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International Institute of Genetics and Biophysics, Consiglio Nazionale delle Ricerche, 80125 Naples, Italy
SUMMARY INTRODUCTION Rhizobium-Plant Interaction Root Nodule Purpose of This Review NH4+ AS A REGULATORY SIGNAL IN SYMBIOSIS Action on Early Steps of Symbiosis Positive and Negative Regulator of Nodule Development Action on Symbiosomes NH4+ METABOLISM NH4+ Uptake Nitrogen Fixation Internal Production from Nitrate and Amino Acids Assimilation NH4+ Efflux REGULATION OF NH4+ ASSIMILATION UNDER FREE-LIVING CONDITIONS ntr Regulatory System PII Regulatory Protein REGULATION OF NH4+ ASSIMILATION IN DIFFERENTIATING SYMBIOSOMES In Situ Detection Expression of Bacterial Genes ntr System N Sources Global Regulation DOWNREGULATION OF NH4+ ASSIMILATION IS REQUIRED FOR EFFICIENT SYMBIOSIS CONCLUDING REMARKS REFERENCES
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| INTRODUCTION |
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Once in the cytoplasm of plant cells, BTs divide a few times, arrest division, and differentiate morphologically and physiologically into a form able to reduce atmospheric N2 and release the fixed N to the plant (for reviews, see references 19, 124, and 197). The organelle-like structure formed by the BT, together with the peribacteroid space and the surrounding plant membrane, is called the symbiosome (SB). After the onset of N2 fixation, SBs continue to differentiate and eventually proceed towards lysis. It was recently shown that, in the symbiosis between Sinorhizobium meliloti and Medicago sativa, undifferentiated bacteria remaining in intracellular ramifications of the ITs infect the cytoplasm of senescent cells growing as free-living bacteria (186). This process occurs after the SBs are degraded. This observation may explain the benefit of the bacterial partner in the symbiosis.
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Nodule organogenesis, such as the formation of other plant organs (flowers, leaves, roots), proceeds through many temporally and spatially regulated developmental steps. It is induced by rhizobia, but the development of a mature and functional nodule requires the exchange of several signal molecules and metabolites between the prokaryotic and eukaryotic partners. Nodule organogenesis is studied with the help of bacterial and plant mutants, which reveal specific patterns of differentiation, and with the in situ study of molecular markers, which in this case are not only eukaryotic, but also prokaryotic. The combined efforts of physiologists, embryologists, molecular biologists, and cytologists are needed to make progress in this field.
The purpose of this review is to show that more detailed studies have revealed a very fine regulation of bacterial NH4+ metabolism during the establishment of the symbiosis. Downregulation of structural and regulatory genes involved in NH4+ assimilation is a key and necessary step for the differentiation of efficient SBs and thus for the development of effective nodules. During SB differentiation, a conserved molecular mechanism acts selectively to switch off gene transcription and to remove the corresponding proteins, either before or when N2 fixation is turned on.
| NH4+ AS A REGULATORY SIGNAL IN SYMBIOSIS |
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NH4NO3 is known to be a potent inhibitor of root hair deformation, initial cortical cell division, and IT formation, but its effect on the Nod factor perception has been excluded (70). The presence of NH4+ in the medium reduces the ability of Sinorhizobium meliloti to produce exopolysaccharides, which play a critical role in the process of nodule invasion (44). Although bacterial lipopolysaccharides are necessary for competitiveness (93) and nodule invasion (23, 203), no reports on the regulation of their structure and synthesis by NH4+ have been reported. Their structure is modified in response to the pH of the medium (175), and the latter is strongly influenced by the N source. Thus, differences in N metabolism may indirectly provoke the structural alterations of the lipopolysaccharides observed in bacteria growing inside the ITs (164). The addition of N compounds, including NH4+, lowers the expression and/or the activity of some nodulins, plant gene products that are either specifically expressed or highly induced in the nodule (27; for a review, see reference 156).
NH4+ produced through the action of the nitrogenase complex appears to be required as a positive signal necessary for normal development of indeterminate nodules (5, 199). Nodules induced by S. meliloti mutants unable to reduce N2 because of a mutation in one of the structural genes for nitrogenase (nifHDK), and thus unable to produce NH4+, show normal early organogenesis, but later on the N2-fixing zone (zone III) of the nodule is not formed, the apical meristem is inactivated, and early nodule senescence is observed (72, 73, 199). Therefore, completion of nodule development and sustained action of the meristem require nitrogenase activity. The regulatory signal for completion of nodule development may be either the NH4+ released by SBs or some other metabolic changes resulting from nitrogenase activity, such as enhanced consumption of O2 and/or reducing power.
