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PHYSIOLOGY AND BIOCHEMISTRY

Effects of spontaneous mutations in PipX functions and regulatory complexes on the cyanobacterium Synechococcus elongatus strain PCC 7942

Javier Espinosa, Miguel Angel Castells, Karim Boumediene Laichoubi, Karl Forchhammer, Asunción Contreras

  • 1División de Genética, Universidad de Alicante, Apartado 99, E-03080 Alicante, Spain
  • 2Lehrstuhl für Mikrobiologie, Organismische Interaktionen, University Tübingen, Auf der Morgenstelle 28 D-72076 Tübingen, Germany
  • Correspondence
    Asunción Contreras
    contrera{at}ua.es

    • Microbiology 2010; 156(5):1517–1526 · https://doi.org/10.1099/mic.0.037309-0

    View at publisher PubMed

    Abstract

    In Synechococcus elongatus sp. PCC 7942, PipX forms complexes with PII, a protein found in all three domains of life as an integrator of signals of the nitrogen and carbon balance, and with the cyanobacterial nitrogen regulator NtcA. We recently showed that previous inactivation of pipX facilitates subsequent inactivation of the glnB gene. Here, we show that the three spontaneous pipX point mutations pipX-92delT, pipX160C>T and pipX194T>A, initially found in different glnB strains, are indeed suppressor mutations. When these mutations were reconstructed in the wild-type background, the glnB gene could be efficiently inactivated. Furthermore, the point mutations have different effects on PipX levels, coactivation of NtcA-dependent genes and protein–protein interactions. Further support for an in vivo role of PipX–PII complexes is provided by interaction analysis with the in vivo-generated PIIT-loop+7 protein, a PII derivative unable to interact with its regulatory target N-acetyl-l-glutamate kinase, but which retains the ability to bind to PipX. The implications of these results are discussed.

    • Two supplementary figures, showing conservation involving amino acid residues 54 and 65 on PipX proteins and yeast two-hybrid interactions involving PII and PipX derivatives, are available with the online version of this paper.

    Edited by: C.-C. Zhang

    INTRODUCTION

    Cyanobacteria are phototrophic organisms that perform oxygenic photosynthesis. Autotrophic growth requires the constant assimilation of ammonium via the glutamine synthetase–glutamine oxoglutarate aminotransferase glutamate cycle, resulting in consumption of 2-oxoglutarate (Muro-Pastor et al., 2005). Due to the lack of 2-oxoglutarate dehydrogenase in cyanobacteria, synthesis of 2-oxoglutarate represents the final step in the oxidative branch of the tricarboxylic acid cycle and directly links 2-oxoglutarate levels to nitrogen assimilation (Muro-Pastor et al., 2001). Thus, 2-oxoglutarate accumulates during nitrogen starvation, making this metabolite an excellent indicator of the intracellular carbon to nitrogen balance (Forchhammer, 2004; Laurent et al., 2005).

    In cyanobacteria, multiple metabolic and developmental processes are induced by nitrogen starvation. NtcA, the global regulator for nitrogen control, activates genes involved in nitrogen assimilation, heterocyst differentiation and acclimation to nitrogen starvation (Herrero et al., 2001; Luque et al., 2001; Sauer et al., 2000). 2-Oxoglutarate, the signal of nitrogen deficiency, stimulates binding of NtcA to target sites (Vazquez-Bermudez et al., 2002), transcription activation in vitro (Tanigawa et al., 2002) and complex formation between NtcA and PipX, a regulatory protein conserved in cyanobacteria (Burillo et al., 2004; Espinosa et al., 2006). The interaction between PipX and NtcA is known to be relevant for maximal activation of NtcA-dependent genes under nitrogen limitation (Espinosa et al., 2007, 2006). PipX-deficient cultures of Synechococcus elongatus sp. PCC 7942 (hereafter S. elongatus) showed reduced activity of nitrogen assimilation enzymes, retarded induction and a slower rate of nitrate consumption and, when subjected to nitrogen starvation, retarded phycobilisome degradation and a faster reduction of the chlorophyll content (Espinosa et al., 2007).

