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Corresponding author: Turlough M. Finan, Department of Biology, McMaster University, 1280 Main St. West, Hamilton, Ontario, L8S 4K1, Canada, finan{at}mcmaster.ca (E-mail).
Communicating editor: P. L. FOSTER
 | ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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We report the isolation of phoB and phoU mutants of the bacterium Rhizobium (Sinorhizobium) meliloti. These mutants form N2-fixing nodules on the roots of alfalfa plants. R. meliloti mutants defective in the phoCDET (ndvF ) encoded phosphate transport system grow slowly in media containing 2 mM Pi, and form nodules which fail to fix nitrogen (Fix-). We show that the transfer of phoB or phoU insertion mutations into phoC mutant strains restores the ability of these mutants to: (i) form normal N2-fixing root-nodules, and (ii) grow like the wild type in media containing 2 mM Pi. We also show that expression of the alternate orfA pit encoded Pi transport system is negatively regulated by the phoB gene product, whereas phoB is required for phoCDET expression. We suggest that in R. meliloti cells growing under Pi limiting conditions, PhoB protein activates phoCDET transcription and represses orfA pit transcription. Our results suggest that there are major differences between the Escherichia coli and R. meliloti phosphate regulatory systems.
THE presence of two phosphate transport systems is a common feature in bacteria, and such systems have been identified in Escherichia coli (
The high-affinity phosphate-specific transport system of E. coli, PstSCAB, is a periplasmic-binding protein, ABC-type transporter, and pstSCAB transcription is induced when cells are starved for phosphate (
In E. coli, the PstSCAB and PhoU proteins appear to be involved in the sensing of environmental Pi, as many pstSCAB phoU mutations result in constitutive expression of the Pho regulon (
Wild-type Rhizobium meliloti form N2-fixing nodules on alfalfa, whereas mutants defective in the phoCDET (originally designated ndvF ) encoded phosphate transport system form nodules which contain few bacteria and fail to fix N2 (Fix-) (
In this article, we report the isolation of phoU and phoB mutants of R. meliloti. The phoU/B mutations are shown by transduction to map to the Class II suppressor locus (sfx2) and also to suppress the Fix- phenotype of phoCDET mutants to Fix+. We show that phoB is required for phoCDET expression, and that phoB regulates expression of the orfA-pit operon in a phosphate-dependent manner.
| MATERIALS AND METHODS |
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Bacterial strains, plasmids, media, and growth conditions:
The strains and plasmids employed in this work are listed in Table 1. The growth media used were Luria-Bertani (LB) or LB containing 2.5 mM MgSO4 and 2.5 mM CaCl2 (LBmc) with antibiotic concentrations as previously described (
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For experiments employing lacZ gene fusions, LBmc-grown cells, supplemented with tetracycline (Tc) 2 µg/ml for strains carrying pMP220-derived plasmids (
The low osmolarity media (GYM) was prepared as stated in
The chromosomal transcriptional lacZ fusions to pit were constructed as outlined in Figure 8. The structure of the recombinants was confirmed by Southern blot of the genomic DNA digested with HindIII, SacI, and HindIII/SacI, and probed with the 4.8-kb HindIII/SacI fragment of pTH90. All ß-galactosidase values reported in Figure 6 were corrected for the ß-galactosidase activity obtained from recombinants with the lacZ gene in the opposite orientation (ort II) to the direction of orf-pit transcription (e.g., RmH695). This background activity remained constant at 10 Miller Units, regardless of the background or whether the fusion was to the sfx1 or wild-type locus.
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DNA manipulation and genetic techniques:
Cloning procedures, including DNA isolation, restriction digests, ligation, and transformation were performed according to
Conjugal mating using MT616 as a helper strain, 
M12 generalized transduction, TnV and Tn5-233 replacement, and determination of the linkage between two markers by transduction were performed as described previously (
TnV possesses the NptII gene (Kmr) and the pSC101 origin of vegetative replication (able to replicate in E. coli but not in R. meliloti), flanked by the inverted repeats IS50L and IS50R (
-competent cells. As TnV does not contain a Sal I restriction site, the religated plasmid carried flanking genomic DNA. Subclones of the resulting plasmids were sequenced using the IS50 primer (5'-TCACATGGAAGTCAGATCCT-3').
