1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References

Two ‘calcium-irreparable’ acid-sensitive mutants were identified after mutagenizing Rhizobium leguminosarum bv. viciae and Sinorhizobium meliloti with Tn5. Each mutant contains a single copy of the transposon which, inserted within the actP gene, prevents expression of a P-type ATPase that belongs to the CPx heavy metal-transporting subfamily. Here, we show that both actP-knockout mutants show sensitivity to copper; omission of this heavy metal from low pH-buffered media restores acid tolerance to these strains. Furthermore, complementation of the mutant phenotype requires only the actP gene. An actPgusA fusion in R. leguminosarum was transcriptionally regulated by copper in a pH-dependent manner. Downstream to actP in both organisms is the hmrR gene that encodes a heavy metal-responsive regulator (HmrR) that belongs to the merR class of regulatory genes. Insertional inactivation of hmrR abolished transcriptional activation of actP by copper ions and increased the basal level of its expression in their absence. These observations suggest that HmrR can regulate actP transcription positively and negatively. We show that copper homeostasis is an essential mechanism for the acid tolerance of these root nodule bacteria since it prevents this heavy metal from becoming overtly toxic in acidic conditions.


  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References

The root nodule bacteria (Bradyrhizobium, Mesorhizobium, Rhizobium, Sinorhizobium) fix atmospheric nitrogen in root nodules to a form that can be used for the growth of the legume host. Soil acidity has a detrimental impact on plant productivity by interfering in the infection process or by decreasing the survival of the microsymbiont (Munns, 1986; Howieson et al., 1988; Coventry and Evans, 1989; Recourt et al., 1989; Robson and Bottomley, 1991). However, it is the long-term persistence of the prokaryotic partner in acidic soils between pasture-growing seasons that governs the regeneration of productive pasture legumes. The response to acidity differs among the strains (see review by Barnet, 1991). Mesorhizobium and Bradyrhizobium are quite acid tolerant and grow readily at pH 4.5 or lower, whereas R. leguminosarum can grow at pH levels down to pH 5.0. Sinorhizobium meliloti is one of the most acid-sensitive root nodule bacteria, growing only at pH levels down to pH 5.5. An understanding of these differences is being investigated at the physiological and genetic level (Glenn et al., 1999).

Rhizobial mutants with acid-sensitive phenotypes have been identified using Tn5 mutagenesis (Goss et al., 1990; Tiwari et al., 1992). Genes (designated act for acid-tolerance) identified in S. meliloti in this manner include actR (the regulator module of a two-component sensor–regulator system; Tiwari et al., 1996a), actS (the sensor module; Tiwari et al., 1996a) and actA (a potential lipid-metabolizing protein; Tiwari et al., 1996b).

It is interesting that each of these mutants displays a pleiotropic phenotype with sensitivity not only to acid but also to azide and/or certain heavy metals. The actR/S mutants are sensitive to acidity, azide, cadmium or zinc (Glenn et al., 1999), whereas a mutation in actA creates sensitivity to acidity, copper and zinc (Tiwari et al., 1996b). Omission of heavy metals from the medium does not alleviate the acid sensitivity of these mutants. However, acid tolerance may be restored to mutants defective in actR, actS or actA by supplementing the growth medium with high concentrations of calcium (Tiwari et al., 1992; Reeve et al., 1993). By using a different screening strategy, we isolated one Tn5-induced mutant of S. meliloti WSM419 (RT3-27) and one of R. leguminosarum bv. viciae WSM710 (WR1-14) that were acid sensitive even in the presence of high concentrations of calcium (10 mM or higher) in low-pH-buffered medium (Reeve et al., 1993). These mutants were therefore labelled ‘calcium-irreparable’ acid-sensitive mutants (Reeve et al., 1993). The growth rates of the mutants at pH 7.0 were similar to those of the wild types, indicating that the acid sensitivity was not due to any simple growth defect. Here, we show that a failure to produce a CPx P-type ATPase results in the calcium-irreparable acid-sensitive phenotype. Furthermore, we demonstrate the importance of this protein for copper homeostasis and highlight how the changes in copper availability resulting from pH changes may affect gene expression and thereby influence acid tolerance. Finally, we demonstrate that transcriptional regulation of actP depends on the heavy metal response regulator HmrR.

Results and discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References

The ‘calcium-irreparable’ acid-sensitive defects are caused by Tn5

A single copy of Tn5 resides in both R. leguminosarum WR1-14 and S. meliloti RT3-27 that has been identified from the hybridization of a digoxigenin (DIG)-labelled Tn5 probe (pTn5-H) (data not shown) to a single fragment of EcoRI restricted genomic DNA from each mutant. These Tn5-containing EcoRI fragments of 11 and 9 kb were cloned from WR1-14 and RT3-27, respectively, and restriction mapped (Fig. 1).