    The homotrimeric PII protein is one of the most conserved and widespread signal transduction proteins in nature and plays key roles in nitrogen assimilatory processes (Leigh & Dodsworth, 2007). PII proteins contain three binding sites (one per subunit) for 2-oxoglutarate and ATP and their primary function is to regulate, by direct protein–protein interactions, the activity of proteins implicated in nitrogen metabolism (reviewed by Forchhammer, 2008). In cyanobacteria, several proteins are known to form complexes with PII. The first two PII receptors were identified in S. elongatus: the enzyme N-acetyl-l-glutamate kinase (NAGK), a PII target conserved across domains of life during the evolution of oxygenic photosynthetic organisms (Burillo et al., 2004; Chen et al., 2006; Sugiyama et al., 2004), and the regulatory factor PipX (Burillo et al., 2004; Espinosa et al., 2006). The non-conserved membrane protein PamA has been identified as a PII receptor in Synechocystis sp. PCC 6803 (Osanai et al., 2005). Structural and functional details are only known for the PII–NAGK complex (Llacer et al., 2007). This complex consists of two polar PII. trimers sandwiching one ring-like hexameric NAGK, with the flexible T-loop, a key element for regulatory interactions, adopting a novel compact shape. Other PII functions for which direct protein–protein interactions have not been reported yet include the control of nitrate transport (Kloft & Forchhammer, 2005; Lee et al., 2000) – nitrate reductase (Takatani et al., 2006) – and the control of inorganic carbon transport (Hisbergues et al., 1999).

    PII proteins bind 2-oxoglutarate and ATP synergistically. In S. elongatus and Synechocystis sp. PCC 6803, the T-loop is phosphorylated at a seryl residue (S49), located at the apex of the solvent-exposed T-loop (Forchhammer, 2004). The phosphorylation status of PII correlates with the 2-oxoglutarate levels, both being maximal during nitrogen starvation. ATP in concert with elevated 2-oxoglutarate levels prevents complex formation of PII with either NAGK or PipX (Espinosa et al., 2006; Maheswaran et al., 2004), suggesting that PipX–PII complexes also have a function under nitrogen-sufficient conditions. PipX does not seem to be required for PII-dependent functions like the ammonium inhibition of nitrate transport (Espinosa et al., 2007) or the stimulation of NAGK activity (Espinosa et al., 2008).

    Recently, we have shown that PII was essential under standard growth conditions in S. elongatus and that PipX was involved in the phenomenon. Here, we show that all three spontaneous point mutations at pipX that we identified in different glnB strains were indeed suppressor mutations contributing to survival in PII-deficient backgrounds and that the spontaneously generated protein PIIT-loop+7 retains the ability to interact with PipX and not with its regulatory target NAGK. The effects of mutations pipX-92delT, pipX160C>T and pipX194T>A on PipX levels, coactivation of NtcA-dependent genes and protein–protein interactions are reported.

    METHODS

    Strains and growth conditions.

    Strains, plasmids and oligonucleotides used in this work are listed in Tables 1⇓ and 2⇓. Constructs and genomic mutations were analysed by automated dideoxy DNA sequencing. All cloning procedures were carried out in Escherichia coli DH5α and GM119 using standard techniques. S. elongatus strains were routinely grown photoautotrophically at 30 °C while shaking under constant illumination (40 μmol photons m−2 s−1) provided by cool white fluorescent lights. Media used were blue–green algae media BG110 (no added nitrogen), BG11 (BG110 plus 17.5 mM NaNO3 and 10 mM HEPES/NaOH pH 7.8) and BG11A (BG110 plus 5 mM NH4Cl and 5 mM HEPES/NaOH pH 7.8). For growth on plates, the media were solidified by addition of 1 % (w/v) agar. Plates were routinely incubated at 30 °C under constant illumination. S. elongatus strains were transformed essentially as described by Golden & Sherman (1984). Whenever used, antibiotic concentrations for S. elongatus were 10 μg kanamycin ml−1, 5 μg streptomycin ml−1 and 5 μg chloramphenicol ml−1.

    Table 1.

    Strains used in this study

    Table 2.

    Oligonucleotides used in this study

    Yeast culture and transformation procedures were performed as described by Ausubel (1999). Interaction signals between pairs of fusion proteins were determined using the three reporters present in PJ696/Y187 diploids as previously described (Burillo et al., 2004).

    Construction of plasmids and strains.