The presence of normal Fix+ nodules inhibits the induction of further nodules on the root. In fact, plants inoculated with Fix- rhizobia show a greater number of nodules than in normal nodulation due to reinfections taking place on secondary roots (72). In conclusion, NH4+ produced in the nodule appears to act locally as a positive regulatory signal for nodule and SB maintenance, whereas it acts systemically as a negative signal inhibiting the reinfection process. Moreover, NH4+ produced in the nodule may repress flavonoid and isoflavonoid production (the latter compounds being involved in the defense reaction [for a review, see reference 16]). In fact, the chs (chalcone synthase) gene, involved in flavonoids and isoflavonoid biosynthesis, is induced in mature and invaded (18 days postinoculation) nodules elicited by a Fix- mutant of S. meliloti and not in nodules elicited by the wild-type strain (67). In nodules elicited by an S. meliloti strain mutated in the nifA regulatory gene, the SBs degenerate at an earlier stage of development than in nodules formed by a nifH mutant (73), indicating that NifA is involved in the expression of bacterial functions that are required for SB viability, in addition to those involving N2 fixation.
A different behavior is observed in the case of determinate nodules. When G. max and P. vulgaris are inoculated with a nifH mutant strain and Vigna unguiculata is nodulated in the absence of N2 (replaced by argon), the resulting nodules are similar (in shape, dimension, and histology) to normal nodules (8, 25, 58). This suggests that NH4+ is not a signal for nodule organogenesis and that younger BTs release NH4+ by a mechanism not involving nitrogenase action, such as the utilization of amino acids as a C source (see below). Instead, if these plants are infected with a nifA mutant strain, SBs are degraded at an early stage (25, 58, 209). This indicates that the nifA regulatory gene, as in the case of elongated nodules, is required for SB maintenance, while nifH, and thus nitrogenase activity, is not (60, 129).
SBs of elongated nodules during nodule development maintain a single BT, which increases up to 30-fold in size and shows polar swellings and Y shapes (199). Moreover, BTs undergo significant changes in DNA content and in the ratio of chromosomal to symbiotic plasmid DNA (14, 202, 210). The morphological changes of SBs become evident after the arrest of BT divisions occurring in the middle of zone II (Fig. 1) and thus before the onset of nitrogenase activity. The same shape modifications are observed in nodules elicited by Fix- strains and when free-living bacteria are treated with cell cycle inhibitors (94) and C4-dicarboxylic acids (193), excluding the involvement of NH4+ in this process. However, the appearance of C4-dicarboxylic acids may provoke, at the corresponding stage of differentiation, severe changes in N metabolism, such as the inhibition of the utilization of amino acids as a C source and thus of NH4+ excretion (see below). Furthermore, the completion of SB differentiation takes place after nitrogenase activation (199).
Globose nodules develop SBs formed by multiple rod-shaped BTs, which do not increase in size (25, 34). The number of BTs per SB appears to increase more by fusion of younger SBs than by BT division inside a single membrane vesicle (25, 55, 127). Moreover, the merging of SBs proceeds through the fusion of their membranes. It increases during nodule senescence, when BT division stops, and appears to be related to the N status of the cell, since it is also accelerated when the plant is infected with Fix- mutants (25). Lack of nitrogenase activity, and therefore of NH4+ production, might cause a change in pH at the cytoplasmic surface of SBs, thus activating a homotypic mechanism of membrane fusion that may involve SNARE proteins (49).
| NH4+ METABOLISM |
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The amtB gene from Azorhizobium caulinodans and Rhizobium etli has been shown to be required for high-affinity NH4+ transport (117, 179). This gene codes for an integral membrane protein highly conserved in many organisms (184, 185) and it is cotranscribed with the glnK gene (coding for a PII-like protein), forming a glnK-amtB operon. This operon is expressed from a promoter located upstream of glnK, whereas no promoter activity was detected in the intergenic region (179). The glnK promoter becomes inactive very early during symbiosis, and in correlation isolated BTs do not show high-affinity NH4+ uptake activity (92, 134, 179, 180). Downregulation of amtB expression is essential for the normal function of BTs (see below).
-proteobacteria (for a review, see reference 6), has not been identified in rhizobia. In free-living diazotrophs such as Klebsiella pneumoniae, the NH4+ produced through the action of nitrogenase is assimilated for bacterial growth. The nitrogenase complex may be induced in free-living rhizobia but, with the exception of A. caulinodans and Rhizobium trifolii strain 0403 (40, 194), most of the NH4+ produced is excreted (132, 194). Therefore, in rhizobia, N2 fixation and assimilation of the NH4+ produced are uncoupled, and this is essential for an effective symbiotic interaction. In fact, plants nodulated by strains unable to express an active nitrogenase complex and unable to release the NH4+ produced show signs of N deprivation, which are relieved by exogenously added combined N (5, 72, 73). However, it has recently been reported that an ntrR mutant strain of S. meliloti induces nodules with an enhanced N2 fixation capacity due to the higher expression of nif genes (135). Moreover, this phenotype is not abolished by the exogenous addition of N, suggesting that in S. meliloti the expression of the nif genes is also N regulated during the symbiosis.