Figure 1. Restriction maps of the Tn5-containing EcoRI fragments cloned from the mutants R. leguminosarum WR1-14 (A) and S. meliloti RT3-27 (B). The NotI site used for insertion of the CAS-GNm cassette (Reeve et al., 1999) to construct a R. leguminosarum actPgusA fusion in the plasmid clone pWR668 has been marked. The site of transposon insertion has been indicated by a Tn5-marked flag (103 bp from the end of actP in R. leguminosarum and 225 bp in S. meliloti). The direction of aacC1 transcription in the CAS-Gm cassette in pWR901 and pWR903 is provided by the direction of the arrow. The restriction map (C) of the actP gene region in S. meliloti WSM419 was derived from the plasmid pRT327-1. The S. meliloti actP gene was inactivated in pNK57 by blunting and religating an intragenic SphI site (represented by an asterisk), whereas the hmrR gene was inactivated in pNK77 by cloning the CAS-GNm cassette into the intragenic BclI site.

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Verification that the single Tn5-induced lesion created the ‘calcium-irreparable’ acid-sensitive defect in each mutant was established using two different methods. A RL38 phage lysate prepared from WR1-14 was used to transduce the kanamycin and streptomycin markers of Tn5 into WSM710; all transductants displayed acid sensitivity. A phage that can be used to transduce S. meliloti WSM419 has not yet been identified; 187 stocks of virulent and temperate phages failed to lyse this strain (C. Defives, personal communication). To reconstruct the mutation in WSM419, marker-assisted exchange was used to replace the wild-type allele with one containing Tn5 using the plasmid pWR327-200. Reconstructed actP::Tn5 mutants displayed an acid-sensitivity profile similar to RT3-27.

The Tn5 insertions in RT3-27 or WR1-14 also cause copper sensitivity

Acid-sensitive mutants of S. meliloti display pleiotropic phenotypic defects, including sensitivities to certain heavy metals (Glenn et al., 1999). The sensitivities of WR1-14 and RT3-27 to heavy metals were therefore compared with their respective wild types. Addition of CuSO4 to TY plates (at pH 7.0) at concentrations above 0.4 mM prevented growth of RT3-27 or WR1-14, whereas WSM419 and WSM710 could tolerate 1 and 2 mM, respectively. The metal sensitivity is restricted to copper since the mutants displayed the same tolerances on tryptone-yeast media (TY) plates as their respective wild types to AgNO3 (25 μM), CdCl2 (200 μM), CoCl2 (500 μM), HgCl2 (10 μM), Ni(NO3)2 (50 μM) or ZnSO4 (500 μM).

The relatively low concentration of copper (2 μM) present in the minimal medium was sufficient to terminate growth of the actP-defective mutants only at low pH; neither R. leguminosarum WR1-14 nor S. meliloti RT3-27 could grow at pH 5.5 or pH 5.7, respectively, unless copper was omitted from a minimal medium prepared with high-purity reagents. The latter finding is a contrast to the observations that acid tolerance cannot be restored to actA, actS or actR mutants by removing heavy metals (Glenn et al., 1999). These data indicate that the disruption of genes in RT3-27 and WR1-14 prevents the expression of a copper-export system. Here, we present an important insight into metal toxicity as the availability of a heavy metal may also change in response to pH-dependent binding.

The Tn5 lesion in RT3-27 or WR1-14 does not impair symbiotic performance

The mutants RT3-27 and WR1-14 produced normal nodules on Medicago murex and Pisum sativum L. (Wirrega pea) respectively. In contrast, no nodules were found on uninoculated seedlings. Retention of Tn5 in nodule occupants was established by reisolating kanamycin-resistant clones. The nodules of inoculated plants were pink–red in colour and plants were healthy and green (in contrast to yellow–light green stunted uninoculated controls), indicative that the mutants were fixing nitrogen. The conditions we have used included a copper supplement of 120 μM in the nutrient solution; under these conditions, the gene that is mutated does not appear to be required for the successful establishment of a normal nitrogen-fixing nodule. It should be noted, however, that the copper concentration in nodules increases with increasing copper application (Snowball and Robson, 1980). The induction of the conserved FixI cation transporter in bacteroids (Kahn et al., 1989) appears to be involved in the uptake and metabolism of copper for the proper functioning of the symbiotically essential cbb3 oxidase (Preisig et al., 1996). It could therefore be envisaged that if excessive copper import occurred via FixI or another transporter a counterbalancing export mechanism might be required. If copper readily crosses the peribacteroid membrane, this situation could potentially be worsened because enveloped bacteroids could be exposed to enhanced copper toxicity in the apparently acidic environment of the symbiosome (Mellor, 1989; Parniske, 2000). In view of the normal nodulation behaviour of the mutants, this appears to be unlikely.

Sequence and analysis

Full sequences for 5.1 and 3.2 kb DNA regions surrounding the Tn5 insertions in the mutants WR1-14 and RT3-27, respectively, showed that the disrupted genes code for cation-transporting ATPases; they have therefore been designated actP (acid-tolerance P-type ATPase). The Tn5 insertions are located 103 and 225 bp from the 3′ end of the actP open reading frame (ORF) in R. leguminosarum and S. meliloti respectively; the sequences immediately surrounding the insertions are not similar. These Tn5 insertions, which are in the C-terminal tenth of the ActP protein in both cases, apparently result in complete inactivation of the protein, possibly suggesting that (if the altered protein is actually produced) the C-terminal domain is functionally or structurally important. Several other genes were identified: hmrR occurred downstream to actP in both organisms while an additional two partial ORFs (treY and ecfR) were located in the R. leguminosarum strain sequence (Fig. 1A).

