World Journal of Microbiology and Biotechnology 9, 444-454

Special Topic Review

Control of the expression of bacterial genes involved in symbiotic nitrogen fixation

M. Megias,* J.L. Folch and C. Sousa

Several genera of NJixing bacteria establish symbiotic associations with plants. Among these, the genus Rhizobiurn has the most signiticant contribution, in terms of yield, in many important crop plants. The establishment of the Rhizobiunrlegume symbiosis is a very complex process involving many genes which need to be co-ordinately regulated. In the lirst instance, plant signal molecules, known to be flavonoids, trigger the expression of host-specific genes in the bacterial partner through the action of the regulatory NodD protein. In response to these signak, RGzo6ium bacteria synthesize llpo-oligosaccharide molecules which in turn cause cell differentiation and nodule development. Once the nodule has formed, Rhizobium cells differentiate into bacteroids and another set of genes is activated. These genes, designated nif and fix, are responsible for N, fixation. In this system, several regulatory proteins are involved in a complex manner, the most important being NifA and a two component (FixK and FixL) regulatory system. Our knowledge about the establishment of these symbioses has advanced recently, although there are many questions yet to be solved.

R$ WOT&: Bacteria, expression, genes, nitrogen fixation, symbiotic

Bacteria-plant interactions range from the pathogenic, in which bacteria parasitize a plant, to the symbiotic, in which two organisms help each other to survive under otherwise difficult environmental conditions. Bacteria of the genus Rhizobium are able to interact symbiotically with specific legume host plants, leading to the formation of N,-fixing root nodules. Nodules are specialized organs, within which the rhizobia, differentiated into bacteroids, utilize plant- provided C compounds and reduce atmospheric N, to ammonia for its assimilation by the plant (Long 1989). In return, the bacteria obtain C sources (citric acid cycle intermediates like malate and succinate). As this association is normally of mutual benefit to both the invader (Rhizobium cells) and the invaded (plant host), it is called symbiosis.

The incorporation of atmospheric N, into organic material which results from the Rhizobiurn-legume symbiosis is estimated to account for one third of the total N needed for world agriculture (Economou & Downie 1992). This unique intracellular association contributes significantly towards agricultural yield. The agronomic importance of this symbiotic association has thus justified extensive research

M. Meglas, J. L. Folch and C. Sousa are with the Departamento de Microbiologla y Parasitologia, Univenidad de Sevilla, Apdo 674, 41012 Sevilla, Spain; fax: +34 64 626162. ‘Corresponding author.

@I 7993 Rapid Communications of Oxford Ltd

over the past 100 years and the association is probably one of the best known of all plant-microbe interactions (Economou & Downie 1992; Fischer & Long 1992).

Nitrogen-fixing bacteria are taxonomically diverse and occupy various ecological niches. Some genera in the Rhizobiaceae (Rhizobium, Brudyrhizobitrm and Az.orhizobium) nodulate legumes whereas other members (Agrobacterium) are plant pathogens (Young & Johnston 1989). In an agricultural context, the most important association is that between members of the Leguminosae and Rhizobium, so not surprisingly this has received the most detailed study and will be the subject of this review. Each bacterial species is restricted to its specific group of host plants, thus defining ‘cross-inoculation groups’ in which the bacteria are classified according to their ability to establish symbiosis with a particular group of plants.

Successful nodulation is a host-specific process in the sense that Pisum and Viciu species are host plants for R. leguminosarum biovar (bv.) viciae, Medicago and Melilotus are hosts for R. meliloti, and Trifolium spp. are hosts for R. legtlminosamm bv. trifolii. Members of the genus Rhizobittm are relatively fast growing strains and have a narrow host range. On the other hand, the genus Bradyrhizobium contains a group of slow-growing rhizobia that generally nodulate quite a wide range of host plants.

Regulation of symbiotic nitrogen fixation

al. 1988; Barbour et al. 1991). Chemoattraction by flavonoids has also been reported (Caetano-Anolles et al. 1988; Barbour et al. 1991). The bacteria attach to the root hair surface probably via the specific Cat+-binding protein rhicadhesin and then more bacteria accumulate on the ones which have already adhered, this process being mediated by bacterial cellulose fibrils (Smit et al. 1987). On the other hand, such an attachment may not be necessary for nodulation to proceed, at least in soybeans.

