A helicase gene (helO) in Rhizobium meliloti WSM419

Authors

  • Wayne G Reeve,

    1. Nitrogen Fixation Research Group, School of Biological and Environmental Sciences, Murdoch University, Murdoch, WA 6150, Australia
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  • Ravi P Tiwari,

    1. Nitrogen Fixation Research Group, School of Biological and Environmental Sciences, Murdoch University, Murdoch, WA 6150, Australia
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  • Michael J Dilworth,

    1. Nitrogen Fixation Research Group, School of Biological and Environmental Sciences, Murdoch University, Murdoch, WA 6150, Australia
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  • Andrew R Glenn

    Corresponding author
    1. Nitrogen Fixation Research Group, School of Biological and Environmental Sciences, Murdoch University, Murdoch, WA 6150, Australia
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Corresponding author. Tel.: +61 (9) 360 6158; Fax: +61 (9) 360 2931; E-mail: arglenn@central.murdoch.edu.au

Abstract

A 2.8 kb Bam HI DNA fragment adjacent to a Bam HI fragment containing actR-actS (a sensor/regulator pair required for low pH tolerance in Rhizobium meliloti WSM419) was cloned and sequenced. A computer predicted protein of 821 amino acids, designated HelO, showed extensive similarity with ‘DEAH’ motif helicases. Expression of helO was higher at pH 7.0 than pH 5.8 and it did not require the product of the actR gene. Inactivation of helO by insertion of a Ω interposon at codon 40 did not affect nodulation, growth or tolerance to low pH, high temperature, osmolarity or elevated levels of copper or zinc.

1Introduction

In the Medicago-Rhizobium meliloti symbiosis the bacterial partner is more sensitive to proton stress than its legume partner[1]. The isolation of acid-tolerant strains of R. meliloti like WSM419 from acidic soils of the Mediterranean[2] has led us to investigate the mechanism allowing these strains to function under acid conditions when conventional inoculants do not [3–6].

A two component sensor-regulator pair (ActS/R) is essential for growth of R. meliloti WSM419 at low pH[6]. Mutations in either the sensor protein, ActS, or the regulator, ActR, result in an acid-sensitive phenotype[6]. In some cases, genes controlled by such two component signal transduction systems are adjacent to the sensor-regulator pair [7, 8]. To explore this possibility in strain WSM419 the DNA directly upstream from actS was cloned, mapped and sequenced. Inactivation and expression studies indicate that a presumptive RNA helicase immediately downstream of actR/actS is not controlled by actR. This novel finding is the first report of a helicase in root nodule bacteria.

2Materials and methods

2.1Bacterial strains, plasmids, and media

Bacterial strains and plasmids are described in Table 1. Strains of Rhizobium were grown at 28°C in JMM minimal medium[9]. Escherichia coli strains were grown in Luria-Bertani (LB) medium at 37°C. Media were supplemented with the following concentrations of antibiotics (μg ml−1): ampicillin (100), chloramphenicol (20), gentamicin (40), kanamycin (50), or tetracycline (20).

Table 1.  List of strains and plasmids used
Strains/plasmidsRelevant characteristicsSource/reference
  1. aWestern Australian Department of Agriculture.

  2. r, resistance to; Ap, ampicillin; Cm, chloramphenicol; Gm, gentamicin; Km, kanamycin; Sp, spectinomycin; Sm, streptomycin; Tc, tetracycline.