    Plasmid pUAGC59 was used as template for QuickChange Mutagenesis in combination with either primers PipX-L65Q-F/PipX-L65Q-R (pipX194T>A change) or PipX-R54C-F/PipX-R54C-R (pipX160C>T change), resulting in plasmids pUAGC387 and pUAGC388, respectively. To obtain plasmid pUAGC389, the pipX-92delT sequence from MP2-A (Espinosa et al., 2009) was amplified by PCR with primers Syn2060-1F and PipX-6R, digested with NruI and SalI, and the corresponding fragment was used to replace the NruI/SalI fragment of pUAGC59. A C.S3 cassette from pUAGC453 was cloned into the Klenow-treated NheI site of plasmids pUAGC59, pUAGC387, pUAGC388 and pUAGC389, giving pUAGC393, pUAGC390, pUAGC391 and pUAGC392, respectively. Stable transformation of S. elongatus with pUAGC393, pUAGC390, pUAGC391 and pUAGC392 was confirmed by PCR with primers PipX-6R and CS3-2F. The presence of pipX wild-type or mutant alleles was checked by sequencing analysis. Subsequently, plasmids pFAM2 and pFAM84W were independently transformed into control and pipX derivatives. Correct integration of reporter PglnB : : luxAB or PglnN : : luxAB fusions into the neutral site NSII was confirmed by PCR with primers NSII-1F and NSII-1R.

    To obtain plasmids that carry ΦC.K1-pipXR54C and ΦC.K1-pipXL65Q, an XhoI–ClaI fragment excised from digested pUAGC388 and pUAGC387 was cloned into XhoI–ClaI-digested pUAGC410, resulting in plasmids pUAGC681 and pUAGC682, respectively. After S. elongatus clones transformed with pUAGC681 and pUAGC682 were selected with kanamycin and the presence of pipX mutant alleles was verified by sequencing analysis, these mutants and the strain SA410 were subsequently transformed with pUAGC702 (Espinosa et al., 2009) bearing glnB : : C.S3(+). Transformants were selected on kanamycin- and streptomycin-containing BG11 plates. To obtain pUAGC613, C.K1 from pRL161 was extracted by HincII digestion and cloned into BamHI/PstI-digested pUAGC623 and Klenow filled.

    Transformation with pUAGC613 and correct integration of the resistance cassette C.K1 into S. elongatus was verified by PCR with primers CK1-2F and PipX-3X-1R. Mutations were verified by automated dideoxy DNA sequencing. Detection of glnB alleles by PCR was carried out with either GlnB-1F or CS3-2F as a forward primer and GlnB-1R as a reverse primer.

    PipX sequences were amplified by PCR from genomic DNA with primers PipX-OV-2F and PipX-3X-1R, the product was cut with EcoRI and ligated to EcoRI-digested pGAD424 and pGBT9, giving plasmids pUAGC471 (GAL4AD : PipX) and pUAGC472 (GAL4BD : PipX), respectively. The pipX160C>T allele (PipXR54C) was amplified by PCR from a MP2 derivative (Espinosa et al., 2009) using primers PipX-OV-2F and PipX-3X-1R and cloned into pGEX-3X vector giving plasmid pUAGC406. pipX160C>T and pipX194T>A sequences were amplified by PCR with primers PipX-OV-2F and PipX-3X-1R from pUAGC406 and genomic sequences, respectively, and cloned into the EcoRI site of pGAD424 and pGBT9, giving plasmids pUAGC497 (GAL4AD : PipXR54C), pUAGC498 (GAL4BD : PipXR54C), pUAGC371 (GAL4AD : PipXL65Q) and pUAGC372 (GAL4BD : PipXL65Q). The glnB133-134ins allele was amplified by PCR with primers GlnB-1F and GlnB-1R from the GlnBS(−)T-loop+7 strain (Espinosa et al., 2009) and introduced into yeast two-hybrid plasmids by recombination cloning (Kolonin et al., 2000). To this end, XhoI-digested pUAGC11 and BglII-digested pUAGC12 were co-transformed with the PCR product into yeast strains PJ696 and Y187, respectively, giving plasmids pUAGC373 (GAL4AD : PIIT-loop+7) and pUAGC374 (GAL4BD : PIIT-loop+7). To obtain pUAGC6 and pUAGC8, the ntcA sequence was amplified by PCR from genomic DNA with primers NtcA-YTH-1F and NtcA-1R and cloned into EcoRI–SalI-digested pGAD424 and pGBT9, respectively. All pGAD derivatives were sequenced using ACTAseq plus GAD-REV primers, and GBT-1F plus GBT-2R primers for pGBT derivatives.

    RT-PCR analysis.