ActP is a P-type ATPase that belongs to the CPx family. The sizes of the ActP proteins deduced from R. leguminosarum and S. meliloti DNA sequences are 88 and 86 kDa respectively. The pI of S. meliloti ActP is 6.42 whereas that for R. leguminosarum is 5.72. Both proteins are very similar over their entire lengths: the two ActP proteins show 49% identity (and 65% similarity) over 664 amino acids.

A BLASTP search of the databases using the R. leguminosarum and S. meliloti ActP proteins revealed considerable similarity to putative cation-transporting P-type ATPases from numerous organisms. The matches to the R. leguminosarum ActP protein include a putative silver ion transporter (SilP; AF067954) from Salmonella typhimurium (53% identity over 808 amino acids), a putative copper ion transporter (PacS; D16747) from Synechococcus spp. (49% identity over 656 amino acids) and a heavy metal ion transporter (U42410) from Proteus mirabilis (47% identity over 671 amino acids). The matches to the S. meliloti ActP protein include the heavy metal ion-transporting P-type ATPase (U42410) from P. mirabilis (66% identity over 806 amino acids), a heavy metal ion-transporting ATPase (Z99121) from Bacillus subtilis (45% identity over 809 amino acids) and the copper ion-transporting ATPase (PacS; D16747) from Synechococcus spp. (47% identity over 756 amino acids). These proteins have been placed into the CPx subfamily of the P-type ATPases that recognize and transport heavy metals and are distinguished from other ATPases by the presence of the CP(C/H/S) motif, the P-residue generally located 43 amino acids upstream to an E1–E2 ATPase phosphorylation signature site (DKTGTLT) (Solioz and Vulpe, 1996). Both rhizobial ActP proteins contain the CPC motif, 41 amino acids distal to the phosphorylation site. This motif is located in the region considered to form an ion channel; members that transport a similar ion share more conserved residues in this region. Hydrophilicity profiles of both rhizobial ActP proteins calculated from the Kyte–Doolittle algorithm show a typical plot for CPx proteins.

A search for domains (using the programs SCANPROSITE and PROFILESCAN) in the ActP proteins has identified a heavy metal-associated (HMA) domain in the ActP protein of S. meliloti (IEGMTCASCVRRVEKAIAAVPGVASANVNL). This sequence matches the consensus found in other CPx-type ATPases involved with heavy metal transport [L, I, V or N]–X2–[L, I, V, M, F or A]–X–C–X–[S, T, A, G, C, D, N or H]–C–X3–[L, I, V, F or G]–X3–[L, I or V]–X9–11–[I, V or A]–X–[L, V, F, Y or S] (Bull and Cox, 1994; Lin and Culotta, 1995); residues underlined indicate the selections from the consensus domain sequence, while those not underlined are spacer residues. Such an HMA domain is absent from the R. leguminosarum ActP. The Escherichia coli HRA-1, HRA-2 and Enterococcus hirae CopB P-type ATPases also lack the HMA domain, but contain histidine- and methionine-rich leader sequences which Trenor et al. (1994) have suggested bind to heavy metals. The R. leguminosarum ActP sequence also contains a histidine-rich leader which may serve a similar function.

In addition, a prokaryotic lipoprotein lipid attachment site [D, E, R or K]–X6–[L, I, V, M, F, W, S, T, A or G]–X2–[L, I, V, M, F, Y, S, T, A, G, C or Q]–[A, G or S]–C– (C is the lipid attachment site; PROSITE database) found in membrane-associated proteins is present within the ActP amino acid sequences of R. leguminosarum (LAAVAVLIIAC) and S. meliloti (VNAVAVLIIAC), although the initial consensus residue is absent.

HmrR is a putative transcriptional regulator. Directly downstream from actP in R. leguminosarum is an ORF that encodes a protein of 14.7 kDa and a pI of 5.78, whereas in S. meliloti this protein is 16.3 kDa and has a pI of 9.53. The two proteins are 55% identical (and contain 74% similarity) over 129 amino acids. It is interesting that the exopoly-saccharide (EPS) biosynthetic regulator ExoR is also more acidic in R. leguminosarum (pI 5.5) than in its S. meliloti counterpart (pI 7.2) (Reeve et al., 1997). This ORF downstream from actP in both species encodes a protein with highest similarity to transcriptional regulators of the MerR family; these proteins contain the MerR family helix–turn–helix DNA-binding signature in the N-terminal region (Helmann et al., 1989) (GEASKVSGVSSKMIRYYEQIGLI and GEASERSGLPSKTIRYYEDIGLI in S. meliloti and R. leguminosarum respectively; residues underlined are invariant across the proteins YBBI, YBBI and PMTR from E. coli, Haemophilus influenzae and P. mirabilis respectively).

The greatest identity (47% over 126 amino acids for R. leguminosarum and 60% over 128 amino acids for S. meliloti) occurs in the merR family with the heavy metal-dependent transcriptional regulator PMTR of P. mirabilis (Noll et al., 1998). The gene directly downstream to actP in the rhizobia has therefore been named hmrR (heavy metal-response regulator).