  3. Acidt or Acids represents acid-tolerant or acid-sensitive, respectively.

Strains
E. coli 
 DH5αF f80dlac ZDM15 rec A1 end A1 gyr A96 thi-1 hsd R17 (rKmK+) sup E44 rel A1 deo R D(lac ZYA-arg F)U169Bethesda Research Laboratory
R. meliloti
 RT40RhelO 40; ΩKm interposon inserted into codon 40 of helO in WSM419; Cmr, KmrThis study
 RT488helO 488; ΩKm interposon inserted into codon 488 of helO in WSM419; Cmr, KmrThis study
 TG5-46actR::Tn5 mutant of WSM419 (Acids); Cmr, Kmr[9]
 WSM419Acidt Sardinian isolate; CmrJ. Howiesona
Plasmids
 pGEM-7Zf(+)Cloning vector; AprPromega Corporation
 pMP220Broad host range lacZ fusion vector; Tcr[12]
 pPH1J1Broad host range IncP plasmid; Gmr, Smr, Spr[15]
 pHP45ΩKmpHP45Ω derivative with Kmr interposon; Apr, Kmr[19]
 pRK2013Helper plasmid; Kmr[13]
 pRT546-15The 0.8 kb Kpn I-Cla I fragment (Fig. 1) cloned in pGEM-7Zf(+); AprThis study
 pRT546-18The 3.5 Kpn I-Eco RI fragment from pRT546-51 was cloned as an Eco RI fragment into pSUP102[21]; TcrThis study
 pRT546-21A Bam HI fragment containing the ΩKm interposon was cloned into the Bgl II site of pRT546-18; Kmr, TcrThis study
 pRT546-51A 5.5 kb Kpn I frgment from pRT546-1[6] was cloned into the Kpn I site of pGEM-7Zf(+); AprThis study
 pRTHEL-1The 9.5 kb Eco RI fragment containing the ΩKm interposon from RT488 was cloned into the Eco RI site of pGEM-7Zf(+); Apr, KmrThis study
 pRTHEL-3The recircularised 8 kb Cla I fragment from pRTHEL-1 containing pGEM-7Zf(+); AprThis study
 pRTHEL-5RA blunted Hin dIII fragment containing the ΩKm interposon cloned at the blunted Kpn I site of pRTHEL-3; Apr, KmrThis study
 pRTHEL-6A 3.5 kb Eco RI/Kpn I DNA fragment cloned from pRTHEL-1 into pMP220; TcrThis study
 pRTHEL-12RA blunted Hin dIII/Eco RI fragment of pRTHEL-5R was cloned at a blunted Bgl II site of pMP220; Kmr, TcrThis study

2.2Studies on stress tolerance

A pH sensitivity assay was performed as described earlier[3] by spotting 104 cells on JMM solid medium at neutral and acidic pH. Stress tolerance was determined by viable count by spreading 102 or 104 cells onto TY[9] plates and TY plates containing copper (1 mM), sucrose (10%) or zinc (0.75 mM) and incubating at 28°C or 37°C.

2.3Nodulation tests

Medicago murex or Medicago sativa seeds (Agriculture-Western Australia) were surface sterilised with 0.2% (w/v) HgCl2 for 3–5 min and washed five times with sterile deionised water. The seeds were germinated on TY agar prior to sowing in a neutral pH local yellow Jandakot sand:clay (50:50 w/w) mixture which had been steam treated twice at 90°C for 90 min. Immediately after planting, germinated seeds were inoculated with R. meliloti WSM419 or RT40R. Inoculated and uninoculated seedlings were incubated at 22°C and the pots watered to 50% capacity. Nodules isolated from 4 week old plants were sterilised, crushed in sterile saline and the suspension replica patched onto solid medium in the presence and absence of kanamycin.