Plant growth, alkaline phosphatase, and ß-galactosidase assays:
Plant growth experiments in a nitrogen-free environment and acetylene reduction assays were performed as stated in
The alkaline phosphatase activity was measured as described in 
T), with T being the reaction time (min).
The ß-galactosidase assay was performed by mixing 0.5 ml of cell, for which OD600 was determined, with 0.5 ml Buffer Z (pH 7; 60 mM, Na2HPO4; 40 mM, NaH2PO4; 10 mM, KCl; 1 mM, MgSO4 and 2.7 ml/liter of 2-mercaptoethanol added just before use), 20 µl chloroform and 10 µl 0.1% SDS. The tubes were equilibrated at 30° for 5 min, and the reaction was started by adding 0.2 ml of 4 mg/ml o-nitrophenyl-ß-D-galactoside (ONPG). When the solution turned yellow, the reaction was stopped by adding 0.5 ml of 1 M Na2CO3. The optical density at 420 nm (OD420) was determined after centrifuging the cells for 5 min at 12,000 rpm. The ß-galactosidase activity in Miller Units was calculated using the formula (1000 x OD420)/(OD600 x T x V), with T being the reaction time (min) and V representing the initial volume of culture used (ml).
| RESULTS |
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| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Strains carrying sfx2 are deficient in alkaline phosphatase production:
Measurements of alkaline phosphatase activity in R. meliloti cells can be used to monitor the physiological status of cultures with respect to phosphate availability (
To examine the effect of the previously isolated phoCDET (ndvF ) symbiotic suppressor mutations (phoCDET background, also lacked alkaline phosphatase activity (AP- phenotype) (Figure 1 and data not shown). The AP- phenotype of strains carrying a Class II mutation, together with the suppression of the mucoid colony and symbiotic phenotypes associated with the phoCDET (ndvF ) mutations (
Isolation of phoUB mutants:
To isolate mutants defective in the phosphate-signaling regulatory pathway, we screened for alkaline phosphatase negative mutants (AP-). Tn5-132 (oxytetracycline resistance) and Tn5 insertion mutants of Rm1021 were plated on LB agar containing the AP chromogenic substrate 5-bromo-4-chloro-3-indolyl phosphate (LBXPhos). One Tn5 insertion mutant (RmH405, pho27 ) and 26 Tn5-132 mutants which formed white colonies (AP-) were identified and purified. To check for linkage of the AP- mutations to the sfx2 locus, we utilized the insertions 
5258::Tn5, 5259::Tn5, and 5263::Tn5-233, which were previously identified to be linked to the sfx2 locus (
Nmr was transduced from strain RmG551 (5258::Tn5 ) into the 26 Tn5-132 AP- mutants, and transductants were screened for the presence of blue colonies (AP+) on LBXPhos plates. Three of the Tn5-132 insertion mutations designated pho10, pho8, and pho3 showed 60, 64, and 66% linkage to 5258::Tn5, respectively. In further transductions, Nmr from insertion 5259::Tn5 (strain RmG552) showed 48, 44, and 42% linkage to the pho10, pho8, and pho3 mutations, respectively. These linkage values were similar to those between the two insertions 5258 and 5259, and sfx2, suggesting that the pho10, pho8, and pho3 insertion mutations and sfx2 map to the same locus (see Figure 2). The remaining 23 AP- (white) Tn5-132 insertion mutants showed no linkage to 5258::Tn5; however, these were 100% linked in transduction to the Tn5 insertion in pho27 (strain RmH405). These mutants were not examined further.
To identify the gene(s) in which the pho10, pho8, and pho3 Tn5-132 insertions were located, we first replaced the Tn5-132 insertions with TnV (
The location of the phoUB locus on the R. meliloti genome was determined by conjugation using seven Tn5-mob insertion strains as described in 5263,Tn5-233, 50% linked to sfx2; Figure 2). Following purification, the resulting transductants were crossed in conjugation with the rifampicin resistant (Rif r) Rm5000 strain. The frequency of Rif r, Gmr Spr colonies placed the phoUB locus between trp-33 and pyr49 on the R. meliloti chromosome (
The phoUB genes are required for phoCDET expression:
We have previously shown that transcription of the pho-CDET transport genes is induced when R. meliloti cells are starved for phosphate, and that the phoC promoter contained two elements which are similar in sequence to the consensus-binding site for the E. coli PhoB protein (
phoU and phoB mutations suppress the symbiotic and associated phenotypes of phoCDET mutants:
The phoU10, phoB8, and phoB3 mutations had similar phenotypic effects to the sfx2 suppressor mutation. As the sfx2 allele was originally identified as a suppressor of the symbiotic Fix- phenotype of phoCDET (ndvF ) mutants, we tested the three phoB and phoU insertion mutations for their ability to suppress the Fix- phenotype of a phoC mutant. We also examined the ability of phoB and phoU mutations to suppress the slow growth of phoCDET mutants in media containing 2 mM Pi, and the mucoid colony phenotype which phoCDET mutants exhibit when plated on low osmolarity media (see below). The phoUB-phoC strains were constructed by transducing the phoU10, phoB8, and phoB3::TnV (Nmr) insertion mutant alleles from strains RmH399, RmH428, and RmH430, respectively, into RmG490 (phoC 490) to create strains RmH623, RmH624, and RmH625, respectively.