The symbiosis may be viewed as an exchange of photosynthate (in the form of C4-dicarboxylic acids) for fixed N, produced by the SBs (80a, 147, 192a). During SB differentiation, BTs of globose nodules accumulate polyhydroxybutyrate and glycogen granules, perhaps because their capacity to fix N2 decreases while C supply is still plentiful. In fact, ineffective nodules of P. vulgaris induced by a Fix- R. etli mutant strain show an earlier and higher accumulation of polyhydroxybutyrate compared to effective nodules (25). On the other hand, nodules induced by a mutant strain blocked in polyhydroxybutyrate biosynthesis show a higher and prolonged nitrogenase activity compared to nodules induced by the wild-type strain, indicating that nitrogenase and polyhydroxybutyrate biosyntheses compete for reducing power (26). Rhizobium tropici mutants unable to synthesize glycogen also show a better symbiotic performance (107a).
NH4+ is also produced from amino acids, which, like NO3-, behave as poor N sources in the presence of a rich C source. Unlike enteric bacteria, free-living rhizobia (and Pseudomonas aeruginosa) can use a single amino acid (such as glutamine, asparagine, or histidine) as the sole source of C and energy (99). Some mammalian cell lines also use glutamine as the principal energy source in the presence of hexoses (for a review, see reference 151). Amino acid utilization requires active uptake, deamination/deamidation, common metabolic pathways, such as the citric acid cycle and gluconeogenesis, and NH4+ excretion.
R. etli mutants showing very low levels of glutaminase, asparaginase, and aspartase activity are Nod+/Fix+ (46, 47, 50, 79). Enzymes involved in gluconeogenesis are expressed in BTs of R. leguminosarum, S. meliloti, and Rhizobium sp. strain NGR234 (57, 111, 137), and R. etli mutants impaired in this pathway induce only a few nodule primordia (R. Tatè, unpublished). Mutant strains altered in sugar catabolism are Nod+/Fix+ (for a review, see reference 204a). In the utilization of glutamine and histidine as a C source, glutamate is produced, which, for further catabolism, requires a dissimilatory glutamate dehydrogenase (GDH), distinct from the assimilatory one (99). Rhizobial mutants lacking this enzyme have not been described, but some mutants altered in glutamate catabolism show an altered symbiotic phenotype (see below). However, since aap (general amino acid permease) mutant strains of R. leguminosarum are unable to grow with glutamate as a carbon source but are Nod+/Fix+, it has been concluded that glutamate is not an important carbon and energy source in BTs of R. leguminosarum (204).
GSI, encoded by the glnA gene, is a dodecameric enzyme similar to the GS present in enteric bacteria. The intracellular concentration of GSI increases two- to fourfold when R. leguminosarum is grown on a poor N source compared to a rich one, while its activity is regulated by reversible adenylylation (153). When R. leguminosarum is switched from N-rich to N-poor conditions, GSI activity is essential to allow an efficient rate of GSII derepression (183). This suggests that under these conditions, GSI is required for a prompt synthesis of glutamine to be used for protein synthesis. Regulation of GSI concentration and activity (adenylylation) is similar in other rhizobial species (for a review, see reference 116).
GSII, encoded by the glnII gene, is a heat-sensitive (relative to GSI) octameric enzyme (103). The expression of the glnII gene is activated under N deficiency (for a review, see reference 54) and requires an upstream activating sequence (140). When R. leguminosarum is switched to an NH4+-containing medium, glnII transcription is 10-fold repressed, but GSII activity and the GSII protein become undetectable (103, 140). That this is due to a posttranscriptional control mechanism of glnII expression has recently been demonstrated (172, 183). The inhibition of glnII mRNA translation is mediated by the product of the gstI gene, located upstream and transcribed divergently of glnII. Under N-excess conditions, glnII transcription is low and gstI transcription is high, while the opposite is found under N-limiting conditions (Fig. 3, right panel).
The gstI gene codes for a small protein (63 amino acids) that inhibits translation of glnII mRNA, probably through direct binding to the 5' untranslated region. In fact, in silico remote modeling predicts that the GstI protein may fold to match a domain of the Escherichia coli glutaminyl-tRNA synthetase. On the other hand, the 5' untranslated region of glnII mRNA may adopt a secondary structure involving two loops, both of which contain the UUG anticodon sequence for glutamine (172, 183). The reason for the presence of such a sophisticated regulation of glnII expression is unknown. It is possible that GstI not only inhibits translation but at the same time stabilizes glnII mRNA for prompt expression when the intracellular concentration of glutamine becomes limiting. Under microaerobic free-living conditions as well as in isolated BTs of B. japonicum, glnII mRNA but neither GSII activity nor GSII protein was detected, and thus a posttranscriptional mechanism of regulation of glnII expression has been hypothesized (108). However, no evidence for the presence of the gstI gene in this bacterium has been reported. GSI but not GSII activity was detected in isolated BTs of R. etli (122).
Very low levels of GS activity are found in isolated N2-fixing SBs, and genetic manipulation indicates that none of the GS isozymes is necessary for effective symbiosis (37, 54, 116, 192). However, a glnA glnII double mutant of S. meliloti induces normal invaded nodules (37), and GSIII activity was detected (162). A glnA glnII mutant of B. japonicum is severely affected in nodulation (22), and GSIII is absent in this species. These results apparently lead to contradictory conclusions that may be clarified if the symbiotic interaction is considered as a multistage process. In fact, while the ability of rhizobia to invade developing nodules may be dependent on the capacity to synthesize glutamine, GS activity appears to be dispensable for BT function.
