From the
MSU-DOE Plant Research Laboratory and the ¶Department of Plant Biology, Michigan State University, East Lansing, Michigan 48824
Received for publication, January 17, 2003 , and in revised form, February 28, 2003.
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ABSTRACT |
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INTRODUCTION |
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Genes involved specifically in heterocyst differentiation and spacing have been cloned and characterized (4, 5, 6, 7, 8, 9). However, no overall regulatory strategy has been identified, and the detailed mechanisms that regulate differentiation remain largely unknown. It has been shown, however, that transcription of hepA, whose product is a member of the family of ABC transporters and is essential for biosynthesis of heterocyst envelope polysaccharide (4), is controlled by (i) HepK, a putative protein-histidine kinase (10); (ii) HepC, which resembles galactosyl-PP-undecaprenol synthetase (10); and (iii) two unusual site-specific DNA-binding proteins (11).
Polysaccharides, a major class of macromolecules, have great structural diversity. Once thought of only as reserve materials or inert structural elements, they are now recognized as playing an important role in the growth and development of plants (12, 13), cell recognition and adhesion (14), hemostasis and lipid metabolism (15, 16), pathogenesis (17), symbiosis (18, 19), and defense responses (20, 21). Different polysaccharides have been linked to carcinogenesis (15, 22) and shown to have anticancer activity in vitro, in vivo, and in human clinical trials (23, 24). Far more research on polysaccharides has emphasized their structure and biosynthesis than the regulation of their biosynthesis. Developing heterocysts provide a model of patterned, functional differentiation in which synthesis of a specific polysaccharide is an essential feature. The structure of heterocyst envelope polysaccharide has been determined for several species (25, 26), the broad outlines of its biosynthesis have been examined (27), and a cluster of genes has been presumptively identified (28, 29) that is involved in its biosynthesis. However, the regulation of its synthesis remains largely uncharted.
Two-component phosphorelay regulatory systems are the principal means for coordinating responses to environmental changes in bacteria and are found also in plants, fungi, protozoa, and amoebae (30, 31, 32). Typically, such a system consists of a membrane-associated sensor kinase and its cognate response regulator. Most known sensor kinases consist of a cytoplasmic N terminus, two transmembrane segments linked by a periplasmic bridge, and a cytoplasmic, C-terminal, transmitter portion with a conserved histidine residue. Signal reception by the kinase stimulates an ATP-dependent autophosphorylation of that histidine. Transfer of the phosphate group to a conserved aspartyl residue of the response regulator renders the latter functional. Most known response regulators function as transcription factors whose effector domains bind DNA and regulate the expression of certain genes, effecting an adaptive response (33). However, some response regulators lack a known effector domain (32), and some effector domains are enzymatically active (34, 35). When signaling subsides, both components undergo dephosphorylation, inactivating the response regulator.
Genomic sequence data have identified no presumptive vertebrate, nematode, or fruit fly genes that encode two-component phosphorelay proteins (36, 37, 38). Because some two-component phosphorelay proteins are essential for the virulence of microbial pathogens including human fungal and bacterial pathogens, novel anti-microbial drugs targeted to two-component phosphorelay systems may prove highly specific for microbial pathogens. Although more than 1000 two-component phosphorelay proteins have been predicted from genomic data, very few have an identified function and have been shown experimentally to participate in phosphorelays. Some predicted homologs of histidine kinases may not be kinases or may not phosphorylate a histidine residue (32). For example, Azoto-bacter vinelandii NifL contains five conserved blocks of amino acids characteristic of histidine sensor kinases (39) but has not been observed to autophosphorylate (40, 41), and DivL (42), pyruvate dehydrogenase kinase (43), and plant phytochromes (44) phosphorylate a tyrosine or serine rather than a histidine.