Subsequently, the root hairs curl (Hat) into a so-called ‘shepherd’s crook’ shape (Smit et al. 1987), thus entrapping the bacteria within the curl (Yao & Vincent 1969; Bhuvaneswari et al. 1980), and the plant responds by forming thick and short roots (Tsr) (Van Brussel et al. 1990).

The infection starts with a very localized hydrolysis of fhe plant cell wall in the root hair curl (Ridge & Rolfe 1986). At the site of cell wall hydrolysis, bacteria enter the root hair cell by invagination of the plasma membrane. Around the invaginated membrane, the plant forms a hollow ingrowth which develops into a tubular tunnel-like structure, the infection thread, which penetrates further into the root hair cell. The bacteria multiply along the growing infect-ion thread and ultrastructural studies show them to be surrounded by a matrix-glycoprotein (plant-derived), their own extracellular polysaccharides and a mucigel composed of cell-wall polysaccharides (VandenBosch et al. 1989;

Economou & Downie 1992). The plant proteins that are specifically synthesized during the formation and function of a root nodule are called nodulins. During these early stages of infection, early nodulins (ENOD) are expressed (Nap & Bisseling 1990). Simultaneously with the growth of the infection thread, some cortex cells start to divide, resulting in a nodule primordium. The infection threads carry

In this symbiosis an exchange of signals between the host plant which releases flavonoids, and the bacterium, which releases so-called Nod metabolites, plays a crucial role. Lately, the Rhizobium-legume interaction has received much attention because of the finding of a small lipo- oligosaccharide molecule (Figure 1 and Table 1) which alone, at a very low concentration (lo-l1 M), causes nodule development in alfalfa plants (Lerouge et al. 1990). The molecule is synthesized in response to flavonoid compounds exuded by the root.

The development of symbiosis fakes place in several stages, confrolled by genes in the bacterium as well as in the plant. Molecular, genetic and cell biological studies revealed three major, somewhat independent series of events: (1) bacterial attachment to the root hairs, followed by root hair deformation and curling; (2) formation and growth of infection threads in which the bacteria multiply; and (3) induction of meristematic activity in the root cortex, leading to nodule development which culminates in N,-fixing symbiosis.

The sequence of events leading to a nodule starts with chemotactic attraction. Amino acids and dicarboxylic acids are probably the most important chemoattractants under field conditions (Giitz et al. 1982; Caetano-Anolles et

Figure 1. Basic structure of Nod factors of Rhizobium strains.

Table 1. Structures of Nod factors produced by Rhizobium strains.

Rhizobium strain Substitutions* References

4

R. me/i/o0 2011 C16:2

R. leguminosarum bv. C18:4 or C16:l viciae RBL5560

R. melilofi AK41 C16:3

Rhizobium sp. NGR234

C18: 1 or C16:O

6. japonicum USDA110 C18:1, C16:O or C16:l and USDA135

A. caulinodans C18:O or C18: 1 ORS571

R2 R3 X Y

-H or -CO-CH, -SO,H -H -H

-CO-CH, -H -H -H

-H --SOaH -H -H

-CO-CH, 2-CTMethylfucose, -CONH? -CH, 3-Gsulphated or CO-acetylated

-H or -CO-CH, P-O-Methylfucose -H -H or -H

-CONH, or -H o-Arabinose or -H -H -CH,

n

2, 3 Lerouge et al. (1990)

2, 3 Spaink et a/. (1992)

1,2, 3 Schultze et al. (1992)

3 Demont et a/. (1992)

3, 4 Sanjuan et a/. (1992)

2, 3 Mergaert et al. (1992)

l &-Fatty acyl residue: vaccenic acid (C18:1), stearic acid (C18:0), palmitic acid (C16:O); R, and Rx-hydrogen or a different substitution; X-hydrogen or carbonyl residue; Y-hydrogen or Kmethyl residue; n-number of glucosamine units.

World Journal of Microbiology and Biotechndo~y, Vol 9, 1993 445

M. Meg&, JL. Folch and C. Sousa

the bacteria from the root hair to the nodule primordia formed in the root cortex (Dudley et al. 1987).