Assimilatory GDH activity (Fig. 3, left panel) is absent in R. leguminosarum, and consequently this bacterium grows better on glutamine than on NH4+ as the sole N source (153). It is also absent in other rhizobial species (18, 76, 100, 114). This suggests that assimilatory GDH activity is not needed for efficient nodulation and function. In contrast, it has been shown that the expression of the E. coli gdhA gene, coding for an assimilatory GDH, reduces nodulation ability when expressed in R. etli, whereas if expressed in N2-fixing BTs, it has detrimental effects on nodule efficiency (see below).
GOGAT activity is present in both free-living rhizobia and isolated SBs (20), and the structural genes, gltB and gltD, coding for GOGAT in R. etli have been isolated (24). Due to the absence of assimilatory GDH activity, mutants lacking GOGAT activity are glutamate auxotrophs (24, 45, 88, 95, 133, 136) (Fig. 3). S. meliloti and R. etli mutants lacking GOGAT are not altered in their symbiotic behavior (24, 95). Thus, during symbiosis, glutamate is provided in the nodule by the plant and is synthesized through a different pathway, such as amino acid degradation. In the case of R. etli, lack of GOGAT activity causes an enhancement of nodulation efficiency, probably correlated to an increased ability of BTs to release fixed N (24). In contrast, strains of B. japonicum and A. caulinodans lacking GOGAT are defective in symbiotic N2 fixation (71, 133).
In the rhizobium-legume symbiosis, most of the N2 fixed in BTs is liberated to the host plant to satisfy its demand for N, and consequently the growth of nodulated plants becomes almost independent of alternative N sources (69). The form (e.g., NH3/NH4+ and organic compounds, such as amino acids) and the mechanism (passive diffusion and transport) by which fixed N is exported are unclear. B. japonicum SBs isolated from crushed nodules have been shown to excrete alanine (206). R. leguminosarum SBs excrete different ratios of alanine and NH4+ according to the experimental conditions used (open or closed systems), but NH4+ excretion prevails in an open system where the supernatant is continuously removed (2, 147). It should be pointed out that in an intact nodule the N compounds excreted by SBs are promptly utilized by the plant, which is known to derepress NH4+ assimilation activities, as for example a nodule-specific GS (for a review, see reference 156). Therefore, the primary form excreted in the symbiosis by SBs is most likely NH4+, whereas other compounds like alanine are synthesized when NH4+ accumulates either in vitro (closed chamber) or in vivo when the NH4+ assimilation capacity of the plant is reduced (2, 13).
It has been proposed (36, 192) that NH3 might passively diffuse across the BT membrane into the relatively acidic peribacteroid space, where it is converted into NH4+. However, the present evidence does not exclude the existence of a channel for NH3 or NH4+ efflux in free-living bacteria and in BTs during symbiosis (Fig. 4, left panel). The NH3/NH4+ diffused or excreted outside the BTs reaches the plant cytosol through a channel located on the SB membrane and capable of transporting NH4+ (190) or through a channel mediating NH3 flux (36). However, the plant gene(s) involved in this process remains unrecognized (81, 107). By complementation of a triple MEP mutant strain of Saccharomyces cerevisiae, genes coding for NH4+ transport systems have been identified in different plants (for a review, see reference 78) and recently in the legume Lotus japonicus (155). One of the MEP genes, coding for the high-affinity NH4+ uptake system of S. cerevisiae, may act as a sensor for extracellular NH4+ (98).
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-ketoglutarate and, through a cascade of regulatory proteins, regulates the activity of different promoters by means of a two-component system, comprising the protein kinase NtrB and the transcriptional regulator NtrC (Fig. 3, right panel). Most studies on the rhizobial ntr system have used the knowledge of the enteric one as a paradigm, even though differences in gene organization and regulation were found. The R. etli ntrBC genes are not cotranscribed with glnA as in enteric bacteria, but with an open reading frame (ORF) that shows significant homology at the amino acid level with an ORF located upstream of the fis gene of E. coli and named yhdG (141). The YhdG protein belongs to the NifR3-SMM1 protein family of unknown function (Prosite PS01136). The same gene organization has been described for Azospirillum brasilense, Mesorhizobium loti, and Rhodobacter capsulatus (62, 82, 102). In the following description, the ORF1-ntrBC operon has been renamed nifR3 ntrBC. The nifR3 ntrBC operon of R. etli is transcribed from a promoter containing a sequence homologous to the -35/-10 consensus, identified by deletion analysis and runoff transcription experiments using specific oligonucleotides as DNA competitors (110). The switch from N-rich to N-poor conditions causes a 14-fold increase in NtrC concentration in enteric bacteria, but no increase (actually a 2-fold decrease) in R. etli. Under N deficiency, the DNA-binding activity of NtrC increases, and as a consequence some promoters (such as glnII) are activated, whereas others (such as gstI) are repressed (Fig. 3) (140-143, 183). This shows that, while in enteric bacteria N availability regulates both the intracellular concentration of NtrC and its activity, in R. etli only NtrC activity is regulated (110, 142). On the other hand, a mutant strain expressing a truncated form of the NtrC protein shows an intracellular concentration of nifR3 ntrBC mRNA at least 20-fold higher than that of the wild-type strain (141). This demonstrates the presence of a mechanism of autogenous regulation acting on either promoter activity or mRNA stability.