Predicted proteins HepK from Anabaena sp. strain PCC 7120 and DevR from Nostoc punctiforme strain ATCC 29133 resemble respectively a sensory protein-histidine kinase (10) and a response regulator that lacks known DNA-binding domains (45). The amino acid sequence of Anabaena sp. ortholog DevRA of DevR but for a possible 30-amino acid, N-terminal extension, is 93% identical and 98% similar to that of Nostoc DevR. Unlike most response regulators, DevR and DevRA have a 7-amino acid peptide, 57SRSVYQG63, in the -turn loop region. DevR, but not DevR
7, from which that peptide was deleted, is weakly phosphorylated in vitro by Escherichia coli sensor kinase EnvZ, a result that supported the interpretation of Hagen and Meeks (46) that DevR is a response regulator in an unidentified phosphorelay system that controls heterocyst maturation in N. punctiforme strain ATCC 29133. However, no cognate histidine kinase was identified for DevR. No gene that evidently encodes a response regulator neighbors hepK. devRA is 1.4 Mb distant from hepK (28). hepK Anabaena sp. and devR N. punctiforme cannot assimilate N2 aerobically (10, 45). We present evidence that Anabaena sp. HepK is a histidine kinase, that DevRA is its cognate response regulator, and that the two comprise part or all of a two-component system that controls the biosynthesis of heterocyst envelope polysaccharide.
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EXPERIMENTAL PROCEDURES |
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DNA ManipulationRecombinant DNA procedures were performed in a standard manner (49). The enzymes were purchased from New England Biolabs, Invitrogen, and Roche Applied Science and used as recommended by the manufacturers.
Construction of Expression PlasmidsTruncated hepK ('hepK) encoding residues 267575 of HepK that lack the two presumptive transmembrane regions and full-length hepK (GenBankTM accession number U68034
[GenBank]
) were amplified by PCR with, respectively, primer pairs 5'-GGAATTCCATATGCGGACTAGTCGAGCGATCGCT-3' and 5'-GAAGATCTTAACTTTGCTCCTGAAGTGGCAGG-3' (CPW99), and 5'-GGAATTCCATATGCAAACTCAGCAGCCGATCCCTG-3' and CPW99 (introduced NdeI and BglII sites are underlined) with pRL2079 (10) as template. The PCR products were cloned between the NdeI and BamHI sites of plasmid pET-14b (Novagen, Inc., Madison, WI), which provided an N-terminal hexahistidine tag. To reduce the extent of PCR product that had to be validated by sequencing, the region from MfeI to BstXI in intact hepK was replaced by a portion of wild-type hepK as was a smaller portion in truncated hepK. The resulting plasmids were denoted pRL2406 and pRL2433, respectively. devR and devR21 (encoding DevR
7) of N. punctiforme were PCR-amplified from plasmids pSCR169 and pSCR343 (Ref. 46; gifts of J. C. Meeks), respectively, as templates with the primers 5'-GGAATTCCATATGAAAACTGTTTTAATTGTC-3' and 5'-GAAGATCTTATGATTGGCCGTCTGTGG-3' and were cloned between the NdeI and BamHI sites of pET-14b, producing pRL2461 and pRL2487. pSCR351, encoding H6DevR-D53Q, was also from Dr. Meeks. devRA, PCR-amplified from total Anabaena sp. DNA with the primers 5'-GGAATTCCATATGACAGCAAAGCATATTGATACTA-3' and 5'-CGCGGATCCTCAGTGGTTGTCACTGGGGAGGAGAG-3', was cloned between the NdeI and BamHI sites of pET-14b to produce pRL2749 for production of H6DevRA. The ompR gene (GenBankTM accession number J01656
[GenBank]
) of E. coli strain MC4100 in plasmid pLAN801 (a gift of Dr. M. Igo) was amplified by PCR with the primers 5'-GGGAATTCCATATGCAAGAGAACTACAAGATTCTG-3' and 5'-CCCAAGCTTTCATGCTTTAGAGCCCTCCGG-3'. The PCR product was first cloned into the EcoRV site of pBluescript SK+ (Stratagene, La Jolla, CA) to produce pRL2483 for DNA sequencing; then an NdeI-BamHI fragment containing ompR was transferred between the NdeI and BamHI sites of pET-14b, yielding pRL2484. All of the above PCR products were proven error-free by DNA sequencing.