    To analyse the abundance of pipX mRNA under nitrate growth conditions, cells were grown under standard conditions until they reached OD750 0.5. Aliquots (50 ml) were removed from the cultures for RNA extraction. The samples were rapidly chilled on ice and centrifuged, and the pellets were stored at −80 °C. Total RNA was isolated using the hot phenol method. RT-PCR analysis of pipX mRNA was performed using 0.5 μg total Synechococcus RNA that was retrotranscribed in a total volume of 30 μl with the RevertAid H Minus M-MuLV reverse transcriptase (Fermentas) using oligonucleotide PipX-3X-1R (for pipX) and Sip1-BTH-R (for sipA) as primers. A 10 μl sample of the retrotranscription reaction was subjected to 30 PCR cycles (94 °C 30 s, 42 °C 30 s and 72 °C 30 s) with primers PipX-OV-2F and PipX-3X-1R using NETZYME DNA polymerase. A total of 24 PCR cycles with primers Sip1-BTH-F and Sip1-BTH-R were used for sipA, used as loading control. For each pair of primers, a parallel reaction was carried out without reverse transcriptase as a control for DNA contamination of RNA preparations.

    Determination of luciferase activity.

    Bioluminescence measurements were determined, essentially, as described previously (Espinosa et al., 2007). Light emission was recorded using a Perkin Elmer Victor 3 microplate luminometer.

    Immunoblot analysis of PipX.

    H6-PipX was overexpressed in XL1-Blue E. coli cells as described previously (Espinosa et al., 2006). Purified protein was sent to Pineda antibodies service in order to produce a PipX antiserum. After 90 days of rabbit immunization, antiserum raised against H6-PipX was obtained.

    To isolate proteins from S. elongatus, whole-cell protein extracts were prepared from 10 ml of cells grown to mid-exponential phase (OD750 around 0.5). Cell pellets were lysed and the supernatant fraction was collected. Soluble protein concentrations were determined by Bradford reagent. Equal amounts of protein (60 μg) from each whole-cell extract sample were separated in a linear gradient (5–20 %) SDS-PAGE for immunoblotting. Proteins were transferred to 0.1 μm PVDF membranes (GE Healthcare) by semi-dry blot transfer, according to the manufacturer's instructions. To verify equal loading and transfer of proteins onto PVDF membranes, staining with Fast Green FCF dye was carried out after blotting.

    Polyclonal rabbit antiserum raised against PipX was used at a 1 : 5000 dilution and PipX was detected using horseradish-peroxidase-conjugated anti-rabbit at dilution 1 : 10 000. Detection was carried out using an ECL Plus Western blotting detection kit (GE Healthcare) and scanning in a Typhoon 9410 fluorescence imaging system (GE Healthcare).

    Multiple alignment and sequence comparison.

    Forty-seven PipX homologues obtained from the National Center for Biotechnology Information (NCBI) database were aligned using clustal_x (Larkin et al., 2007) with default settings. RefSeq numbers are: YP_172742.1, Synechococcus elongatus PCC 6301; YP_001865314.1, Nostoc punctiforme PCC 73102; NP_484529.1, Nostoc sp. PCC 7120; ZP_01632661.1, Nodularia spumigena CCY9414; CAO89102.1, Microcystis aeruginosa PCC 7806; ZP_02976743.1, Cyanothece sp. PCC 7424; YP_001804130.1, Cyanothece sp. ATCC 51142; ZP_00514538.1, Crocosphaera watsonii WH 8501; NP_001035873.1, Synechocystis sp. PCC 6803; ZP_01624711.1, Lyngbya sp. PCC 8106; YP_001519639.1, Acaryochloris marina MBIC11017; ZP_01726412.1, Cyanothece sp. CCY0110; YP_001735159.1, Synechococcus sp. PCC 7002; ZP_02943302.1, Cyanothece sp. PCC 8801; NP_682399.1, Thermosynechococcus elongatus BP-1; YP_001660607.1, Microcystis aeruginosa NIES-843; NP_894038.1, Prochlorococcus marinus str. MIT 9313; YP_001018149.1, Prochlorococcus marinus str. MIT 9303; YP_476982.1, Synechococcus sp. JA-2-3B'a(2-13); YP_382322.1, Synechococcus sp. CC9605; YP_729816.1, Synechococcus sp. CC9311; YP_376658.1, Synechococcus sp. CC9902; NP_896752.1, Synechococcus sp. WH 8102; ZP_01086530.1, Synechococcus sp. WH 5701; YP_001224293.1, Synechococcus sp. WH 7803; ZP_01081458.1, Synechococcus sp. RS9917; YP_724299.1, Trichodesmium erythraeum IMS101; ZP_01472290.1, Synechococcus sp. RS9916; YP_001550275.1, Prochlorococcus marinus str. MIT 9211; YP_475449.1, Synechococcus sp. JA-3-3Ab; YP_001226724.1, Synechococcus sp. RCC307; NP_923420.1, Gloeobacter violaceus PCC 7421; ABE10750.1, uncultured Prochlorococcus marinus clone ASNC1092; ABE10884.1, uncultured Prochlorococcus marinus clone ASNC2259; YP_001517213.1, Acaryochloris marina MBIC11017; NP_874784.1, Prochlorococcus marinus subsp. marinus str. CCMP1375; YP_001090637.1, Prochlorococcus marinus str. MIT 9301; YP_292919.1, Prochlorococcus marinus str. NATL2A; YP_001010771.1, Prochlorococcus marinus str. MIT 9515; NP_892511.1, Prochlorococcus marinus subsp. pastoris str. CCMP1986; YP_001483672.1, Prochlorococcus marinus str. MIT 9215; ZP_01124790.1, Synechococcus sp. WH 7805; NP_925339.1, Gloeobacter violaceus PCC 7421; YP_396886.1, Prochlorococcus marinus str. MIT 9312; ZP_01085880.1, Synechococcus sp. WH 5701; YP_001014274.1, Prochlorococcus marinus str. NATL1A; ABE11268.1, uncultured Prochlorococcus marinus clone HF10-88F10. This alignment was used to generate a sequence logo, created with WebLogo (/logo.cgi) (Crooks et al., 2004).