Function of TreY and EcfR. Upstream of actP in R. leguminosarum is a partial ORF (treY) that encodes a protein 34% identical over 346 amino acids to a malto-oligosyl trehalose synthase from Brevibacterium helvolum (accession number AAB95368), Arthrobacter sp. (accession number BAA09667) and Rhizobium sp. (accession number BAA11186). Malto-oligosyl trehalose synthase catalyses the conversion of maltodextrins to malto-oligosyl trehaloses through a transglucosylation reaction (Maruta et al., 1996). The match with TreY occurs over the entire length of the partially sequenced R. leguminosarum protein.

Downstream from hmrR in R. leguminosarum is an ORF for a protein with strong similarity to sigma factors of the extracytoplasmic factor (ECF) subfamily. The start codon for this ORF (ecfR) in R. leguminosarum could be an ATG codon or an upstream GTG; both are potential initiation codons, but only the GTG codon is preceded by a ribosome binding site (RBS). EcfR shows the greatest identity to the sigma-E factors (sigma-24) of Mycobacterium tuberculosis (32% over 162 amino acids of SigH; CAB08314), Streptomyces coelicolor (32% over 161 amino acids of SigR; CAB09088), Mycobacterium smegmatis (31% over 163 amino acids of SigH; AAD41810) and Treponema pallidum (33% over 145 amino acids of RpoE; AAC65087). In addition, the BLASTP search revealed 36% and 34% identity to a stretch of 144 amino acids of SigE from M. smegmatis (AA45268) and M. avium (AAC45219).

Typical for ECF sigma factors, the EcfR protein from R. leguminosarum is shorter than other classes of sigma factors and it contains a fis-type (Kostrewa et al., 1992) helix–turn–helix DNA-binding motif EEQREALHLVAIEDLYSQEAAQ (residues underlined are invariant).

These sigma-24 proteins transcribe genes whose products are required for responses to oxidative stress in S. coelicolor (Paget et al., 1998) and both heat shock and oxidative stress in M. tuberculosis (Fernandes et al., 1999). Interestingly, SigE in M. smegmatis has been shown to promote survival against acidic pH in addition to high temperature, detergents and oxidative stress (Wu et al., 1997).

The phenotypic defects are not caused by a polar effect on hmrR expression

Since the hmrR gene is located downstream to actP in both organisms and is transcribed in the same direction, it became prudent to investigate whether the acid and copper sensitivity was caused by a Tn5 polar effect on hmrR expression.

Complementation of RT3-27. Ten individual clones were isolated from a DNA library of S. meliloti WSM419 that could complement both the acid- and copper-sensitive phenotype of RT3-27. Digestion of each complementing plasmid with EcoRI released a 4.1 kb fragment that was cut by HindIII into 2.5 and 1.6 kb fragments. The pattern observed corresponds to that obtained from the rhizobial DNA region in the clone pRT3-27 (Fig. 1C). A 4.8 kb HindIII fragment (in pNK52; Fig. 1) restores copper tolerance to RT3-27 but a 0.9 kb deletion between the EcoRV sites (in bold in Fig. 1) prevented complementation revealing that the actP ORF does indeed extend into the EcoRV fragment, as predicted by sequence analysis.

Disruption of hmrR does not prevent complementation by the plasmid pNK77 while the generation of a frame shift mutation in actP (actP SphI site inactivated; Fig. 1C) destroyed the ability of pNK57 to complement.

Mobilization of pNK52 (containing S. meliloti actP and hmrR) into WR1-14 restores growth of this mutant on JMM plates buffered at pH 5.5 (containing 2 μM CuSO4) and also on TY plates at pH 7.0 containing 0.75 mM CuSO4. Despite the difference in the proposed metal-binding domains for the ActP proteins, it is evident that ActP possessing the HRA domain may be substituted with ActP containing a HMA domain to serve the same function in R. leguminosarum.

Complementation of WR1-14. The plasmid pWR880 was obtained by forcing spontaneous precise excision of Tn5 from actP by selecting for copper-tolerant colonies of WR1-14 that originally contained pWR873. Using this strategy, precise excision (identified from the number of copper-tolerant colonies) could be detected approximately once in every 107 cells. All the copper-tolerant colonies had lost Tn5 from the plasmid, indicating that multiple copies of the wild-type actP gene provided a growth advantage to the cells on copper-containing plates over those cells that lost Tn5 from the single copy actP lesion already in WR1-14. Since the hmrR gene may be co-transcribed with actP in R. leguminosarum, we examined the complementation pattern of plasmids derived from pWR880. The plasmids pWR901 and pWR903 contained only a functional actP gene and were still able to restore the wild-type phenotype to WR1-14.

These observations indicate that the phenotypic defects in both organisms result directly from the loss of the ActP protein and not from a polar effect on the expression of the hmrR gene.

Expression of actP

An actPgusA fusion present in the plasmid pWR668 was transcriptionally induced by copper in R. leguminosarum grown in MJMM-S (see Experimental procedures) containing glutamate as the nitrogen source (Fig. 2A). Transcription of actP increased further at low pH, paralleling the observed increase in copper sensitivity of the mutants at low pH. The expression appears to be specific to copper since no such response was observed after cell exposure to silver or zinc (data not shown). The lack of induction in response to silver indicates that the considerable similarity of ActP to SilS does not extend to function, in line with the lack of any increased sensitivity to silver for both actP mutants.