Site-directed Mutation of hepKPrimer-mediated PCR mutagenesis (QuikChange site-directed mutagenesis; Stratagene) was used to change the conserved His348 to Ala at the presumptive site of phosphorylation of HepK to produce a mutant truncated HepK protein, which we denote 'HepK-H348A. The mutagenic oligonucleotides 5'-ATTTCTTGCCAACGTTAGTGCTGAGTTGCGTAC-3' and 5'-GTACGCAACTCAGCACTAACGTTGGCAAGAAAT-3' (codon 348, CAT GCT) with pRL2406 as template were used for PCR; the product was cloned in pET-14b, producing pRL2454. The sequence of the 310-bp BstEII-BstXI fragment of pRL2454 was shown to be identical to the corresponding fragment of pRL2406, except for the H348A substitution, by DNA sequencing. Transfer of the BstEII-BstXI fragment from pRL2454 to pRL2406 produced pRL2457, from which H6'HepK-H348A was produced and purified as had been H6'HepK.
Test of Complementation of hepK Mutant Y7 by hepK-H348ATransposon-generated hepK mutant Y7 cannot grow aerobically on N2, but Y7 (pRL2078) can (10). pRL2480, identical to pRL2078 but for an H348A change in hepK, was constructed by transferring the 4.9-kb, hepK-bearing BamHI DNA fragment of pRL2078 to pRL57 (50), which lacks BstEII and BstXI sites; replacing the 310-bp BstEII-BstXI fragment of hepK by the corresponding fragment of pRL2457; and then replacing the 4.9-kb BamHI fragment of pRL2078 with the resulting BamHI fragment. pRL2480 was transferred by conjugation (51) into Y7 with selection on AA plus nitrate agar plates supplemented with 30 µg of neomycin sulfate ml1 to select for Y7 and 10 µg of spectinomycin sulfate ml1 to select for pRL2480. 10 days later, 10 resulting colonies were restreaked onto the same medium for proliferation and were then spotted onto AA agar plates without or with nitrate (the latter was supplemented with neomycin and spectinomycin as before).
Production and Purification of Hexahistidine-tagged ProteinsTo overproduce H6'HepK, H6HepK, H6'HepK-H348A, H6DevRA, H6DevR, H6DevR7, H6DevR-D53Q, H6OmpR, and H6EnvZc, E. coli strain BL21 (DE3) was transformed with pRL2406, pRL2433, pRL2457, pRL2749, pRL2461, pRL2487, pSCR351, pRL2484, and pPH001 (a gift of M. Inouye) (52), respectively. Each transformant was grown in 500 ml of LB medium supplemented with 100 µg of ampicillin ml1 at 37 °C to an A600 of 0.50.6, 0.5 mM isopropyl-
-D-thiogalactopyranoside was added, and incubation was continued for 3 h. The His6-tagged proteins were purified to homogeneity as follows. E. coli suspended in 50 mM sodium phosphate, 100 mM NaCl, pH 7.0, was broken with a French press (American Instrument Co. Div. Travenol Laboratories, Inc., Silver Spring, MD). The supernatant solution from centrifugation for 20 min at 20,000 x g at 4 °C was applied to a cobalt-based resin column (Clontech), which was then washed thrice with 50 mM sodium phosphate, 300 mM NaCl, 5 mM imidazole, pH 7.0. The intended protein, eluted with 50 mM sodium phosphate, 300 mM NaCl, pH 5.5, was loaded onto a Sephadex G-100 gel filtration column (Amersham Biosciences) from which it was eluted at 4 °C with 50 mM sodium phosphate, 100 mM NaCl, pH 7.0, at 20 ml h1.
Assays of Phosphorylation and TransphosphorylationUnless otherwise specified, phosphorylation reaction mixtures contained 25 mM Tris-HCl, pH 8.0, 50 mM NaCl, 5 mM MgCl2,1mM dithiothreitol, 0.5 mM EDTA, and 3080 µM [-32P]ATP (6,000 Ci mmol1). H6HepK, H6DevR, or derivatives of them, H6DevRA,H6EnvZc, and H6OmpR were added to final concentrations of 2.5 µM or as indicated. The reactions were initiated by the addition of the radioactive substrate, incubated at room temperature (approximately 24 °C) for different times, and stopped by the addition of an equal volume of 2x concentrated SDS-PAGE loading buffer (50 mM Tris-HCl, pH 6.8, 4% SDS, 10% glycerol, 200 mM dithiothreitol, 0.003% bromphenol blue). The reactions, once stopped, were immediately subjected to SDS-PAGE. After completion of electrophoresis, the gels were electroblotted onto polyvinylidene difluoride membranes (Bio-Rad) at 200 mA in protein-transfer buffer (25 mM Tris, 250 mM glycine, 5% methanol, 0.1% SDS, pH 8.3) at 4 °C for 1 h, and the membrane was then air-dried at room temperature. The radioactivity of proteins attached to polyvinylidene difluoride membranes was determined qualitatively by autoradiography with Hyperfilm (Amersham Biosciences) or quantitatively with a PhosphorImager model V5.6 (Molecular Dynamics, Sunnyvale, CA). To show the protein bands, the polyvinylidene difluoride membrane was later stained with Coomassie Brilliant Blue R-250 for 2 min and then destained with 50% methanol. In time course experiments, portions of a sample were removed, mixed with loading buffer, and kept on ice until all of the samples were taken.