    RESULTS AND DISCUSSION

    Point mutations R54C and L65Q or a lower level of PipX suffice to overcome lethality in PII-deficient backgrounds

    Spontaneous mutations in the pipX gene were found in PII-deficient and PII-null derivative strains (Espinosa et al., 2009). In addition to one internal deletion, which is likely to cause a complete loss of function, we found three point mutations. pipX-92delT, located upstream of the coding region, is likely to decrease expression of the pipX gene, while the effect of mutations pipX160C>T and pipX194T>A encoding PipXR54C and PipXL65Q, respectively, could not be directly anticipated. To obtain indications of the possible effect of the R54C and L65Q substitutions on PipX functions, we analysed PipX sequences available at the NCBI database. A consensus sequence derived from a multiple alignment of the more relevant C-terminal sequences of PipX is shown in Supplementary Fig. S1, available with the online version of this paper. Residues R54 and L65 do not belong to any of the invariant positions in PipX, but both are moderately conserved. Position 54 is occupied in most PipX orthologues by either Arg or Tyr. Position 65 is always occupied by a hydrophobic residue, with Leu being the most frequent amino acid. Thus, the non-conservative nature of the amino acid substitution and the relative conservation of Arg54 and Leu65 strongly suggested that mutations R54C and L65Q could alter PipX properties.

    To confirm that the spontaneous point mutations found in PII-deficient backgrounds were indeed suppressing lethality, mutations pipX-92delT, pipX160C>T and pipX194T>A were introduced at their original chromosomal location, by allelic replacement, into a wild-type background. To this end, the streptomycin resistance cassette C.S3 was used as a linkage marker and placed 121 bp upstream (from the initial ATG) of the corresponding pipX alleles (Fig. 1a⇓). To exclude polar effects and minimize possible artefacts due to the presence of the C.S3 cassette, a streptomycin-resistant control strain retaining the wild-type pipX allele (CS3X) was generated in parallel to mutant strains (CS3XP↓, CS3XR54C and CS3XL65Q). Homozygosity for C.S3 alleles was promptly achieved and it was confirmed that the presence of the streptomycin resistance cassette C.S3 did not confer significant phenotypic differences to the wild-type S. elongatus strain under standard or stress conditions (data not shown). RT-PCR analyses of CS3XP↓ and the control strain showed that there were lower transcript levels in the mutant (Fig. 1b⇓), thus confirming that the negative effect on pipX transcript levels observed in the MP2-A strain (Espinosa et al., 2009) was indeed caused by the pipX-92delT change.

    Figure image not available in archive
    Fig. 1.