In R. etli the ntrC gene does not affect the utilization of either glutamine or histidine as the sole N source when added at 1 g/liter (123). Moreover, an ntrC mutant strain grows on 10 mM nitrate, but not on 3 mM (123). The same phenotype was observed in ntrC mutant strains of B. japonicum and S. meliloti (108, 174). It is possible that lack of growth at low nitrate concentrations is a consequence of a reduced bacterial capacity to maintain NH4+ inside the cells and/or to assimilate it. In fact, the genes involved in NH4+ uptake and assimilation (like amtB and glnII) are highly expressed in an NtrC-dependent mode in bacteria grown on nitrate (140, 143, 179). Moreover, it has been reported that the majority of the N limitation-induced genes are not regulated by the ntr system (119). It seems, therefore, that in rhizobia the main function of NtrC is the transcriptional regulation of genes involved in NH4+ metabolism.
In free-living diazotrophs, NtrC is necessary for expression of the nifA gene, which in turn activates expression of the other nif promoters (43), and as a consequence ntrC mutants are unable to grow when N2 is the sole N source. By contrast, different rhizobia mutated in the ntrC gene are Nod+ Fix+ (108, 123, 174), showing that NtrC is not essential either for activation of nif and fix genes or for an efficient symbiosis. However, P. vulgaris plants nodulated with an R. etli ntrC mutant are invaded faster (R. Tatè, unpublished) and accumulate more N than plants nodulated with the wild-type strain (165). This suggests that NtrC exerts a negative control on other functions related to the symbiotic interaction. In fact, in S. meliloti, NtrC regulates nod gene expression as a function of the NH4+ concentration (48). In R. leguminosarum, NtrC represses the expression of the aap (general amino acid permease) operon (204), and in R. etli NtrC prevents the expression of the symbiotic terminal oxidase (167) and affects the degree of exopolysaccharide polymerization (C. Giordano and E. J. Patriarca, unpublished data). Moreover, NtrC is necessary for the alteration of nodulation exerted by the ectopic expression of the amtB and gdhA genes (114, 180).
In R. leguminosarum the PII protein is essential to inactivate (dephosphorylate) NtrC under conditions of N excess, but it is dispensable for the activation (phosphorylation) of NtrC, which, in the absence of PII, becomes active independently of the N conditions (Fig. 3, right panel). In fact, transcription of the glnII gene (requiring NtrC for expression) is constitutive in a glnB mutant (3). In a glnD mutant lacking uridylyl removing/uridylyltransferase (UR/UTase), the enzyme catalyzing uridylylation/deuridylylation of PII, the latter is constitutively deuridylylated, and therefore NtrC cannot be activated. As a consequence, the glnII promoter also remains silent under N-limiting conditions (159). An R. leguminosarum glnB mutant induces normal early organogenesis (3), but nitrogenase is only weakly expressed compared to the level in nodules induced by the wild-type strain (183). In contrast, a glnD mutant strain elicits the formation of normal, N2-fixing nodules (159). Altogether, these data indicate that PII, in its deuridylylated form, is essential for normal levels of nitrogenase expression, most probably due to the inactivation exerted by PII on NtrC and/or GSI, ensuring a reduced NH4+ assimilation capacity of BTs (see below). This is supported by the observation that NtrC-activated promoters are switched off, in a PII-dependent manner, very early during symbiosis (183).
In S. meliloti PII is instead essential to activate (phosphorylate) NtrC under conditions of N starvation, and as a consequence, NtrC-dependent promoters are inactive in a glnB mutant (5). When M. sativa is infected with this mutant strain, most ITs abort early. Since strains lacking NtrC are able to induce efficient nodules, the abnormal phenotype of the glnB mutant cannot be explained by a requirement for NtrC phosphorylation. Therefore, PII should be involved in the regulation of another bacterial function(s) essential at the onset of nodulation (growth in the ITs), such as the activation of genes needed for the synthesis of Nod factors, exopolysaccharides, and lipopolysaccharides. Otherwise, a constitutively dephosphorylated NtrC might repress and alter the expression of the above-mentioned genes. The phenotype of the S. meliloti glnB mutant strain cannot be explained by an action of the PII protein on GSI adenylylation, since a glnA mutant strain impaired in adenylylation of GSI shows a normal symbiotic phenotype (4).
In the R. leguminosarum glnB mutant, the NtrC-activated genes (such as glnII and glnK-amtB) are highly expressed independently of the N source, but this strain does not grow on nitrate (3). It appears therefore that PII activates another function, as yet unknown, required for nitrate utilization.