The symbiotic phenotype of the various mutants was determined from the analysis of alfalfa plants 28 days after inoculation with the various R. meliloti strains. Plants inoculated with the phoC mutant were small and chlorotic, and showed little evidence of N2-fixation as measured by acetylene reduction and plant dry weight determinations (Table 2). Plants inoculated with the phoC, phoU/B double mutants had shoot dry weight and acetylene reduction values comparable to the wild-type strain Rm1021 (Table 2). Thus, the phoU10, phoB8 and phoB3 mutations suppressed the Fix- phenotype of the phoC to Fix+. We also note that the individual phoB, and phoU insertion mutants showed no reduction in symbiotic effectiveness compared to the wild-type strain.
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The ability of the various R. meliloti strains to grow in MOPS-buffered minimal media containing 2 mM Pi was determined (Figure 5). While the phoC mutant grew poorly, the single phoB, phoU or sfx2 mutants, and the double mutants carrying phoC together with phoU10, phoB8, phoB3 or the sfx2 allele grew as well as the wild-type strain Rm1021 (Figure 5, data not shown). Given that phoU/B mutants do not express the phoCDET genes (Figure 4), it appears that the transport system(s) which allows Pi uptake into phoU/B mutants may also be responsible for allowing the phoU/B, phoC double mutants to grow normally in media containing 2 mM phosphate.
In summary, the results presented in this section show that phoB and phoU mutations suppress the symbiotic and phosphate-dependent phenotypes associated with mutations in the phoCDET locus.
Mechanism of phoUB mediated suppression of phoCDET mutants:
In view of the above results, we suspected that phoB and/or phoU mutations led to increased expression of the recently characterized orfA-pit locus, which appears to encode an alternate phosphate transport system (S. D. BARDIN, R. VOEGELE, N. FALCIONI and T.M. FINAN, unpublished results). To investigate this possibility, we transduced phoB, phoU, and sfx2 mutations into strain RmH662, which carries a chromosomally located transcriptional lacZ fusion to the wild-type orfA-pit locus [to give strains RmH754 (phoU10), RmH755 (phoB8), RmH756 (phoB3), and RmH765 (sfx2)], and into strain RmH771, which carries the lacZ fusion to sfx1 orfA-pit locus [to give strains RmH861 (phoU10), RmH862 (phoB8 ), RmH863 (phoB3), and RmH860 (sfx2)]. We note that the lacZ fusion construct employed in these experiments was prepared so that lacZ was inserted immediately downstream of the pit translational stop codon (see MATERIALS AND METHODS and Figure 8 for details of this construction). Thus, in the wild-type and sfx1-directed gene fusions, both the orfA- and pit-encoded proteins should be fully functional. (This was confirmed as the sfx1 constructs in a phoC mutant background grew like wild type in MOPS medium containing 2 mM Pi, data not shown.) The level of orfA-pit expression (ß-galactosidase activity) was measured after culturing the cells for 32 hr in MOPS-buffered medium with no added Pi (P0) and with 2 mM Pi added (Figure 6B). To monitor the physiological status of the cells with respect to phosphate, we also measured alkaline phosphatase activity (Figure 6A).
Strains with a wild-type background (Figure 6A, datasets 1 and 6) showed the expected high AP activity in P-starved cells and low AP activity in cells grown with excess Pi (2 mM). As expected, the various phoU/B and sfx2 mutant strains contained minimal AP activities regardless of the level of Pi in the growth medium.