2.4DNA preparation and manipulation

Plasmid DNA isolation and manipulation techniques and transformation were as described earlier [10, 11]. Restriction and modification enzymes were purchased from either Life Technology Inc. or Boehringer Mannheim Ltd. Genomic DNA was isolated from 3 ml of a log phase culture of Rhizobium grown in TY. The culture was transferred into two tubes and centrifuged. Cells were washed with 1 ml of TES (30 mM Tris-HCl pH 8.0, 5 mM EDTA and 50 mM NaCl) buffer, pelleted and resuspended in 0.5 ml TES buffer. The latter step was repeated and 100 μl of lysozyme solution (10 mg ml−1 in TES buffer) was added and the mixture was incubated at 37°C. After 30 min, 75 μl of SDS (10% solution) and 30 μl proteinase K (6 mg ml−1 in TES) were added and the mixture incubated at 45°C for 1 h. Another aliquot of 30 μl proteinase K was added and the mixture incubated at 55°C for 1 h. Two successive extractions were performed with phenol:chloroform:isoamyl alcohol (25:24:1, by volume) followed by chloroform:isoamyl alcohol (24:1, by volume). DNA was precipitated with the addition of 0.1 volume of 3 M sodium acetate (pH 5.2) and an equal volume of room temperature isopropanol. The mixture was left for 1 h, centrifuged and the pellet resuspended in TE buffer.

2.5DNA sequencing and analysis

DNA sequencing was carried out by using an Applied Biosystems Prism Ready Reaction kit and automated DNA sequencer. DNA sequences were analysed using the MacVector DNA software analysis package. Databases (GB and EMBL) were searched using the ANGIS facility at the University of Sydney.

2.6Hybridisation techniques

The plasmid pHP45ΩKm was labelled with digoxigenin-11-dUTP (Boehringer Mannheim) as described by the manufacturer and used as a probe in hybridisation studies to detect allele exchange in RT40R and RT488. Southern hybridisation was performed as described earlier[5]. The construct pRT546-15 containing the intragenic DNA sequence from helO was used as a probe to determine the copy number of helO in the genome of R. meliloti WSM419.

2.7Expression studies

The 3.5 kb Eco RI-Kpn I rhizobial fragment containing the start codon of helO and the upstream DNA was cloned into the Eco RI-Kpn I sites of the promoterless lacZ broad host range expression vector pMP220[12] to construct pRTHEL-6. The plasmid pRTHEL-6 was mobilised into WSM419 (wild-type) and TG5-46 (actR::Tn5) using the helper plasmid pRK2013[13]. β-Galactosidase was assayed as described earlier[14] and protein concentration was measured using a Bio-Rad protein assay kit. β-Galactosidase specific activity was expressed as nmol o-nitrophenol produced min−1 mg protein−1 at 28°C.

2.8Allele exchange

Insertional inactivation of helO was performed by cloning an Ω-Km interposon into unique intragenic Kpn I or Bgl II sites.

The 3.5 kb Eco RI/Kpn I rhizobial DNA fragment containing helO was cloned as an Eco RI fragment from pRT546-51 into pSUP102 to construct pRT546-18. A Bam HI fragment containing an Ω-Km interposon was cloned from pHP45Ω-Km into the unique Bgl II site of pRT546-18 to construct pRT546-21. This insertionally inactivated helO gene was designated as helO488::ΩKm. The plasmid pRT546-21 was mobilised into cells of R. meliloti WSM419 using the helper plasmid pRK2013. Kanamycin resistant colonies that were tetracycline sensitive were selected from JMM plates.

A blunted Hin dIII fragment containing an Ω-Km interposon was cloned in a blunted Kpn I site present within the 4.2 kb Eco RI/Cla I rhizobial DNA fragment in the plasmid pRTHEL-3 to create pRTHEL-5R. This insertionally inactivated helO gene was designated helO40R::ΩKm. The insertionally inactivated helO gene was delivered into cells of R. meliloti WSM419 using pRTHEL-12R and the helper plasmid pRK2013. Selection for marker exchange was forced using the incompatible plasmid pPH1J1[15]. Gentamicin and kanamycin resistant colonies that were tetracycline sensitive were selected from JMM plates.

Allele replacement of the wild-type helO of WSM419 with helO40R::ΩKm or helO488::ΩKm created strains RT40R or RT488, respectively. Verification of allele replacement was performed by Southern hybridisation and by cloning, restriction mapping and partially sequencing the Eco RI fragments containing the kanamycin resistance determinant from RT40R and RT488.