As mentioned above, a second glnB-like gene, glnK, located upstream of the amtB gene is present in different rhizobia (6, 82, 179). In R. etli the glnK-amtB operon is transcribed from a 
54-dependent promoter requiring NtrC for activity (179). This promoter does not show a clear-cut sequence for NtrC binding, and therefore its activation by NtrC may be indirect. In some bacterial species, glnB and glnK are functionally equivalent (for a review, see reference 6). In rhizobia the function of GlnK is unknown, although it has recently been shown that in E. coli and Azotobacter vinelandii, GlnK binds to the membrane in an AmtB-dependent manner and that GlnK acts as a negative regulator of the transport activity of AmtB (33a). This interaction represents a novel form of signal transduction pathway sensing extracellular NH4+.
In A. caulinodans, a second two-component system (NtrY/NtrX) showing considerable homology to NtrBC has been identified (145) and found to regulate the expression of NtrC and NifA. A. caulinodans is a diazotrophic bacterium in which NtrC mediates the induction of nifA transcription under N limitation. Homologous genes are also present in M. loti, R. etli, and S. meliloti (64, 82; E. J. Patriarca and M. Merrick, unpublished data), but no evidence for their presence has been reported in the case of enteric bacteria. Sequence analysis predicts that NtrY (the homologue of the protein kinase NtrB) may be membrane associated through a transmembrane element. Therefore, NtrY may modulate the activity of NtrX (the NtrC equivalent) as a function of the extracellular N. If so, its function might be linked to that of the glnK-amtB operon.
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Studies on the pattern of gene expression in developing nodules clarify the above contradictions. Although in situ localization of bacterial mRNA has been reported (158, 212), the histochemical localization of the product of a reporter gene (gfp, gusA, and lacZ) is more generally used because of the simplicity and efficacy of the method (7, 63, 161). Most studies make use of lacZ fusions because the protocol used to lower the background of ß-galactosidase activity (7) and the use of stable plasmids (170, 208) permit clear establishment of when and where during nodulation a bacterial promoter is active.
As shown below, the in situ analysis of the activity of different promoters in the elongated nodule confirms and extends the conclusions reached by histological analysis (186, 199). In fact, with respect to the regulation of NH4+ metabolism in V. hirsuta nodules, this kind of analysis confirms the activation of nif gene expression in BTs of SBs located at the II-III interzone (144, 179) and allows the description of two previously unrecognized metabolic events (Fig. 1). The first one is the downregulation of genes required for NH4+ uptake and assimilation, observed when bacteria are released, by endocytosis, from the ITs into the meristematic host cells. The second one takes place later on, a few cell layers before the II-III interzone, where promoters of several N-regulatory genes (including gstI, ntrC, and glnB) are switched off.
In the globose nodule of Proteus vulgaris, the in situ analysis of bacterial promoters, together with earlier evidence of gradients of expression of eukaryotic gene products in this nodule (176), has been instrumental in revealing a spatial component of development. In particular, downregulation of the nifR3-ntrBC promoter clearly defines a previously unrecognized developmental zone (144). Furthermore, the analysis of the nifHc promoter indicates that nif genes are activated much earlier in the globose nodule than in the elongated nodule (180). Table 1 lists the topological expression of bacterial genes in nodules.
The gstI promoter in free-living R. leguminosarum is induced in an N-rich medium, while it is repressed under N-poor conditions (183). In developing nodules of V. hirsuta, the gstI promoter is active in SBs released in the first ICs of the inner cortex during primordium formation. In 1-week-old nodules, a decreasing distal-proximal gradient (with respect to the root) is observed, which becomes more evident later on, when the activity is restricted to the SBs present in the most distal cells of the invasion zone (zone II according to reference 199) (Fig. 1). The gstI promoter becomes inactive before transcription of the nif genes is activated (183). The pattern of gstI expression suggests that dividing BTs located in the distal part of zone II of elongated nodule sense a signal of N excess, whereas the most proximal ones are under severe N limitation and are unable to activate the gstI promoter. In the presence of GstI, glnII expression in BTs may be inhibited not only at the transcriptional level but also at the posttranscriptional level, as occurs under free-living conditions (183).
The R. etli nifR3-ntrBC promoter in V. hirsuta nodules is active in bacteria growing in ITs and in dividing BTs located in differentiating host cells of the distal zone II. It becomes inactive in the SBs of the last two or three cell layers before nif gene expression is switched on (Fig. 1) (144). The same pattern of expression is observed in Fix- nodules, confirming that downregulation of the nifR3-ntrBC promoter is not due to the synthesis of NH3 produced through nitrogenase activity. In globose nodules of P. vulgaris, the nifR3-ntrBC promoter is active in bacteria growing in the ITs, in the BTs released in the first ICs of the primordium, and in all ICs of early emergent nodules. In developed nodules, expression is limited to the BTs located in the two and three most peripheral layers of ICs (Fig. 2) (144). Immunolocalization of NtrC confirms this pattern of expression and shows that not only is transcription of the nifR3-ntrBC operon switched off but also that, since BTs at this stage do not divide, the NtrC protein is removed.