The level of orfA-pit expression (ß-galactosidase activity) directed from the sfx1 promoter was higher than the level directed from the wild-type orfA-pit promoter (Figure 6B, datasets 1–5 vs. 6–10); and in the wild-type background, both wild-type and sfx1-directed orfA-pit expression was phosphate-regulated as the ß-galactosidase activity was two- to fourfold higher in cells grown with 2 mM phosphate compared to phosphate-starved cells (Figure 6B, datasets 1 and 6). In the phoU, phoB, and sfx2 backgrounds, orfA-pit expression increased 4.5–5.5-fold when the cells were cultured in the absence of added Pi, and 1.5–2-fold when the cells were grown with excess Pi (2 mM) relative to orfA-pit expression in a wild-type background (Figure 6B, datasets 1 vs. 2–5, and 6 vs. 7–10). These data suggest that phoB (phoU ) is negatively regulating orfA-pit expression. Moreover, in the phoB and sfx2 backgrounds the phosphate-dependent regulation of orfA-pit expression was dramatically reduced, suggesting that phosphate regulation of orfA-pit expression was probably mediated via PhoB. The results outlined above were obtained from the chromosomal orfA-pit ::lacZ gene fusions. We obtained similar data when pit expression from a plasmid-borne gene fusion was assayed in these strains (data not shown).
As phoB and phoU mutations suppressed the Fix- and phosphate-growth phenotype of phoCDET mutants, we wished to examine the influence of the phoU, phoB, and sfx2 mutations on the level of orfA-pit expression observed in a phoCDET mutant background. In these experiments, expression from plasmid-borne wild-type and sfx1-directed orfA-pit::lacZ fusions in phoC phoB and phoC phoU backgrounds was monitored, and again AP activities were measured as controls (Figure 7, a and b). We note that phoC mutant cells behave as if they are phosphate-starved, even in the presence of excess phosphate (Figure 7A, dataset 2; Figure 1). In the phoC background, under both culture conditions, orfA-pit expression directed from the wild-type promoter was as low as its expression in phosphate-starved wild-type cells (Figure 7B, datasets 1 and 2). The introduction of a phoB or phoU mutation resulted in a greater than fivefold increase in the level of orfA-pit expression, suggesting that the repression of orfA-pit expression in phoC cells was mediated by PhoB or PhoU (Figure 7B, dataset 2 vs. 3–5). In the case of sfx1-directed orfA-pit expression, the level of expression observed in the phoC phoB and phoC phoU double mutants was two- to fourfold higher than the basal level observed in phosphate-starved wild-type cells and in phoC cells (Figure 7B, datasets 8–10 vs. 6 and 7). Thus, under phosphate starvation conditions, PhoB and/or PhoU appear to repress sfx1-directed orfA-pit expression. In cells cultured in 2 mM Pi, the level of sfx1-directed orfA-pit transcription was slightly higher in the wild-type and phoC background than in the phoC phoB and phoC phoU double mutants (Figure 7, datasets 6 and 7 vs. 8–10) Thus, the combination of sfx1 and phoB or phoU does not have an additive effect on the level of pit transcription. Lastly, we note that in a phoC background, sfx1-directed orfA-pit expression, but not AP synthesis, was responsive to the media phosphate concentrations (Figure 7, a and b, dataset 7). This difference between wild-type vs. sfx1-directed orfA-pit expression was previously observed (S. D. BARDIN, R. VOEGELE, N. FALCIONI and T.M. FINAN, unpublished results).
| DISCUSSION |
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| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Defined phoB and phoU mutants of R. meliloti were isolated and used to examine the role of phoB and phoU in the expression of the phoCDET and orfA-pit genes. Expression of both phoD::lacZ and phoE::lacZ gene fusions was completely abolished in phoB and phoU mutant strains (Figure 4). We showed earlier that phoCDET expression is strongly activated in response to Pi starvation, and had noted that two appropriately positioned PhoB-like binding sites are located in the phoC promoter (
Our data also suggest that phoB plays an important role in determining the level of orfA-pit transcription (Figure 6B and Figure 7). In Pi-starved cultures, transcription of pit and orfA was clearly higher in the phoB and phoU mutants than in the wild type (Figure 6B, dataset 1 vs. 2–5, data for orfA not shown). In addition, while the wild-type pit fusions showed increased expression in Pi-sufficient compared to Pi-deficient cells, no such Pi regulation was observed when phoB or phoU mutants were examined (Figure 6B, dataset 1 vs. 2–4). While we assume that PhoB is directly interacting with the orfA-pit promoter, our evidence is indirect.