Figure 2. Transcriptional activation of a plasmid-borne R. leguminosarum actPgusA fusion (pWR668) in WSM711 in response to copper.

A. MJMM-S containing 3 mM glutamate as the nitrogen source at pH 7.0 (filled circles) and pH 5.5 (filled squares).

B. MJMM-S containing 10 mM ammonium chloride as the nitrogen source at pH 7.0 (filled circles) and pH 5.5 (filled squares).

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Transcription is enhanced at low pH presumably because greater protonation of glutamate at acid pH than at neutral pH results in a decrease in copper chel-ation and increased free copper concentrations. In a liquid minimal medium containing 3 mM glutamate and 10 μM copper, the free copper concentration has been calculated to change from 0.00021 to 0.052 μM with a pH shift from 7.0 to 5.5. For the same total concentration of copper in the medium, but with 10 mM NH4Cl replacing the glutamate, the range of free copper concentrations has been calculated to change from 0.47 to 8.7 μM for the same pH change (P. May, personal communication). In line with the theoretical calculations, cells were far more sensitive to added copper in a medium containing NH4Cl, where 10 μM copper prevented growth, than one containing glutamate, which allowed growth in the presence of 100 μM copper.

When glutamate was replaced by NH4Cl in the liquid minimal medium, expression of actPgusA in R. leguminosarum (Fig. 2B) was still pH dependent. In these conditions, however, the fusion was activated in response to far lower concentrations of total copper (compare Fig. 2B and Fig. 2A).

HmrR transcriptionally regulates actP

HmrR and PMTR are both MerR-like transcriptional regulators that have putatively been assigned to the CopR (also called CueR) subgroup (Petersen and Moller, 2000) on the basis of sequence similarity. The role of CopR has been elucidated: it is a copper-responsive transcriptional activator of copA in E. coli (Outten et al., 2000; Petersen and Moller, 2000; Stoyanov et al., 2001). It has been speculated that the members of the CopR subgroup are all copper-responsive transcriptional activators (Petersen and Moller, 2000).

To investigate the role of hmrR in the copper-responsive regulation of actP, we constructed mutant strains by exchanging the wild-type allele for hmrR:CAS-Gm in R. leguminosarum (Fig. 3). The targeted disruption of hmrR created strains that were mildly copper sensitive, the mutants showing no growth at pH 7.0 on TY plates containing 1.75 mM CuSO4 whereas the wild type could grow at 2 mM. The transcriptional activation of the actPgusA fusion in response to copper was eliminated in the hmrR background (Fig. 4). Interestingly, in this background the basal level of expression of the actPgusA fusion increased at both neutral and acidic pH in the absence of copper ions. In contrast, the level of expression of a copAlacZ fusion in an copR mutant of E. coli did not change the activity of β-galactosidase in cells cultured in non-inducing conditions, unlike the wild-type background (Outten et al., 2000; Petersen and Moller, 2000). Our research reveals that HmrR negatively regulates the transcription of actP in the absence of copper ions but positively in the presence of copper ions. The increase in transcription of actP in the HmrR mutant would explain why this mutant tolerates almost as much copper as the wild type. HmrR is therefore the second member of the MerR family that responds to copper ions and therefore does indeed fall into the CopR subgroup initially proposed by Petersen and Moller (2000).


Figure 3. Insertional inactivation of hmrR.

A. Transposon excision. Tn5 was excised from pWR873 as described in the Experimental procedures section to construct pWR880.

B. Cassette insertion and subcloning. A 1.3 kb blunted HindIII fragment containing the CAS-Gm cassette was subcloned from pWRTACT-G2 into the blunted SphI site of hmrR in two orientations. The NotI fragments containing the hmrR:CAS-Gm mutation were subcloned into pJQ2ΩS (filled arrows represent residual aacC1 gene) to form pWR1033 and pWR1037.

C. Marker exchange. The latter plasmids were mobilized into WSM710 and a double crossover forced by plating the mating mixture onto JMM minimal medium containing gentamicin and sucrose (5% w/v). HindIII-restricted genomic DNA from the five streptomycin and spectinomycin-sensitive mutants WR1880 (1), WR1881 (2), WR1882 (3), WR1884 (4), WR1885 (5) and WSM710 (6) was hybridized to DIG-labelled pWR114L–Hp/E to probe for hmrR (D) and DIG-labelled pJQ200SK to probe for the gentamicin resistance gene aacC1 (E). Disruption of hmrR with CAS-Gm should result in a 1.2 kb increase (evident in D and E, lanes 2–5) in size of the 4.0 kb wild-type HindIII fragment (C; and D, lane 6). The size of the 1.2 kb HindIII fragment (C; lower hybridizing band in D) should not be disrupted during the crossover process. The size of fragments of the molecular weight marker from the top are approximately 23, 9.4, 6.6, 5.1, 4.9, 4.4, 4.2, 3.5, 2.3, 2.0, 1.9, 1.5, 1.3, 0.9, 0.8 and 0.5 kb.