2.9Nucleotide sequence accession number

The nucleotide sequence of the 2.8 kb Bam HI fragment containing the putative helO gene has been lodged with the GenBank DNA sequence database (accession number U49051).

3Results and discussion

3.1Cloning, sequencing and analysis of the gene upstream and adjacent to the actR/S sensor-regulator pair

The 8.75 kb DNA fragment from R. meliloti WSM419 containing actS/R[6] was restriction mapped (Fig. 1A). The 2.8 kb Bam HI fragment from this region (dashed line in Fig. 1A) was sequenced. Analysis of the DNA sequence (GenBank accession number U49051) identified a potential open reading frame (ORF) transcribed in the same orientation as actS and actR (Fig. 1). This ORF encodes a computer predicted protein of 821 amino acids with a molecular mass of 88 915 Da.

Figure 1.

A: Restriction map of the 8.75 kb DNA fragment containing helO and actS/R. The RIME1 (Rhizobium-specific intergenic mosaic element) sequence[20] is represented as a hatched box. The single Cla I site not affected by the dam methylation system is marked by an asterisk. The dashed line between two Bam HI sites represents the DNA sequenced in this study. B: The 3.5 kb Eco RI-Kpn I fragment (shown by the boxed region) was fused to a promoterless lacZ in the plasmid pMP220 to construct pRTHEL-6. C: The clone pRT546-21 was derived by inserting the ΩKm interposon into the unique Bgl II site (marked by a triangle) present within the 3.3 kb Kpn I-Eco RI fragment. D: The ΩKm interposon was inserted at the Kpn I site (marked by a triangle) of the 4.2 kb Eco RI-Cla I fragment to construct the plasmid pRTHEL-12R.

3.2HelO is homologous with proteins of the helicase family

FASTA analysis showed that this protein has significant homology to helicases (Table 2) carrying the specific ‘DEAH’ signature in motif II (Fig. 2). Therefore it has been designated a putative helicase (HelO). It showed maximum homology (44% identity and 66% similarity over 758 amino acids) with HrpB of E. coli (unpublished; Swiss Prot accession number P37024). Proteins showing extensive similarity with HelO belong to superfamily 2 (SF2;[16]) and are closely related to the ‘DEAD’ protein family of putative RNA helicases[17]. All seven conserved motifs for ‘DEAH’ helicase proteins are present in HelO (Fig. 2). Motifs I and II (containing the Walker-type nucleotide triphosphate (NTP)-binding pattern) are present not only in helicases, but also in a wide variety of other NTPases and are therefore thought to directly interact with NTP substrates[16]. Motif VI is part of a basic domain suggested to interact with RNA[17]. Mutations in the distal segment of this motif result not only in impaired RNA helicase activity, but also in decreased ATPase activity[16]. No DNA or RNA dependent ATPase activity could be detected for the Lhr protein of E. coli even though it contained all seven conserved motifs[18].

Table 2.  Specific activity of β-galactosidase from cells grown in JMM minimal medium at different pH
  1. β-Galactosidase activity is expressed as nmol o-nitrophenol produced min−1 mg protein−1 at 28°C. The background β-galactosidase activity, contributed by pMP220 in WSM419 grown in similar conditions, has been subtracted.

ClonepH 7.0pH 5.6
WSM419/pRTHEL-6 (PhelO)1050655
TG5-46 (WSM419 actR::Tn5)/pRTHEL-6 (PhelO)1080522
Figure 2.

Alignment of HelO and related DEAH helicases. Only the seven conserved motifs (designated I–VI as described by[17]) from CLUSTAL W analysis are presented. The numbers indicate the distances between motifs and the distance from protein termini. All of the residues conserved among the response regulators are highlighted with an asterisk. Similar residues are marked with a dot. The plasmids pRTHEL-12R and pRT546-21 were mobilised into R. meliloti WSM419 and transconjugants containing the correct allele replacement were named RT40R and RT488, respectively. Arrows present within the boxed regions represent the direction of transcription of the neomycin phosphotransferase II (nptII) gene. Proteins are from the following sources: Ce, Caenorhabditis elegans; Ec, Escherichia coli; Hi, Haemophilus influenzae; Hs, Homo sapiens; Rm, Rhizobium meliloti; Sc, Saccharomyces cerevisiae; Sp, Saccharomyces pombe.