The bacteria are released from the tips of the infection threads into the host cytoplasm by a process that resembles endocytosis. After their release into the cytoplasm of the plant cell, rhizobia differentiate into bacteroids. The bacteroids lie alone or together, surrounded by a plant membrane derived from the Golgi and endoplasmic reticulum, this membrane is termed the peri-bacteroid membrane (PBM). The bacteroids are pleirnorphic and the fate of the PBM varies from legume to legume. In the case of the pea, the PBM has a volume approximately T-fold that of free-living bacteria. This membrane forms an interface of key importance, essential for the transfer of metabolites between the plant and bacteria. The mature bacteroids are now capable of using nitrogenase to reduce N,, so fixing N in the form of ammonia. Around the start of N, fixation, plant genes encoding so-called late nodulins are expressed. The best known and the most abundant of these is leghaemoglobin (Lb). Lb is an oxihaemoprotein which is localized in the peribacteroid space, with a high 0, affinity, and which resembles the vertebrate globins. Lb controls the concentration of free 0, in the nodule and forms a filter protecting the very O,-sensitive nitrogenase (Nap & Bisseling 1990).

Bacterial genes involved in the nodulation process are: (I) the nod genes (genes involved in host recognition and nodule formation) and also nol genes (for nodulation related); (2) ezo genes, encoding exopolysaccharides, and lipopolysaccharide-encoding genes; and (3) fiz and nif genes, involved in carrying out and supporting N, fixation.

Nodulation Genes

Nodulation genes in Rhizobium are involved in the early steps of the nodulation process. These genes are regulated

by plant-derived signal molecules called flavonoids, through their interaction with the nodD gene product (Rossen et al. 1985; Djordjevic et al. 1987; Horvath et al. 1987).

In Rhizobitrm species, the majority of genes for nodulation and nitrogen fixation are located on high molecular weight Sym (for symbiotic) plasmids aohnson et al. 1978; Nuti et

al. 1979). Other symbiotic loci map on the chromosome (Dylan et al. 1986) or in other plasmids (Toro & Olivares 1986). In contrast, in Bradyrhizobium and Azorhizobittm species, the nod genes are correlated with the chromosome (Van Den Eede et al. 1987).

The organization of nod and no1 genes in several species and biovars is shown in Figure 2. Nodulation genes are involved in the synthesis and transport of small lipo-oligosaccharide molecules which are recognized in a specific manner by the host plant and which by themselves start the organogenesis of the nodule (Lerouge et al. 1990; Spaink et al. 1991, 1992) (Figure 1 and Table 1). In some cases, complete nodule development can be achieved simply by adding these purified factors to the roots of uninoculated alfalfa plants.

The nodABCIj genes were designated as ‘common’ because they can be complemented by the corresponding genes from other strains or species. All the other nod genes, which, unlike the common nod genes, are not’ found in all strains and species and cannot be complemented by nod genes from other species, were designated host-specific nodulation genes (hsn) (Kondorosi et al. 1984). Table 2 presents a summary of the roles that many of the nod genes have.

Regulation of the nodI3 Gene

The nod genes are arranged in several different transcriptional units and these operons are co-ordinately regulated by the nodD product and specific plant signal

Rhizobium melilori

0 TNM. L EF. 0 ASCIJ -ml-n- .,,,,I, .m

+ -d--c-

Rhizobium leguminosarum bv. viciee

X NM, LREF 0 A8CIJ T I I I I II I, ,,.(gn.r I I I I ,I I - -cIcp-

Rhizobium legominosarum bv. trifolii

L Bradyrhizobium japonicum IWIA &I$, 01 rA 6 csu I JNNO Z v w

I I I II I I - C +-L---C----L ---t” -

Figure 2. Genetic organization of the nod and nol genes of R. meliloti, R. leguminosarum bv. viciae, R. leguminosarum bv. trifolii and Bradyrbizobium japonicum. Arrows indicate orientation of nod and no/ gene expression. l Position of the nod boxes; 0 nod genes; n no/ genes; q transcriptional regulatory nodD and syrM genes.