    Effects of point mutations on pipX transcription and PipX accumulation. (a) Strategy used to construct strains CS3X, CS3XP↓, CS3XR54C and CS3XL65Q carrying Φ(C.S3–pipX), Φ(C.S3–pipX-92delT), Φ(C.S3–pipXR54C) and Φ(C.S3–pipXL65Q), respectively. Mutant pipX alleles linked to C.S3 are collectively designated here pipX*. The position of C.S3 in relation to the pipX gene and flanking genomic regions is shown. (b) Effect of pipX-92delT mutation on pipX transcript levels. Amplification of pipX and sipA (used as a loading control) by RT-PCR in the CS3X and CS3XP↓ strains. A representative experiment from two independent RNA extractions is shown. Molecular sizes (bp) are given on the left. (c) Effect of pipX mutations on PipX protein accumulation levels. Immunodetection was performed from cells grown in the presence of nitrate (NO3−), ammonium (NH4+) and in a medium lacking combined nitrogen for 24 h (−N). Immunodetected PipX is indicated by an arrowhead. The protein loading control for each lane is shown. Strains used were wild-type (WT), PipX-null mutant (strain SA591) and CS3X derivatives.

    To determine the effect of point mutations on the levels and/or stability of PipX in S. elongatus, cultures of strains CS3X, CS3XP↓, CS3XR54C and CS3XL65Q were obtained under different nitrogen regimes (ammonium, nitrate and 24 h after nitrogen depletion) and analysed by Western blots (Fig. 1c⇑). In close agreement with the RT-PCR data, reduced protein levels were observed in strain CS3XP↓; the differences from the control strain were small but significant under all tested conditions. On the other hand, no significant differences were observed between CS3XR54C and the control strain CS3X, while the level of protein in CS3XL65Q was significantly reduced under nitrogen-deprivation conditions. In summary, two of the three spontaneous point mutations affected PipX levels in at least one of the environmental conditions analysed.

    Because cyanobacteria contain multiple chromosome copies, the persistence, in the course of genetic inactivation trials, of wild-type alleles under selective conditions indicates that the targeted gene is essential. In this context, we showed that, while glnB was readily inactivated in pipX-null mutants, the presence of pipX alleles prevented homozygosis of cassette-inactivated alleles of glnB in the two S. elongatus strains used as wild-type controls. In the control strain SA410, carrying the marker fusion Φ(C.K1–pipX), transcription of the pipX gene takes place from a constitutive promoter present in the C.K1 cassette and results in twice the level of PipX protein, irrespective of whether cultures were grown with nitrate or ammonium, or supplemented with kanamycin (Fig. 2a⇓).

    Figure image not available in archive
    Fig. 2.

    Genetic inactivation of glnB in pipX mutants. (a) In vivo over-expression of PipX mediated by the C.K1 promoter. Immunodetection of PipX in strains grown to mid-exponential phase in the presence of either nitrate (NO3−) or ammonia (NH4+) in the presence (+) or absence (−) of kanamycin. Immunodetected PipX is indicated by an arrowhead. The protein loading control for each lane is shown. Strains: wild-type (WT), SA410 [Φ(C.K1–pipX)] and WTK (C.K1 inserted into Neutral site I). (b) Segregation of glnB alleles, verified by PCR, with primers GlnB-1F and GlnB-1R in three independent clones of strains carrying Φ(C.K1–pipX) (SA410), Φ(C.K1–pipXR54C) (CK1XR54C) and Φ(C.K1–pipXL65Q) (CK1XL65Q) after transformation with glnB : : C.S3(+) and three consecutive transfers onto selective media. Lane L, size marker (GeneRuler 100 bp plus DNA ladder, Fermentas). PCR products corresponding to specific alleles are indicated to the right, and relevant marker sizes (bp) are shown to the left.

    The levels of PipX protein were slightly but consistently lower in strains carrying the Φ(C.S3-pipX) allele (compare WT and CS3X in Fig. 1c⇑). Interestingly, the small decrease in PipX levels caused by the upstream insertion of the C.S3 facilitated rapid glnB inactivation in strain CS3X (data not shown). This supports the notion that a relatively small decrease in pipX gene dosage was enough to suppress lethality, as could be inferred by the occurrence of the pipX-92delT change in strain MP2A (Espinosa et al., 2009) and the present results with CS3XP↓ (Fig. 1c⇑).