The R. leguminosarum glnBA promoter in V. hirsuta nodules shows a pattern of activity quite similar to that of the nifR3-ntrBC and gstI promoters, since it is active in bacteria growing inside the ITs and in BTs released in the first ICs of the inner cortex during primordium formation. In more mature nodules it is switched off, in correlation with the disappearance of NtrC, a few cell layers before nif gene expression (52).
In nodules of P. vulgaris, the R. etli nifHc promoter is induced at a very early stage of nodule invasion, namely, in BTs just released in the plant cells (180). This promoter is induced in a NifA-dependent manner both under free-living microaerobic conditions and during symbiosis (195). Instead, in elongated nodules the R. etli nifHc promoter is switched on abruptly in SBs located at interzone II-III (144), exactly where predicted by an in situ hybridization analysis revealing the endogenous nifH mRNA (212). In interzone II-III, the ICs are filled with nondividing, almost completely differentiated SBs (199), and a sharp drop in O2 tension takes place (168) (Fig. 1). The earlier timing of NifA activation, and therefore of nifHc expression, in globose versus elongated nodules may be related to differences either in the organizational structure of the parenchyma (211) or in plant and/or bacterial metabolism, which generate a low O2 tension environment (91, 120).
That the invasion of globose nodules takes place under microaerobic conditions is supported by the following data. First, in the youngest ICs of developing nodules of P. vulgaris, harboring only a few SBs formed by dividing BTs, a large number of condensed (crista-rich) mitochondria, which cluster, aggregate, and divide, are observed (25). Second, a terminal oxidase complex, which is expressed under microaerobic conditions in free-living bacteria, is required for the invasion process, since a cytochrome c-deficient mutant of R. etli induces empty nodules (166). Third, nitrogenase activity (requiring microaerobiosis) is detected in young soybean nodules, in which most of the BTs in the host cells are still actively dividing (12). These data indicate that the nitrogenase complex is induced in young dividing BTs during the invasion of globose nodules, even though the analysis of nif mutants indicates that nitrogenase activity is not essential for invasion.
During symbiosis, the N status of rhizobia is determined by the composition of the medium in which they are immersed. The analysis of ntr-regulated promoters indicates that during endocytosis, the N metabolism of BTs changes from a condition of N deficiency to one of N richness (and C deficiency, which would result in a relative abundance of available N). The movement of the bacterium from a C-rich environment, the cell wall-like matrix of the ITs, into the intracellular environment of the BTs, which are surrounded only by the SB membrane, may reduce C availability. A condition of C limitation and N excess may also be generated by the utilization of amino acids as a C source (see below). A second factor causing a condition of C deficiency is the permeability of the SB membrane. The latter arises from an invagination of the plasma membrane, so that if it does not invert the polarity of its transport systems, the peribacteroid space is, at least initially, equivalent to the extracellular space. This implies that the IC may take up solutes, such as sugars, not only from the extracellular space but also from the peribacteroid space of the youngest BTs. However, later on during differentiation, the structure and function of the SB membrane as well as the composition of the peribacteroid space gradually change (200, 201). For instance, the induction of a C4-dicarboxylate carrier on the SB membrane (138) may drastically change the physiology of the SBs.
N-containing compounds such as purines and pyrimidines are not provided by the corresponding plant partners to B. japonicum, R. etli, Rhizobium fredii, and R. leguminosarum, even in the ITs, since auxotrophic strains induce only empty pseudonodules (128, 130). Mutants of S. meliloti induce Fix- nodules, but a histological analysis was not performed (85).
R. etli strains defective in the biosynthesis of amino acids (such as aromatics, aspartate, cysteine, glutamate, glycine, histidine, isoleucine-valine, leucine, lysine, phenylalanine, and tryptophan) with the exception of an arginine and a methionine auxotroph, are able to induce nodules (24, 56, 178, 181, 182; S. Ferraioli, unpublished data). Analogous observations have been reported in the case of S. meliloti. In fact, except for an isoleucine-valine auxotroph (1), all other mutants analyzed (such as those requiring asparagine, leucine, methionine, ornithine, tryptophan, and tyrosine) are able to induce nodules on M. sativa (10, 85, 148, 188). With minor differences, the same conclusion was reached by analyzing amino acid-requiring mutants of R. leguminosarum (139). In most cases, however, the resulting nodules are partially or totally ineffective, but without histological observations, the nature of these alterations in most cases remains unrecognized.
Auxotrophic strains of R. etli incubated in minimal medium in the absence of their nutritional requirements are unable to produce Nod factors, but when inoculated on the roots of P. vulgaris without exogenously added amino acids, they are able to induce nodules (56, 178, 181, 182). Since Nod factors are essential for nodule induction, the plant must provide the necessary amino acids to the bacterium. The presence of amino acids in the root exudates of some legumes has been demonstrated (196), and it has been proposed that they are produced by lysis of the root cells detached from its cortex due to the outgrowth of developing lateral roots. However, it is not possible to exclude that root cells even under N starvation (a condition necessary for symbiosis) release amino acids to stimulate rhizobia both to divide and to produce Nod factors. Chemotaxis towards root exudates has been demonstrated (21).