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Figure 4. The actP gene is transcriptionally regulated by HmrR in R. leguminosarum in response to copper. β-Glucuronidase expression from the actPgusA fusion located on the broad host range plasmid pWR668 was measured from the wild-type (PW711) and hmrR mutants (WR1880 and WR1885) in the absence (non-filled bars) and presence (filled bars) of 50 μM CuSO4.

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For a constant copper content, higher free copper concentrations will occur at lower pH, presumably resulting in greater influx into the cell. Copper homeostasis at low pH therefore becomes an important component of the resistance of these bacteria to low pH alongside actual resistance to proton stress per se. The rhizobial ActP protein would therefore be the means for exporting excess copper from the cell, forming a vital line of defence against a toxic copper intake. The capacity to produce the required activity of ActP to resist acid-associated copper toxicity is dependent on HmrR being able to induce actP expression.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References

Bacterial strains, plasmids and media

Bacterial strains and plasmids used in this study are listed in Table 1. Escherichia coli strains were grown at 37°C on Luria–Bertani (LB) medium (Miller, 1972) or antibiotic medium no. 3 (AM3) medium (Oxoid) when using gen-tamicin. Sinorhizobium and Rhizobium strains were grown in either TY medium (Beringer, 1974) or JMM minimal medium (O’Hara et al., 1989). Modified JMM (MJMM) was prepared by omitting the trace metal salts MnSO4, ZnSO4, Na2MoO4 and CuSO4 from JMM. Buffers and carbon sources were incorporated into MJMM after they had been passed through a CPG/8-hydroxyquinoline column, as described by Carson et al. (1992) to prepare MJMM-S. This medium contained either glutamate (3 mM) or NH4Cl (10 mM) as the nitrogen source. Media were supplemented with the following concentrations of antibiotics (μg ml–1): ampicillin (100), chloramphenicol (20), gentamicin (30; 7.5 for E. coli), kanamycin (50), streptomycin (200; 30 for E. coli) and tetracycline (20; 12.5 for E. coli). Agar was added at a concentration of 1.5% (w/v) to solidify media.

Table 1. List of strains and plasmids used
Strains/plasmidsRelevant characteristicsSource/reference
  • a.

    Centre for Rhizobium Studies.

  • Acid tolerant (Acidt); Cu sensitive (Cus) or tolerant (Cut); resistance to ampicillin (Apr), chloramphenicol (Cmr), gentamicin (Gmr), kanamycin (Kmr), streptomycin (Smr), sucrose (Sucs) or tetracycline (Tcr).

E. coli
BW20767RP4-2-tet:Mu-1kan::Tn7 integrant leu-63::IS10 recA1 Metcalf et al. (1996)
  creC510 hsdR17 endA1 zbf-5 uidAMluI):pir+thi
DH5αFφ80dLacZΔM15 recA1 endA1 gyrA96 thi-1 hsdR17 (rKmK+) supE44 relA1 deoRΔ(lacZYAargF)U169Life Technologies
DH10BFmcrAΔ(mrrhsdRMSmcrBC) φ80dLacZΔM15 lacX74 endA1 recA1 deoRΔ(ara, leu)7697 araD139 galU galK nupG rpsLLife Technologies
HB101Fthi-1hsdS20 (rBmB) supE44 recA13 ara-14 leuB6 proA2 lacY1 rpsL20 (Smr) xyl-5 mtl-1 Boyer and Roulland-Dussoix (1969)
MT616 pro-82 thi-1 hsdR17 supE44 endA1 recA56 (pRK600); Cmr Finan et al. (1986)
S. meliloti
RT3-27 actP::Tn5 mutant of WSM419; Cus, Cmr, KmrThis study
WSM419Acidt Sardinian isolate; Cmr, CutJ. Howiesona
R. leguminosarum bv. viciae
PW711Spontaneous Smr mutant of WSM710 Reeve et al. (1999)
WR1-14 actP::Tn5 mutant of WSM710; Cus, Kmr, SmrThis study
WR1870 hmrR:Gm mutant of WSM710; Cut, GmrThis study
WR1871 hmrR:Gm mutant of WSM710; Cut, GmrThis study
WR1880WR1870 (pMUE668); Tcr, Gmr, KmrThis study
WR1885WR1875 (pMUE668); Tcr, Gmr, KmrThis study
WSM710Acidt Japanese isolate; CutJ. Howieson
pBR01pFUS1 derivative constructed by replacing the PstI/NotI fragment containing gusA with the PstI/NotI MCS from pUK21; TcrBeau Fennera
pBR322Cloning vector; Apr Tcr Bolivar et al. (1977)
pCRS433Broad host range vector; Tcr Reeve et al. (1999)
pCRS487pUT::mTn5-GNm; Apr, Kmr Reeve et al. (1999)
pFUS1pCRS433 derivative containing a promoterless gusA; Tcr Reeve et al. (1999)
pJQ2ΩSpJQ200SK derivative constructed by replacing a blunted EcoRV/SphI fragment containing aacC1 with a blunted HindIII fragment containing ΩSm/Sp; Sm/Spr, SucsSophie Rome
pJQ200SKGene replacement vector; Gmr, Sucs Quandt and Hynes (1993)
pNK52pSW213 containing actP and hmrR on a 4.8 kb HindIII fragment; TcrThis study
pNK57pFUS1 containing a mutated actP and wild-type hmrR on a 4.2 kb HindIII/ApaI fragment (Fig. 1); TcrThis study
pNK77pFUS1 containing a 4.8 kb S. meliloti HindIII fragment carrying actP and hmrR:CAS-GNm (inactivation of the hmrR BclI site); Tcr, KmrThis study
pRK2013Helper plasmid; Kmr Figurski and Helinski (1979)
pRT327RT3-27 EcoRI fragment containing Tn5 cloned in pBR322; Apr, KmrThis study
pRT327-1Complementing plasmid clone from WSM419 genomic library; TcrThis study
pSW213Broad host range cloning vector; Tcr Chen and Winans (1991)
pTn5-H HindIII fragment containing nptII from Tn5 in pUC18; Apr, KmrThis study
pWR053 HindIII fragment of pWR114 containing right inverted repeat of Tn5 and associated rhizobial flanking sequence in pBR322; AprThis study
pWR114Tn5-containing EcoRI fragment of WR1-14 in pBR322; Apr, Kmr, TcrThis study
pWR114L–Hp/E HpaI–EcoRI fragment containing a portion of Tn5 IS50L and associated rhizobial DNA from pWR1-14 in pGEM-7Zf(+); AprThis study
pWR327-200Tn5-containing EcoRI fragment from RT3-27 in pJQ200SK; Gmr, KmrThis study
pWR657 NotI fragment containing CAS-GNm cloned from pCRS487 into the NotI site of pWR053; Apr, KmrThis study
pWR668 HindIII–EcoRI blunted fragment containing the actPgusA fusion from pWR657 in a blunted XhoI site of pCRS433; Tcr, KmrThis study
pWR873 EcoRI fragment containing Tn5 from WR1-14 in pBRO1; Tcr, KmrThis study
pWR880pWR873 devoid of Tn5; TcrThis study
pWR901pWR880 derivative containing actP and hmrR:CAS-Gm (inactivation of the hmrR SphI site); Tcr, GmrThis study
pWR903pWR880 derivative containing actP and hmrR:CAS-Gm (inactivation of the hmrR SphI site); Tcr, GmrThis study
pWR1033 NotI fragment containing CAS-Gm cloned from pWR901 into NotI pJQ2ΩS; Sucs, Gmr, Smr, SprThis study
pWR1037 NotI fragment containing CAS-Gm cloned from pWR903 into NotI pJQ2ΩS; Sucs, Gmr, Smr, SprThis study
RL38Generalized transducing phage Buchanan-Wollaston (1979)