446 World ]oumd of Microbiology and Biotechnology. VOI 9, 1993

Regulation of symbiotic nitrogen fixation

Table 2. Predlcted biochemical function of nod and no/ genes.

nodF””

nodG” nodH”’

nodLmvf

nodM”* nodNmVt nodO’

nodPOmP”

nodT* nods@ no/R”

Predicted function

Proposed to determine the production of Nod factor precursor

Polymerizes the oligosaccharide backbone Transcriptional activator of inducible nod genes Proposed to synthesize Nod factor acyl chain

Proposed to synthetize Nod factor acyl chain

Involved to modify Nod factor fatty acyl side chain Proposed to attach activated sulphate to the

Nacetylglucosamine polymer Proposed to be involved in the excretion of the

signalling molecule Proposed to add an acetyl group to Nod factor

Proposed to synthesize Nod factor sugar subunits Involved in Vicia hirsu~a nodulation Proposed to intern the lipo-oligosaccharide into the

plant cell They could function in the production of an activated

form of sulphate for transfer to Nod factor Proposed to be membrane protein Proposed to add a methyl group to Nod factor Repressor of nodD

Reference

Schmidt et al. (1988), Vargas et a/. (1990), VIzquez et a/. (1991)

Bulawa 8 Wasco (1991), Rodriquez-Quifiones .et a/. (1989) Schlaman et a/. (1992) D&bell& & Sharma (1986). Bibb et a/. (1989),

Spaink et a/. (198ga), Vargas et al. (1990) Shearman et a/. (1986), Sheldon et a/. (1990),

Geiger et al. (1992) Spaink et al. (1991) Schwedock & Long (1990)

Evans & Downie (1986), VAzquez et a/. (1992)

Downie (1989), Bloemberg et a/. (1992), Baev & Kondorosi (1992)

Baev et a/. (1991) Fisher & Long (1992) Downie et a/. (1992)

DBbell& 8. Sharma (1986), Cervantes et a/. (1989), Schwedock & Long, (1990), Folch ef a/. (1992)

Canter-Cremers et a/. (1989) Holsters et al. (1992), Geelen et a/. (1992) Schlaman et al. (1992)

* nod genes are present in R. melilofi (“), R. leguminosarum bv. viciae (‘), R. leguminosarum bv. frifolii (‘), R./eguminosarum bv. phaseoli (‘). R. fropici (“), R. fredii (‘), 6. japonicum (I) and A. caulinodans (“). nodY and nodZ are found in 6. japonicum, nodX has only been found in R. leguminosarum bv. viciae strain TOM. Other genes, including nodK, nodU, no/A, no/E, no/F, no/G, no/H, no// and no/P, have been identified but their possible functions have not been described.

molecules. The nodD gene has been detected in all rhizobia tested so far. In some Rhizobium strains nodD is present as a single copy whereas in others, multiple (two or three) different alleles were found (Rodriguez-Quiiiones et al. 1987). The NodD protein is a transcriptional activator of the nod genes and has different specificity for flavonoids depending on the Rhizobium species from which it comes. This protein shows homology with the LysR family of regulatory proteins. On its amino terminal region there is a sequence which resembles a DNA-binding domain and also a so-called receiver module domain which could allow protein-protein interactions. The carboxy terminus, which varies more than the amino terminus in different species, has been reported to be involved in flavonoid recognition. There is evidence, however, which suggests that this protein does not have two separate functional domains, but rather that the whole tertiary structure is involved in nod gene activation (Spaink et al. 1989b; Sousa et al. 1993).

Transcription of the nodD gene is usually constitutive, but several factors, including combined N, plant signal compounds and regulatory proteins [e.g. the nod repressor protein (NolR), the syrA4 gene product and NodD itself], can regulate its expression (Figure 3) (Rossen et al. 1985;

Horvath et al. 1987; Banfalvi et al. 1988; Dusha et al. 1989;

Kondorosi et al. 1989; Davis & Johnston 1990; Maillet et al. 1990). In this respect, we still know little about the complex regulatory system that controls nodD expression. For example, in R. meliloti groEL mutants (a molecular chaperon) the expression of nodD3 is prevented and that of the nodD1 and nodD2 is reduced when they are expressed constitutively (Fischer & Long 1992).