To compare transfer of 32P from H6'HepK-32P to H6DevR and to H6DevR7, one 13-µl fraction and two 40-µl fractions were removed from a solution of 9 µg of phosphorylated H6'HepK in 100 µl of standard phosphorylation buffer. Six µg of H6DevR or H6DevR
7 in 12 µl of standard phosphorylation buffer were added to separate 40-µl fractions of phosphorylated H6'HepK. At the indicated reaction times, 15-µl portions of samples were rapidly transferred to an equal volume of 2x concentrated SDS-PAGE loading buffer. The mixtures were kept on ice until all of the portions had been removed and were then subjected to SDS-PAGE.
Stability of Phosphate Linkages in Phosphorylated H6'HepK, H6DevRA, and H6DevR, and Effects of Nucleoside Diphosphates on Dephosphorylation of H6'HepK-32PTo examine the chemical stability of their phosphate linkages, 30 pmol of H6'HepK or 50 pmol of H6DevRA or H6DevR with 20 pmol of H6'HepK were phosphorylated for 30 min in 30 µl of reaction mixture. The reactions were terminated by adding an equal volume of 2x concentrated SDS-PAGE loading buffer. Samples of 10 µl were pipetted into microcentrifuge tubes containing 2 µl of 1 N HCl, 0.15 N HCl, water, or 3 N NaOH, giving final pH values of 0.9, 2.7, 7.2, or 12.5, respectively. pH values were confirmed with Color pHast® indicator strips (EM Science, Darmstadt, Germany). The mixtures were incubated for 60 min in a water bath at 42 °C. Another sample, to which 2 µl of water was added, was kept on ice. The samples were separated by SDS-PAGE and analyzed for radioactivity.
The kinetics of loss of 32P from H6'HepK-32P and H6DevR-32P at pH 12.5 were assessed as follows. H6'HepK (35 pmol) was phosphorylated for 30 min in a 30-µl reaction mixture; in some experiments the 35 pmol of H6'HepK-32P in 20 µl was then used to phosphorylate 35 pmol of H6DevR for 30 min in a 30-µl reaction mixture, and the protein or proteins were then denatured at neutral pH by the addition of 30 µl of 2x concentrated loading buffer (pH 6.8). Each resulting 60-µl mixture, prewarmed for 2 min at 42 °C, was adjusted to pH 12.5 by addition of 12 µl of 3 N NaOH. pH values were confirmed with Color pHast® indicator strips. At intervals thereafter (see Fig. 6), an 8-µl sample was withdrawn and introduced into a microcentrifuge tube containing 1.33 µl of 3 N HCl to readjust the pH to 7.0. The samples were immediately placed on ice until sampling was completed. All of the samples were separated by SDS-PAGE and analyzed for radioactivity.
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ADP, CDP, GDP, or TDP (sodium salts; Sigma; sodium ADP was also purchased from TCI America for confirmation) at a final concentration of 0.5 mM was incubated for 5 min in a 15-µl phosphorylation reaction mixture containing 2 µg of H6'HepK-32P. The reactions were stopped by addition of 2x concentrated SDS-PAGE loading buffer. The samples subjected to SDS-PAGE were analyzed for radioactivity.