    To investigate the effects of mutations R54C and L65Q on PipX function, we turned to strain SA410, in which the levels of PipX do interfere with inactivation of glnB, to construct isogenic strains encoding the mutant proteins PipXR54C or PipXL65Q. Cultures from SA410 and mutant derivatives CK1XR54C and CK1XL65Q were then transformed in parallel with allele glnB : : C.S3(+) and analysed by PCR as described previously (Espinosa et al., 2009). As expected, wild-type glnB alleles persisted in all independent transformant clones from the control strain bearing Φ(C.K1–pipX) (Fig. 2b⇑, lanes SA410). In contrast, homozygosis for glnB : : C.S3(+) was easily achieved in both Φ(C.K1–pipXR54C) and Φ(C.K1–pipXL65Q) backgrounds, since only the longer glnB : : C.S3(+) alleles could be amplified from the different transformants tested (Fig. 2b⇑, lanes CK1XR54C and CK1XL65Q). Therefore, each one of the original spontaneous point mutations (pipX160C>T and pipX194T>A) suffices to suppress the lethality associated with glnB inactivation.

    Effect of pipX mutations on NtcA-dependent activation of reporter genes

    The effects of the same three spontaneous point mutations on the function of PipX as a co-activator of NtcA were analysed in strains containing the NtcA-dependent promoter derivatives PglnB : : luxAB and PglnN : : luxAB. Each of the two gene fusions carries a single NtcA-dependent promoter with distinct nitrogen-dependent induction. In these constructs, reporter expression is strictly dependent on NtcA but exhibits different induction profiles (Aldehni & Forchhammer, 2006; Aldehni et al., 2003). Reporter expression was determined by bioluminescence measurements from control and pipX-mutant-derivative strains, grown to mid-exponential phase in the presence of ammonium or nitrate and after cultures were shifted from ammonium-containing to nitrogen-depleted medium. Results for mutant activity levels are presented relative to their appropriate wild-type control (Fig. 3⇓).

    Figure image not available in archive
    Fig. 3.

    Effect of pipX mutations on PglnB : : luxAB and PglnN : : luxAB expression. Bioluminescence of mutant strains, expressed as percentages of glnB (left) and glnN (right) promoter activities, were determined in cells grown in the presence of ammonium or nitrate and 24 h after cultures were shifted from ammonium-containing to nitrogen-depleted medium, taking as a reference (100 %) the activity of the control strain CS3X. Mean values (±sd) from at least three independent experiments performed in duplicate are plotted. Strains: CS3X, 1; CS3XP↓, 2; CS3XR54C, 3; CS3XL65Q, 4; SA591, 5. Relative activities [RLU ml−1 OD750−1 (10 s)−1] of the control strain grown in ammonia or nitrate and after cultures were shifted from ammonium-containing to nitrogen-free media were 618±124, 62 292±17 059, 964 170±543 051, respectively, for PglnB : : luxAB and 1095±425, 4384±70, 118 195±51 184, respectively, for PglnN : : luxAB.

    When CS3X and its derivatives were compared, results indicated that the three point mutants were affected in activation of PglnB : : luxAB and PglnN : : luxAB reporters, the defects being more evident after nitrogen depletion. However, none of these was as impaired as the pipX null mutant control. Taking into account the reduced protein levels shown in the Western blots by strain CS3XP↓ under all conditions tested, its reduced level of activity could be attributable to lower levels of protein rather than to a specific defect in NtcA co-activation. The same would apply to the strain expressing PipXL65Q, which under nitrogen limitation showed lower levels of PipX. In contrast, the lower activity of strain CS3XR54C did not correlate with reduced protein levels, suggesting that PipXR54C is specifically impaired in NtcA co-activation. To take into account possible artefacts related to gene dosage differences, the same experiments were performed with strain SA410 and mutant derivatives. In close agreement with results obtained from CS3X derivatives, strains expressing mutant proteins PipXL65Q and PipXR54C were both distinctly affected in activation of PglnB : : luxAB and PglnN : : luxAB reporters. Again, during nitrogen depletion, but not in nitrate-containing media, PipXL65Q was a less effective co-activator than PipXR54C (data not shown)

    Complex formation by the mutant proteins PipXL65Q, PipXR54C and PIIT-loop+7

    We next tested the ability of the two mutant proteins PipXR54C and PipXL65Q to interact with NtcA and PII in the yeast two-hybrid system, previously used to provide evidence of the specificity of interactions mediated by PipX (Espinosa et al., 2006). Here, we generated additional wild-type and mutant constructs and also included the PII derivative PIIT-loop+7 (Espinosa et al., 2009), a mutant protein that would allow us to explore the effect of a drastic disruption of the T-loop of PII in interactions with PipX, in these analyses. Expression of reporters was determined in Y187/PJ696 diploids as described previously (Burillo et al., 2004). In all cases, mutant and control proteins were fused independently to both GAL4BD and GAL4AD domains. The results of the yeast two-hybrid analysis, shown in Supplementary Fig. S2, available with the online version of this paper, are schematically summarized in Fig. 4⇓.