Most of the different nod operons have a 50 bp (Schofield & Watson 1986) conserved sequence upstream of the promoter region which is the nod-box and to which the nodD protein binds in order to activate transcription. It has been shown by Long (1992) that the nodD protein binds only to one side of the double helix on the nod boxes and causes DNA to bend, resembling the action of the IHF regulatory protein found in other bacterial species (Kustu et al. 1992). It is still not clear whether the NodD protein acts as a monomer or as a multimer in order to activate transcription, although there is some evidence that indicates it could interact with other proteins or with nodD itself to do so (Long 1992). In this respect, a characteristic structure has been found in all nod boxes studied which favours the hypothesis that NodD binds as a tetramer to the nod-box. Other studies involving nod-box deletion mutants also support this model.

World Journal of Microbiology and Bmtechndogy, Vol 9, 1993 447

- Nod factors -

~I”\ ,erJ independent

Figure 3. Cascade regulation of nod gene expression. Arrows indicate regulatory routes. +-Positive controls; controls (Fisher & Long 1992).

NodD protein activity as a tr~s~ptioM1 activator of the nod regulon depends on the protein’s interaction with flavonoids, although it has not been proven that these molecules bind directly to the NodD proteins (Firmin et nl. 1986; Redmond et al. 1986; Bassam et al. 1988; Peters & Long 1988; Zaat et af. 1989; Davis & Johnston 1990). As distinct plant species release different sets of signal molecules (flavones, isoflavones, flavanones and chalcones), NodD proteins can only activate transcription when the right host plant is present, so determining the host specificity (Gyargypal ef al. 1988; Spaink et af. 1989b). Other factors, such as combined nitrogen, can also influence nod gene expression, thus regulating the Rhizobitrtn- legume interaction (Dusha et al. 1989).

Nitrogen Fixation Genes

The genetic basis of symbiotic N, fixation in the Rhiiobiaceae has been the objective of extensive research. The ability of rhizobia to induce N,-fixing nodules requires the specific expression of certain bacterial genes. Those genes homologous to N, fixation genes in Klebsiella peumoniae are referred to as nifgenes. Others, also essential for symbiotic N, fixation but sharing no homology to PC ~~~~iae, are called f;x genes. The presence of these /ix genes correlates with the specific environmental conditions in which symbiotic nitrogen-fixing microorganisms act. This suggests that N, fixation within a root nodule requires a regulatory track different from that in K. ~eumuniae.

The structure and the biochemical properties of the nitrogenase complex are highly conserved, though a marked characteristic of diazotrophs is that they differ in the physiological conditions under which they fix N,. Thus, K. pn~~niae is an enteric bacterium able to use atmospheric N, for growth in the absence of 0, and combined N. On

--negative

the other hand, bacteria of the genus ~~jz~b~u~ fix N, only in symbiotic conditions.

Studies on N, fixation in Rhizobium and Bradyrhizobittm species were facilitated by the previous identification and characterization of 21 contigous nif genes in I( pneumoniae (De Bruijn et al. 1990). These genes are grouped on the chromosome or pSym in Rhizobictm and are organized in different transcriptional units. nif genes of Rhizobitrm which are homologous to those of Klebsiella include nifHDK, the structural genes that encode for the polypeptide subunits of the nitrogenase complex; nifA, the product of which is a positive transcriptional activator for nifHDK and other nif

and fix genes (Ruvkun & Ausubel 1980; Szeto ef al. 1984); nip, nifnr and nifE, genes are involved in the synthesis and processing of the nitrogenase iron-molybdenum cofactor CFeMocof (Norel et al. 1984; Hennecke et al. 1985); and nifF (Hontelez et af. 1984) and nifl (Norel et al. 1984), are genes which encode for nitrogenase-specific electron transport proteins.

Divergent to nigh and upstream of nifA in the R. meli~~fi megaplasmid (pSym1) are the MBCX genes which are transcribed as a single operon from firA to fix (Better et al. 1985; Earl et al. 1987). The fixABCX genes do not exhibit homology to Klebsialla nif genes, and their products may be involved in nitrogenase-specific electron transport. The R. ~elilofj m promoter contains the characteristic nif consensus promoter sequence (Better et a?. 1985) which indicates that it is probably recognized by the product of ntrA and is positively controlled. The f;xX gene is highly homologous to ferredoxin found in other nitrogen-fixing species such as ~ofobacfer vine~an~i~ (Buck & Cannon 1987).