Inactivation of devRAA 1415-bp DNA fragment containing devRA and DNA downstream from it was amplified by PCR from genomic DNA of Anabaena sp. with the primers 5'-TAAATAAAATTCTCATGAAAACTG-3' and 5'-GAGCAAGACTGGCTAGCCAGTT-3' (BspHI site underlined). The PCR product was first cloned into the EcoRV site of pBluescript SK+ to produce pRL2478 for DNA sequencing. Then a BspHI-BamHI fragment containing the PCR product, which was free of errors, was transferred between the BspHI and BglII sites of pRL278 (70) to produce pRL2479. devRA was inactivated by insertion of a 2.7-kb, SmaI-gfp-omega-PvuII cassette from pRL2379 (8) into the unique NruI site of pRL2479, 110 bp 5' from the stop codon of devRA. The resulting plasmid, denoted pRL2481a, was transferred to wild-type Anabaena sp. by conjugation, and a double recombinant (devRA) strain denoted DR2481a was isolated as described (50). The structure of the recombinant was confirmed by diagnostic PCR using primers 5'-CGGGATCCCGTGGAGAGAAGGTTTAA-3' and 5'-CGGGATCCTCAGTGGTTGTCACTGGGGAG-3' and, as templates, total DNA isolated from the wild-type strain and DR2481a.
Construction of hepK::gfp and Fluorescence MicroscopyA 4.9-kb BamHI fragment of pRL2078, containing the 1.7-kb hepK-encoding region and upstream sequence, was cloned into the BamHI site of shuttle vector pRL57 (50) to produce pRL2419. The gfp-omega cassette from pRL2379 (8) was inserted into the blunted, unique BstXI site 1.2 kb downstream from the start codon of hepK in pRL2419. The resulting plasmid, denoted pRL2437, was transferred into wild-type Anabaena sp. strain PCC 7120 by conjugation (51). Fluorescence microscopy and photography of the hepK::gfp strain was performed as described (53).
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RESULTS |
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Autophosphorylation of H6'HepK and Transfer of Its Phosphate Residue Very Efficiently to H6DevRA and H6DevR but Not Measurably to H6OmpRUnlike full-length H6HepK (Fig. 2B, lane 2), H6'HepK autophosphorylated when incubated with [-32P]ATP (Fig. 2B, lane 1). H6DevR (Fig. 3, C and D, lanes 3) and H6DevRA (data not shown) did not do so detectably. The upper weak band with a molecular mass of about 72 kDa in Fig. 2B (lane 1) may be derived from the phosphorylated dimer of H6'HepK. 32P-Labeled H6'HepK (H6'HepK-32P) transferred 90% of its radioactivity to H6DevRA (Fig. 3A, lane 2)orH6DevR (Fig. 3C, lane 2) within 20 s. Whether the phosphotransfer from phospho-H6'HepK to H6DevRA is specific was examined by use of OmpR, a response regulator whose cognate histidine kinase is EnvZ (54). Derivative H6EnvZc, which lacks the transmembrane regions of EnvZ, autophosphorylated (Fig. 4B, lane 2) and then served efficiently as 32P donor to H6OmpR (Fig. 4B, lane 3), whereas transfer of 32P from H6'HepK-32P to H6OmpR was not observed (Fig. 4, A and C, lanes 2 and 3).
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Previously autophosphorylated H6'HepK was mixed with H6DevR and H6DevR7; 85% of 32P from H6'HepK-32P was transferred to H6DevR within 30 s, 97% within 5 min, and an even greater percentage was transferred upon incubation for 1 h (Fig. 5B, lanes 13). H6DevR
7 was phosphorylated at least 10-fold more slowly, with approximately 30% of the 32P transferred by 1 h (Fig. 5B, lanes 57; PhosphorImager quantitation not shown). H6DevR
7 alone was not detectably phosphorylated when incubated with [
-32P]ATP for 1 h (data not shown).