Seeds of Pisum sativum L. (Wirrega pea) or Medicago murex were surface sterilized, germinated and sown as described by Reeve et al. (1999). Immediately after planting, pea seedlings were inoculated with a Rhizobium culture (either WSM710 or WR1-14) and medic seedlings with a Sinorhizobium culture (either WSM419 or RT3-27). Plants were watered with sterile nutrient solution, and isolates were recovered from surface-sterilized crushed nodules as described by Reeve et al. (1999).

DNA manipulation and analysis

Plasmid or genomic DNA isolation, transformation and DNA manipulation techniques have been described previously (Reeve et al., 1999). DNA sequencing and analysis were as reported by Tiwari et al. (1996b). Potential proteins were identified using the BLASTP program and the algorithm generated by Altschul et al. (1997). Protein motifs were identified using the programs SCANPROSITE and PROFILESCAN located on the ExPASy Molecular Biology Server provided as a service by the Swiss Institute of Bioinformatics (SIB).

Plasmid mobilization

Plasmid DNA was mobilized from non-RP4-containing E. coli strains (such as DH5α or DH10B) into Rhizobium or Sinorhizobium by performing a triparental mating with either HB101 (pRK2013) or MT616. Plasmids were mobilized from BW20767 using a biparental mating. Both methods used the conjugation protocol described by Reeve et al. (1999).

Phage transduction

The method of Buchanan-Wollaston (1979) was used to prepare phage RL38 stocks and transduce WSM710. EcoRI-digested genomic DNA of the wild-type and kanamycin-resistant transductants was probed with DIG-labelled pWR114 to show that the mutants contained a single copy of Tn5 in actP.

Reconstruction of a S. meliloti actP mutant

An EcoRI digest of pRT327 was ligated to a partial EcoRI digest of pJQ200SK and the reaction mixture transformed into E. coli BW20767. Transformants were selected for gentamicin and kanamycin resistance and replica patched for sucrose sensitivity. In this way, the 9.0 kb EcoRI fragment containing Tn5 (from pRT327) was cloned into the EcoRI site in the multiple cloning site of pJQ200SK to construct pWR327-200. The latter construct was mobilized from BW20767 into WSM419 to exchange the wild-type actP gene in S. meliloti WSM419 with actP::Tn5 via homologous recombination. Transconjugants were selected on JMM plates (pH 7.0) containing chloramphenicol, streptomycin, kanamycin and sucrose (10% w/v) and then patched for gentamicin sensitivity. The replacement of actP for actP::Tn5 was confirmed by hybridization of EcoRI-digested DNA of putative actP mutants to DIG-labelled pRT327.