A second fir gene cluster of R. melilofi constitutes the four operons fixL], firK, fixNOQP and fixGH1.S (David et al. 1988;

Batut et al. 1989; Kahn et al. 1989); firLJ and fi& are regulatory genes. fiGHIS and ~x~OQP gene products are thought to be membrane located. FixG has homology with

Regulation of symbiotic nitrogen fixation

nifHDKE I I- NifA --I

k -------) +------ -w+ +

k k fixABCX nifA I

nifBQ ’ Other niflfix genes l -o- o-o-

4\ FixL

t\ Y2

Periplasmic space

Inner membrane

Cytoplasmic space

o fixGH$J

FixJ

Figure 4. The organization of the nif and fixclusters and a model of nif regulation in R. meliloti. Solid arrows are used to indicate the individual transcription units. 0, 0 and 0-Promoters activated by NifAINtrA, FixJ and FixK, respectively. Top lines denote regulatory routes. +-Positive control (gene activation); + ?-the fixGHS/ promoter has a potential FixK binding site but it has ._ not been shown that its activation is FixK-mediated.

bacterial ferredoxins and Fix1 is likely to be the catalytic subunit of a cation pump required specifically for symbiotic N, fixation (Kahn et al. 1989). FixP has homologies with c-cytochromes. f;N expression is induced under free-living microaerobic conditions and symbiotic conditions (Batut et al. 1989) (Figure 4).

In K. pneumoniae, the expression of nif genes is regulated at two distinct levels (Buck & Cannon 1987). The first level involves a centralized nitrogen regulation system (the ntr system). The second level involves the specific control of nif operons by the nifA and nifL. products. The synthesis, processing, and activity of the nitrogenase complex are controlled by NifA, a transcriptional activator, and nip, which decreases NifA activity in the presence of oxygen and fixed N,. Expression of the nifLA operon itself is controlled by the level of N by a two-component regulatory system, NtrB/NtrC. At high ratios of a-ketoglutarate to glutamine, the NtrB protein phosphorylates NtrC, which then activates the transcription of the nifL4 operon (Gussin et al. 1986).

NifA belongs to a group of bacterial regulatory proteins which activate transcription from promoters requiring the alternative sigma factor NtrA (RpoN) (Gussin et al. 1986). In addition to nifA, which activates structural genes required for N, fixation, members of this group include NtrC, which activates genes required for N assimilation, and DctD, which activates the C,-dicarboxylate transport in Rhizobium (Ronson et al. 1987). These three regulators share structural

features, including a strongly conserved block of 238 aminoacids in the central region and a less well conserved block of 45 aminoacids at the C-terminal end that contains a helix-turn-helix DNA-binding motif (Drummond et al.

1986). An upstream activating sequence (UAS) with the consensus sequence 5’-TGTCG-N,-ACACA-3’ is present 90 to 150 bp upstream of the NtrA consensus promoters activated by NifA (Gussin et al. 1986). For NifA, the requirement for a UAS associated with the target promoter varies between species of N,-fixing bacteria. For K. pneumoniae nif promoters, the requirement for a UAS is strong, whereas for the R. meliloti nigh promoter the requirement is weak in symbiosis (Better et al. 1985).

The R. melilofi nifA protein activates the symbiotic transcription of a set of genes containing their corre- spondent nif promoter in free-living N,-fixing bacteria (Szeto et al. 1984; Gubler & Hennecke 1986). Under conditions of N limitation, in free-living R. meliloti, the transcription of the nigh and @A promoters could be induced by NtrC (Szeto et al. 1987). However, ntrC seems

to play no significant function in nodules formed by R. meliloti (Szeto et al. x987), whereas nifA is essential for nif gene symbiotic activation (Ditta et al. 1987). In asymbiotic conditions, nifA expression is 0, regulated and can be induced in microaerobiosis (Virts et al. 1988). Furthermore, R. melilofi NifA’s function as a transcriptional activator is inactivated by 0, (Huala & Ausubel 1989).