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Stability of Phosphate Linkages to H6'HepK, H6DevRA, and H6DevRThe chemical stability of the phosphoryl bonds in phospho-H6'HepK, -H6DevRA, and -H6DevR was tested. Phosphorylated amino acid residues in proteins are commonly classified into O-phosphates, phosphomonesters formed by phosphorylation of amino acids Ser, Thr, and Tyr; N-phosphates, phosphoramidates, produced by phosphorylation of Arg, His, and Lys; and acyl phosphates, phosphate anhydrides generated by phosphorylation of Asp and Glu. Phospho-His and -Lys are stable under alkaline conditions but sensitive to mildly acidic conditions, whereas the opposite is true for phospho-Asp and -Glu (which are, however, unstable at pH 0.9); and phospho-Arg is unstable under both conditions (55, 56). H6'HepK-32P and H6DevR-32P were both dephosphorylated within 1 h at the highly acidic pH, 0.9 (Fig. 6, A and B, lanes 2). H6'HepK-32P lost more than 90% of its phosphate within 1 h at pH 2.7 (Fig. 6A, lane 3) but was relatively stable at pH 12.5 (Fig. 6A, lane 5). H6DevR-32P (Fig. 6B, lane 3) and H6DevRA-32P (Fig. 6C, lane 2) were relatively stable at pH 2.7 but unstable at pH 12.5 (Fig. 6, B, lane 5, and C, lane 4). H6DevRA-32P (data not shown), like H6DevR-32P (Fig. 7B), showed no residual radioactivity after 2 min at 42 °C and pH 12.5. H6'HepK-32P did not lose significantly more radioactivity during 260 min at pH 12.5 and 42 °C (Fig. 7A, lanes 37) than at pH 7.0 and either 42 or 0 °C (Fig. 7A, lanes 1 and 2). At neutral pH and room temperature (approximately 24 °C), the half-life of isolated H6'HepK-32P was at least 24 h (data not shown).
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H6'HepK-H348A and H6DevR-D53Q Were Not Phosphorylated, and HepK-H348A Failed to Complement a hepK Mutant Sequence alignments (10, 45) and the chemical stability tests described above suggested that His348 of HepK and Asp53 of DevR and of DevRA are phosphorylated. This inference was tested by use of H6DevR-D53Q (46) and construction and use of H6'HepK-H348A (the numbers 348 and 53 refer to the untruncated amino acid sequences in the absence of the hexahistidine tag). Unlike H6'HepK (Fig. 8, A and B, lane 1), H6'HepK-H348A overexpressed in E. coli and purified to homogeneity (Fig. 8B, lanes 3 and 4) failed to autophosphorylate (Fig. 8A, lane 3). H6DevR-D53Q, similarly overexpressed and purified to homogeneity (data not shown), unlike H6DevR, did not accept 32P (Fig. 8C, lanes 2 and 3). hepK mutant Y7 of Anabaena sp. is neomycin-resistant and unable to assimilate N2 aerobically (10) and is spectinomycin-sensitive and therefore unable to grow in the presence of neomycin, spectinomycin, and fixed nitrogen (Fig. 9C, circle). Plasmid pRL2078, which bears wild-type hepK (10) and confers resistance to spectinomycin, complemented Y7, as shown by the dark green growth of Y7 (pRL2078) (Fig. 9D, circle) in the absence of fixed nitrogen. Plasmid pRL2480 is identical to pRL2078 except that it encodes a HepK-H348A variant of HepK. In contrast to pRL2078, pRL2480 failed to complement Y7, as shown by the lack of growth in the absence of fixed nitrogen (Fig. 9D) of 10 randomly chosen derivatives of Y7 that bear pRL2480 and are therefore able to grow in the presence of fixed nitrogen, neomycin, and spectinomycin (Fig. 9C).
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ADP Elicits Dephosphorylation of H6'HepK-32P but Has No Evident Effect on Phosphotransfer from H6'HepK-32P to DevRBecause autophosphorylation of 'HepK presumably produces ADP, we tested whether ADP affects autophosphorylation of, or phosphate transfer from, H6'HepK-32P. Unlike incubation with CDP, GDP, or TDP (Fig. 10A, lanes 1 and 35), incubation of a solution of phosphorylated H6'HepK for 5 min with 0.5 mM ADP resulted consistently in a loss of 90% of the 32P from H6'HepK-32P (Fig. 10A, lanes 1 and 2). The loss was even greater after 0.5 h (Fig. 10B, lanes 3 and 4), whereas ADP negligibly affected net phosphate transfer from H6'HepK-32P to H6DevR (Fig. 10B, lanes 1 and 2). In the absence of H6'HepK and the presence of 0.5 mM ADP, H6DevR was not phosphorylated by [
-32P]ATP (data not shown).