The ability of most of the amino acid-auxotrophic strains to invade developing nodules indicates that the plant provides the amino acids necessary for bacterial growth occurring inside the ITs. The origin of the amino acids in the ITs is unknown, but, similar to what happens in the case of pollen tubes, membrane vesicles may fuse with the tip of developing ITs, allowing their elongation (16, 68). In this way they would provide nutrients in the form of amino acids and proteins. This may explain why bacteria grow only at the tip of ITs (63). After endocytosis, the composition of the peribacteroid space, as well as the composition of the SB membrane, is modified by fusion of the SB with secretory vesicles formed by budding of the Golgi cisternae and/or originated from the endoplasmic reticulum (201). A variety of hydrolytic enzymes, including proteases and protease inhibitors, have been identified in the peribacteroid space (113). Since BTs divide several times before differentiation and the SB membrane is relatively impermeable to amino acids (192), proteolytic activity may generate a mixture of free amino acids from proteins released through vesicle fusion.
Different lines of evidence indicate that undifferentiated BTs may use amino acids as a source of C and energy and that C4-dicarboxylic acids are the C source utilized after SBs begin to differentiate (51, 198). An S. meliloti mutant strain altered in proline catabolism shows reduced nodulation efficiency and competitiveness, and the promoter for the putA (proline utilization) gene is active during root invasion and nodule formation, but not in N2-fixing BTs (80). An S. meliloti mutant strain missing succinic semialdehyde dehydrogenase (involved in the 
-aminobutyrate bypass), and therefore impaired in glutamate catabolism, shows reduced nitrogenase activity and thus biomass-accumulating ability (61). The -aminobutyrate bypass is responsible, even though partly, for the catabolism of glutamate in BTs of B. japonicum (90). A mutant of S. meliloti missing aspartate aminotransferase (aatA gene) induces Fix- nodules in which only a few BTs are present in the ICs (207). Since proline catabolism and aspartate aminotransferase activity generate glutamate, together these data suggest that glutamate catabolism plays an important role in the dividing BTs.
A very strong inactivation of the glnII promoter (NtrC activated) and derepression of the gstI promoter (NtrC repressed) are observed in free-living R. leguminosarum when an amino acid, such as glutamine and glutamate, is used as the sole C source (see above) (183). Therefore, the almost complete inactivation of NtrC taking place during endocytosis supports the notion that BTs use amino acids as a C source. Moreover, when rhizobia utilize an amino acid as a C source, they excrete NH4+, and if under these conditions amtB expression is forced by means of an inducible promoter, bacterial growth is inhibited (E. J. Patriarca, unpublished data). NH4+ excretion by young BTs may prepare the SBs and the cytoplasm of ICs, through the induction of specific plant genes, for the transport and assimilation of the NH4+ produced later by the nitrogenase.
With almost the same pattern, the lipA (lipoic acid synthetase), nod (Nod factors), putA (proline utilization), and ropA (outer membrane protein III) genes are also downregulated (38, 80, 158, 161, 177). Over 350 proteins, including several proteins involved in N metabolism and the cell cycle, were found to be expressed at lower levels or undetectable in isolated SBs of S. meliloti compared to free-living bacteria (126). These include proteins encoded by genes that are either never expressed or inactivated very early (in the ITs) during symbiosis. This proteome analysis, however, reflects the pattern of expression obtained from a mixture of BTs at different stages of differentiation. Therefore, proteins encoded by genes that are downregulated (even abruptly) later on during BT differentiation, such as in interzone II-III (Fig. 1) could be considered as not altered. Therefore, the number of genes downregulated may be higher. Downregulation is extensive, involving genes related to different metabolic pathways, uncorrelated with respect to regulation and function. It seems, therefore, that in differentiating SBs there is a global mechanism for inactivation of gene expression.
It is known that in bacteria one of the mechanisms used to exert a global control of gene expression is the modification of the chromosome structure. DNA supercoiling varies as a function of the cellular energy status and plays an essential role in bacterial adaptation and survival to suboptimal growth conditions, such as stationary-phase growth, osmotic stress, and aerobic to anaerobic transition (for a review, see reference 157). It has been shown that before the sharp drop in O2 concentration taking place at interzone II-III of nodules, a shallow gradient of decrease is observed in zone II (168) and, in addition, that the DNA superhelicity increases in BTs (66). The energy status (the ADP/ATP ratio) of BTs may change as a function of the decrease in the O2 tension that is encountered inside the nodule, thus altering the superhelical density of the chromosome.
Another event taking place at this stage, in the middle of zone II, is the transition from dividing BTs (distal zone II) to nondividing differentiating BTs (proximal zone II). In bacteria sigma factors are considered global regulators of gene expression. The sigA gene, coding for the putative vegetative sigma factor of R. etli, has been identified (101). The intracellular levels of sigA, lipA, and nifR3-ntrBC transcripts are very high just after bacterial dilution in complete medium, decrease during bacterial divisions, and become very low, almost undetectable, as bacterial growth continues (101, 144, 177). In a similar manner, sigA may be downregulated du