A R. meliloti gene that is also expressed under both

M. Megias, J.L. Folch and C. Sousa

microaerobic and symbiotic conditions, fixK, represses nifA transcription and is a positive regulator of the jixN operon (Batut et al. 1989). firK is homologous to the E. coli regulatory gene frzr which controls genes expressed under anaerobic conditions. Homologues of the R. meliloti JixK gene have been identified in R. leguminosarum bv. viciae (Colonna-Roman0 et al. 1990), A. caulinodans (De Bruijn el al. 1990) and B. japonicum (Anthamatten & Hennecke 1991). However, unlike these homologues, R. meliloti has FixK which lacks the N-terminal sequence which is thought to be responsible for the 0, sensitivity of other Fnr-like proteins (Batut et al. 1989).

The symbiotic and microaerobic expression of firK and n&A in R. meliloti is positively controlled by a pair of regulatory proteins, FixL and FixJ (David et al. 1988; De Philip et al. 1990). FixJ, the response regulator, is activated by FixL, the 0, sensor, in response to conditions of low 0, tension and then activates expression of nif and fix genes. Sequence data have led to the prediction that FixL and FixJ belong to a family of two-component regulatory systems [see review by Stock et al. (1989)]. FixL is an oxygen-binding haemoprotein and senses oxygen through its haem moiety, which is probably contained between residues 86 and 219 of the polypeptide (Gilles-Gonzhlez et al. 1991). This part of the R. meliloti FixL polypeptide is very well conserved in A. caulinodans (Kaminski & Elmerich 1991) and in B. japonicum (Anthamatten & Hennecke 1991). In addition, in its N-terminus, FixL has two sequences which represent potential transmembrane helices as found in many sensor components of two-component regulatory systems. Look- ing at the properties of most members of the two- component systems (Ronson et al. 1987; Albright et al.

1989), FixL could be proposed as a transmembrane sensor protein, which reacts to a change in the bacterial environment by phosphorylating the cognate activator protein FixJ, which in turn promotes the transcription of nifA and fixK genes. A model of the regulation of N, fixation in R. meliloti is presented in Figure 4. FixJ shows similarity to the N terminus of the regulator class components over the first 120 aminoacids (David et al. 1988; Albright et al.

1989). In E. coli, in an heterologous system, fixK requires higher

levels of the activator FixJ than nifA for activation of transcription. However, the biological significance, if any, of the differential expression of nifA and fixK remains to be elucidated (De Philip et aI. 1990). In vitro transcription studies of fixK promoter showed that it was dependent on 07’ holoenzyme. Therefore, the activity of R. meliloti nifA promoter is also probably 0”-dependent (Batut et al. 1991).

Oxygen tension seems to be the main signal for nitrogen fixation gene expression within the nodule. However, there are indications that other signals are needed for effective symbiotic gene expression. Thus R. meliloti jixA promoter is weakly activated in microaerobiosis but strongly in

symbiosis (Ditta et al. 1988; A. Cebolla, personal communication). Although induction of nigh expression in microaerobiosis is strongly dependent on UAS, this sequence is not necessary to achieve high levels of expression during symbiosis (Ditta et al. 1988). No activation of nif gene expression was found, either in free-living microaerobic or symbiotic R. meliloti, when a gene involved in the transcriptional activation of the C,-dicarboxylate transport gene was mutated (Birkenhead et al. 1990). On the other hand, in free-living R. meliloti, nifA expression is sensitive to fixed N, levels but not fixK (Nooman et al. 1992). Therefore the expression of structural genes for nitrogen fixation in R. meliloti may then be controlled through other physiological signals in addition to the oxygen status.

In summary, many signals are involved in the expression of the bacterial genes required for the establishment of a mature nodule. Some of them have been elucidated, but others remain to be found. Once all the signals have been discovered a more integrated view of the complex phenomenon of Rhizobium-legume symbiosis will be possible.

Acknowledgements

The authors are grateful to A. Cebolla for critically reading the manuscript. This work was supported, in part, by grants from DGICYT, Spain (BIO 90-S20-c02-01). JLF and CS were supported by fellowships from Ministerio de Asuntos Exteriores, Spain, and Consejeria de Educaci6n y Ciencia, Junta de Andalucia, Spain, respectively.

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transduction mediated by sensor and regulatory protein pairs. Annual Reviezo of Genetics 23, 311-336.

Anthamatten, D. & Hennecke, H. 1991 The regulatory status of the /i.rL- and jixJ-like genes in Bradyrhizobium juponicum may be different from that in Rhizobium melilofi. Molecular and General Genetics 225, 38-48.

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