987). In concert with P encoded by rpoNI or rpoN, NifA mediates the microaerobic induction of the N,-fixation genes nifDKEN, nipfrxA, fixBCX, nif.5, nigh and fixA (Hennecke 1990; Kullik ef al. 1991). Although the gene product of fixR shows homology to dehydrogenases involved in acetate metabolism (Morett et al. 1993), no function could be assigned to this protein. fixR is not essential for symbiotic N, fixation (Thiiny et al. 1987). The fixRnifA operon is subject to a dual positive control. It is expressed at a moderate level during aerobic growth and is 5-fold induced under microaerobiosis by a mechanism that involves the NifA protein (Thijny et al. 1987, 1989). Aerobic expression of fixRnifA does not result in the production of active NifA protein since B. japonicum NifA is irreversibly inactivated under these conditions (Kullik et al. 1989). Expression of fixRnifA depends on two overlapping promoters (Morett et al. 1993). NifA-independent transcription, occurring independently of the oxygen status of the cell, is probably controlled by @‘, the housekeeping sigma factor of B. japonicum, and depends on th e presence of an upstream activator sequence (Thijny ef al. 1989; Morett et al. 1993). Auto-activaticn of the fixRnifA promoter by NifA occurs from a - 24/ - 12 promoter and is partly independent of the presence of promoter upstream DNA (Thiiny et al. 1989; Morett et al. 1993). Regulation of N,-fixation Genes in R. leguminosarum bv phaseoli The expression of nif and fix genes in R. leguminosarum bv pkaseoli is controlled by two, largely independent, regulatory cascades. The $A regulatory cascade controls the

1. Michiels

and ]. Vaanderleyden

expression of several N,-fixation genes (Michiels & Vanderleyden 1993; Michiels et al. 19%). Although the R. leguminosarum bv phaseoli nifA gene is expressed aerobically and microaerobically under free-living conditions, nifA-dependent nif gene activation is only observed under conditions of low 0, tension (Michiels et al. 1994). Recently, we have identified fill]-homologous genes in R. legwninosarwn bv phaseoli (I. D’hooghe unpublished work). The R. leguminosarum bv phuseoli firL] genes are not involved in the activation of nifA transcription, as it is the case in R. meliloti, but control the expression of the nigh genes under free-living microaerobic conditions. The fact that a R. leguminosarwn bv phaseoli jixL] mutant strain is still able to form N,-fixing nodules may suggest that regulatory mechanisms may differ between free-living and symbiotic conditions.

Concluding

Remarks

References Agron, P.G., Ditta, G.S. & Helinski, D.R. 1993 Oxygen regulation of nifA transcription in vitro. Proceedings of the National Academy States of America

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(Received

in revised

form

27]uly

1994;

accepted

3 August

1994)