3.3Expression of pRTHEL-6 in the wild-type R. meliloti WSM419 and the Tn5-induced derivative TG5-46 (actR::Tn5)

To investigate whether the helO gene was induced at low pH and/or regulated by the ActR protein, a 3.5 kb Eco RI-Kpn I rhizobial DNA fragment (Fig. 1B) containing 3.38 kb of DNA upstream to the ATG start codon of helO was inserted in front of a promoterless lacZ. This construct (pRTHEL-6) was mobilised into WSM419 (wild-type) and TG5-46 (the mutant defective in the acid-tolerance regulator gene actR; actR::Tn5). The β-galactosidase activity of this helO-lacZ fusion was monitored at pH 7.0 and 5.6. β-Galactosidase activity from pRTHEL-6 in the wild-type WSM419 was about two-fold higher in cells at pH 7.0 compared with those grown at low pH. This suggests that the helO gene is not induced at low pH (Table 2). Comparison of β-galactosidase activity in cells with and without a functional actR suggested that actR did not play a pivotal role in helO expression (Table 2).

3.4Insertional inactivation of helO in R. meliloti WSM419 by allele exchange

To determine whether HelO is essential for cell growth under a variety of conditions the wild-type helO gene was insertionally inactivated by allele exchange. An Ω-Km interposon was inserted into the unique intragenic Kpn I and Bgl II site which corresponded to codon positions 40 and 488 of helO, respectively (Fig. 2). Allelic replacement of the wild-type helO with helO488::ΩKm and helO40R::ΩKm created strain RT488 and RT40R, respectively. Our previous experience using the ΩKm interposon indicated that transcription of nptII did not stop at the terminator downstream to nptII. The mutant strains RT40R and RT488 have an Ω interposon inserted such that nptII transcription is in the opposite direction to helO so that it does not cause any downstream transcription.

3.5helO is a single copy gene in R. meliloti WSM419

An intragenic helO probe hybridised to a 7.3 kb Eco RI genomic DNA of WSM419 (Fig. 3) and it is similar to the Eco RI fragment from the mapped helO gene region in Fig. 1. The helO probe also hybridised with a single Eco RI genomic fragment of 9.5 kb from the mutant RT40R (Fig. 3), it confirmed that the mutated helO fragment containing the 2.2 kb interposon had replaced the wild-type allele. These results indicate that the helO gene is present in a single copy in R. meliloti WSM419.

Figure 3.

Southern blot of Eco RI-digested genomic DNA probed with the DIG-labelled Cla I/Kpn I intragenic probe for the helO gene from R. meliloti WSM419. Lanes: 1, R. meliloti WSM419 (wild-type); 2, R. meliloti RT40R (helO::ΩKm); 3, DIG-labelled Hin dIII λ marker.

Both helicase mutants grew in a manner indistinguishable from the wild-type at pH 7.0 and pH 5.7 and were therefore unchanged in their acid-tolerance. The RT40R strain was chosen for further detailed analysis of its phenotype relative to the wild-type WSM419. This mutant produced normal nodules on both Medicago murex and Medicago sativa. No nodules were found on uninoculated seedlings. In addition, RT40R was unaffected in its ability to grow in the presence of other adverse environmental factors such as elevated temperature, high osmotic pressure or increased concentrations of heavy metals like zinc and copper. In this respect the helO defective mutant of R. meliloti WSM419 appears to be similar to mutants in the helicase gene (lhr) in E. coli[18] which appeared to be unaffected in their response to external stresses. The role of these presumptive RNA helicases in eubacterial cells is yet to be identified.

Acknowledgements

This work was supported by the Australian Research Council.

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