DNA hybridizations

DNA labelling, Southern hybridization and probe detection were as described previously (Tiwari et al., 1996b).

Complementation analysis

S. meliloti. A DNA library of WSM419 (Tiwari et al., 1996b) was mobilized from DH10B into S. meliloti RT3-27. Sinorhizobium transconjugants were selected on TY plates containing chloramphenicol, kanamycin, tetracycline and 0.75 mM CuSO4. Plasmid DNA was isolated from Sinorhizobium transconjugants using alkaline lysis (Sambrook et al., 1989) and then transformed into BW20767. Plasmid DNA was extracted from these transformants, purified by CsCl isopycnic centrifugation and mapped for restriction sites. One complementing plasmid (designated as pRT327-1) that contained the S. meliloti actP wild-type gene was mobilized from BW20767 into S. meliloti RT3-27 and R. leguminosarum WR1-14. Transconjugants were selected on JMM plates containing kanamycin and tetracycline. The ability of plasmid pRT327-1 to complement the actP defect in the two mutants was assessed by comparing phenotypes of transconjugants against the wild type on JMM plates in the presence and absence of copper or on low-pH-buffered JMM plates.

R. leguminosarum. The EcoRI fragment containing actP::Tn5 was subcloned from pWR114 into the broad host range vector pBRO1 to construct pWR873. The latter plasmid was mobilized into WR1-14 and the transconjugants grown to saturation in TY containing tetracycline and streptomycin. Aliquots of the culture were spread onto TY plates containing tetracycline, kanamycin and 0.75 mM CuSO4. Plasmid DNA was extracted from 25 copper-tolerant colonies and transformed into E. coli DH5α. Transformants were replica patched onto LB containing tetracycline and then onto LB containing tetracycline and kanamycin. Plasmid DNA was extracted from tetracycline-resistant and kanamycin-sensitive colonies and the restriction profile compared with pWR873 to ensure that Tn5 had excised from actP. One of the complementing plasmids (pWR880) was chosen for further work.

Construction of an actPgusA fusion

A NotI fragment containing CAS-GNm (Reeve et al., 1999) was cloned from pCRS489 into the NotI site of pWR053 to construct pWR657. A HindIII–EcoRI fragment containing the actPgusA fusion from pWR657 was end-filled and then cloned into a blunted XhoI site of the broad host range plasmid pCRS433 to create pWR668. The latter plasmid was mobilized from E. coli BW20767 into R. leguminosarum bv. viciae PW711, WR1870 or WR1875 by selecting for tetracycline-resistant transconjugants on JMM plates.

Expression studies

Cultures containing pWR668 were inoculated into 5 ml of MJMM (glutamate or ammonium chloride as the nitrogen source) at pH 7.0 containing kanamycin. Cultures were grown at 28°C to an optical density at 600 nm (OD600) of approximately 0.4–0.8. Suspensions were centrifuged, concentrated and resuspended in MJMM-S (with kanamycin) in the presence or absence of copper at pH 7.0 or 5.5 to obtain an OD600 of approximately 0.25–0.5 after overnight incubation at 28°C.

Measurement of GUS activity in microplates

A sample of each culture (3 ml) was centrifuged and resuspended in saline (0.9% NaCl, w/v) to an OD600 of approximately 1. Aliquots (80 μl resuspended culture with 120 μl saline) were dispensed into flat-bottomed microplate (Sarstedt Australia) wells and the OD595 measured in a Bio-Rad Model 550 Microplate Reader controlled by MICROPLATEMANAGER software.

For the assay, aliquots of 50 and 125 μl were dispensed in duplicate into four Eppendorf tubes for each culture. The volume was adjusted to 125 μl using saline. β-Glucuroni-dase (GUS) buffer (500 μl) and toluene (two drops) were dispensed into each tube and vigorously vortexed for exactly 10 s (Reeve et al., 1999). The step was performed in Eppendorf tubes to ensure complete cell permeabilization by toluene. These Eppendorf tubes were incubated at 37°C for 30 min with the lids open to evaporate toluene. The tubes were then transferred to 28°C for 5 min. An aliquot of 200 μl from each Eppendorf tube was dispensed into a flat-bottomed microplate well. To start the reaction, aliquots of 5 μl of p-nitrophenyl-β-D-glucuronide (pNPG; 35 mg ml –1in water) were dispensed into the wells using a multichannel pipette. The OD405 was measured every 2 min over 1.5 h using a Bio-Rad Model 550 Microplate Reader controlled by MICROPLATEMANAGER software.

Four replicates per strain were assayed from two separate experiments. β-Glucuronidase specific activity was ex-pressed as nmol p-nitrophenol (pNP) produced per min per OD595 at 28°C.

Accession numbers

The nucleotide sequences derived from R. leguminosarum bv. viciae WSM710 and S. meliloti WSM419 have been lodged with GenBank and have been assigned the accession numbers AF127795 and AF129004 respectively.


  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References

This work was generously supported by the Australian Research Council (ARC). N.B.K. was funded by the John Crawford Scholarship Award and AusAid. We would like to thank Peter May for the calculation of the free copper concentrations in our media, Claude Defives for extensively testing WSM419 for phage susceptibility and Sophie Rome for the plasmid pJQ2ΩS.


  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References
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