    Figure image not available in archive
    Fig. 4.

    Yeast two-hybrid interactions involving NtcA, PII and PipX mutant derivatives. Schematic representation of interacting proteins is shown. The number of arrowheads indicates the relative strength of the interaction as described previously (Burillo et al., 2004).

    The PipXR54C protein was affected in yeast two-hybrid interactions with NtcA, but not with PII, suggesting that residue R54 may interact with NtcA to assist with transcriptional activation of NtcA-dependent promoters. On the other hand, the PipXL65Q protein was not affected in its ability to interact with PII or NtcA in the yeast two-hybrid system. Although the in vivo defect of PipXL65Q may be just due to decreased stability in S. elongatus (see above), other functional defects cannot be excluded at present.

    The presence of seven extra residues at the T-loop of PIIT-loop+7 did impair interactions with NAGK in the yeast two-hybrid system; this was anticipated, since the T-loop forms one of the main interaction surfaces in the NAGK–PII complex (Llacer et al., 2007). Interestingly, the PIIT-loop+7 protein, in spite of the anomalous and longer T-loop, was still able to interact with both PII and PipX protein derivatives, implying that the T-loop does not play a relevant role in formation of PipX–PII complexes. The mutant protein PIIT-loop+7, fortuitously detected in S. elongatus in the course of a previous study, could confer a selective advantage in a PII-deficient background (Espinosa et al., 2009). If that was the case, the results obtained in the interaction analysis could indicate that complex formation between PII and PipX, but not between PII and NAGK, is important to prevent lethality associated with PII deficiency in S. elongatus.

    Point mutations R54C and L65Q and suppression of PipX lethality in PII-deficient backgrounds

    It is now clear that a small reduction in PipX levels suffices to overcome the toxic effect of PipX in S. elongatus. Direct evidence is provided by the fact that in strain CS3X, with lower level of pipX expression than the wild-type control, glnB is readily inactivated. The finding in PII-deficient strains (Espinosa et al., 2009) of mutations that reduce the level of pipX gene products (Fig. 1b⇑) is another indication. However, the question regarding whether the other two known suppressor mutations work in the same way remains. That is, are PipXR54C and PipXL65Q just less active proteins with no specific defects or are they affected in specific functions? In this context, we have shown that the two mutant proteins have different properties. Although neither of them was affected in PII binding, and therefore they retained this specific function, both of them were somehow limited in their ability to activate NtcA. However, the molecular basis of this defect appeared different. PipXR54C, but not PipXL65Q, was impaired in yeast two-hybrid interactions with NtcA, providing a rationale for the reduced NtcA-dependent activation of reporters. On the other hand, PipXL65Q, but not PipXR54C, appeared unstable under nitrogen limitation (Fig. 1c⇑), and thus a correlation was found between the levels of PipXL65Q and its ability to activate NtcA-dependent reporters.

    It should be noted that relatively high 2-oxoglutarate levels are required for binding of PipX to NtcA (Espinosa et al., 2009), arguing against the theory that complex formation between NtcA and PipX plays a major role in the toxic effect observed in PII deficient nitrogen-containing cultures. Although assays for NtcA binding and NtcA activation with the mutant proteins PipXL65Q and PipXR54C showed differences between PipXR54C and PipXL65Q, they did not provide conclusive evidence concerning or excluding a role in toxicity of the NtcA–PipX complexes. Therefore, in light of these results, we cannot yet exclude a role for NtcA–PipX complexes in toxicity. However, we were unable to obtain genetic evidence supporting the implication of NtcA in toxicity (Espinosa et al., 2009) and thus we are inclined to think that PipX toxicity may be related to its binding to an as-yet unknown partner at the relatively low 2-oxoglutarate levels typical of nitrogen-rich cultures. It is tempting to speculate that PipXL65Q and PipXR54C are both specifically affected in that currently unknown interaction.

    Acknowledgments

    This work was supported by grants BFU2006-12424, BFU2009-07374, ACOMP06/083, PR2009-0378 and HA2007-0074. M. A. C. is the recipient of a predoctoral fellowship from the Universidad de Alicante. We thank Jitka Hájková and Paloma Salinas for construction of strains and plasmids and technical help, Alexandra Fokina for protein purification, and Ray Dixon for constructive discussions.

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