We found that phoB, phoU, and sfx2 mutants, which are phenotypically phoCDET - (Figure 4 and data not shown), formed wild-type Fix+ nodules on alfalfa and grew like wild type in media containing 2 mM Pi (Table 2; Figure 5). This indicated that an alternative Pi transport system was induced in these mutants. Our data on the expression of pit ::lacZ fusions in the phoB and phoU mutants (Figure 6B) also strongly suggest that the alternate uptake system is encoded by the orfA-pit locus. We recently demonstrated that the kinetics and specificity of Pi uptake in phoB mutant cells and in phoC sfx1 mutant cells is very similar (
To account for the reciprocal pattern of orfA-pit and phoCDET expression observed in wild-type cells cultured in the presence and absence of Pi, we suggest that when the concentration of Pi is high (2 mM), PhoB is inactive and orfA-pit is derepressed (expressed), and the phoCDET system is not expressed. Conversely, under conditions of phosphate starvation, PhoB is activated, orfA-pit expression is repressed, whereas phoCDET expression is activated by PhoB. The pattern of phoCDET and orfA-pit expression reflects different physiological characteristics of the two transport systems. Thus, the OrfA-Pit system is a low-affinity Pi transporter and the PhoCDET system has a high-affinity for Pi (
When R. meliloti phoC insertion and phoCDET deletion mutants are cultured in media containing 2 mM Pi, the cells contain high AP activity and thus physiologically behave as though they are starved for phosphate (Figure 7A, dataset 2, data not shown). Under these conditions, the orfA-pit genes are repressed and the cell is then phenotypically both orfA-pit and phoCDET negative. The poor growth of phoCDET mutants in media containing 2 mM Pi therefore indirectly results from the lack of orfA-pit expression; and wild-type growth is restored via mutations which increase orfA-pit expression, such as sfx1, phoB or phoU.
Why phoCDET mutations lead to reduced orfA-pit expression is not clear. In E. coli, mutations in the PstSCAB transport system generate a Pho constitutive phenotype, which is not unlike that observed for phoCDET mutants of R. meliloti. The PstSCAB system is thus believed to be part of the Pi sensory system (
We draw attention to the fact that the phoB gene is located 111 bp downstream from phoU in R. meliloti (GenBank accession no. M96261). As both of these genes are transcribed in the same direction, it is probable that phoU and phoB are transcribed as a single mRNA, in which case phoU insertion mutants would also be phenotypically PhoB-. If this is the case, elucidation of the role of phoU in R. meliloti will require the construction of defined phoU - phoB+ strains. The low Pi-regulated expression of phoD and phoE observed in a phoU10 mutant background (Figure 4, dataset 2), together with the apparent Pi-dependent regulation of sfx1-directed orfA-pit expression also observed in the phoU10 background, (Figure 6, dataset 7) suggest that the phoU10 ::TnV insertion may allow a low level transcription of phoB.
The AP- phenotype of the R. meliloti phoB mutants is similar to that observed for phoB mutants in other bacteria (
Our screening experiment did not result in the isolation of any phoR mutants. It is possible that in the absence of PhoR other kinases cross-activate PhoB as observed in E. coli (
Most studies of the Pho regulon have focused on genes whose expression increases in response to Pi limitation. However, over 20 years ago,
sfx1 and sfx2 were originally identified as mutations which suppressed the symbiotic Fix- phenotype of phoCDET(ndvF ) mutants (
| ACKNOWLEDGMENTS |
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We are grateful to BELINDA SCHOEMAN for technical assistance, to MICHAEL HYNES for the use of plasmid pLMS prior to publication and to RALF VOEGELE, MAGNE OSTERAS and ALISON COWIE for comments and discussions. This work was supported by operating and strategic grants from the Natural Sciences and Engineering Research Council of Canada to T.M.F.
Manuscript received September 10, 1997; Accepted for publication December 22, 1997.
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), RmG490 (phoC, 
), RmH625 (phoB3 phoC, 
) and RmH838 (phoB3, 
). The growth characteristics of RmH623 (phoU10 phoC ), RmH624 (phoB8 phoC ), and RmH363 (sfx2 phoC ) were similar to the growth of RmH625 and are not presented here to simplify the figure. Each data point represents the mean of triplicate values.