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Inactivation of devRA, devR, or hepK Blocks Formation of the Heterocyst Envelope Polysaccharide LayerHeterocysts of hepK mutant Y7 of Anabaena sp. form a laminated layer of glycolipids but no polysaccharide layer and fail to fix N2 aerobically (10). Inactivation of devRA by double reciprocal recombination with plasmid pRL2481a (see "Materials and Methods") was confirmed by diagnostic PCR (data not shown). devRA Anabaena sp. also cannot grow aerobically on N2. As in hepK Anabaena sp. (10), heterocyst envelopes of devRA Anabaena sp. (Fig. 11, C and D) and devR N. punctiforme (Fig. 11, G and I) retain a laminated glycolipid layer but lack a polysaccharide layer (Fig. 11, compare A, B, E, and F, respectively).
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In Vivo Localization of Expression of hepKPlasmid pRL2437 bears a hepK::gfp transcriptional fusion (gfp encodes green fluorescent protein) (57). Fluorescence from that transcriptional fusion was observed primarily in single proheterocysts (Fig. 12D, arrow P) but sometimes in paired cells (Fig. 12, B and C, arrow P), of which at least one was a proheterocyst. Slight fluorescence, of questionable significance in this highly autofluorescent organism, originated from vegetative cells (Fig. 12, AD) and mature heterocysts (Fig. 12B, arrow H).
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DISCUSSION |
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Protein-histidine kinases VirA, ArcB, and EnvZ autophosphorylate in vitro when truncated by removal of transmembrane regions but not when intact (54, 55, 58). Similarly, we observed phosphorylation in vitro by truncated but not by intact H6HepK (Fig. 2). Perhaps intact HepK cannot fold properly in vitro; when overexpressed in E. coli, H6HepK formed mostly inclusion bodies, whereas H6'HepK did not. Alternatively, intact HepK may autophosphorylate only in response to a signal specific to heterocyst development that is lacking in vitro. Sequence alignments (10, 45) suggested that His348 of HepK and Asp53 of DevRA and DevR may be phosphorylated. Concordantly, linkage of 32P to H6'HepK is unstable at pH 2.7 and stable at pH 12.5 (Figs. 6 and 7), behavior typical of phosphorylated histidine or lysine residues, whereas H6DevRA-32P and H6DevR-32P are moderately stable at pH 2.7 and unstable at pH 12.5 (Figs. 6 and 7 and data not shown), as is characteristic of acyl phosphates (55, 56). Dephosphorylation of both histidine N-32P (as observed for H6'HepK-32P; Fig. 6A, lane 2) and aspartate acyl-32P (as observed for H6DevR-32P; Fig. 6B, lane 2) at pH 0.9 is expected (55).
Response regulators such as DevRA and DevR that lack a known C-terminal effector domain may (i) interact with a downstream target only in response to phosphorylation (59) or (ii) act as an intermediate in a multicomponent phosphorelay system (33, 60). Both DevR-D53Q (glutamine is uncharged and cannot be phosphorylated) and DevR-D53E (glutamate may structurally mimic phosphorylated aspartate) lack DevR function in vivo in N. punctiforme (46), suggesting that the function of DevR may depend on a role of DevR-Asp53 as a phosphotransferase intermediate. That is, DevR, like Spo0F, may phosphorylate a hitherto unidentified DNA-binding protein (46).
The genome of Anabaena sp. strain PCC 7120 contains 73 genes for putative simple His kinases, 53 more for putative hybrid His kinases, and 77 genes for putative simple response regulators, in addition to other kinds of protein kinases (61). Few of these have a known function, and interacting pairs have not been demonstrated. Specificity of interaction of a particular regulator with a particular sensor in response to a given environmental stimulus may often be essential to avoid inappropriate responses, although certain response regulators may have to receive input signals from more than one sensor. Although there is very weak cross-talk in vitro between the non-cognate histidine kinase EnvZ, whose cognate response regulator is OmpR (54), and H6DevR, no cross-talk was observed with H6DevR7 (46).1 Whereas the 7-amino acid peptide, SRS-VYQG, located in DevR and DevRA is absent from most known response regulators, a similar insertion is present in FruA, a putative response regulator in Myxococcus xanthus (62). Our biochemical data (Fig. 5) showed that deletion of that peptide from the
-turn loop region of H6DevR greatly decreased the rate and extent of phosphotransfer from H6'HepK to H6DevR
7 in vitro and so may influence the rate, and perhaps the specificity, of phosphotransfer from HepK in vivo. The lack of cross-talk between HepK and OmpR (Fig. 4A) confirmed the specificity of recognition between HepK and DevRA.
Although the presence of ADP results in dephosphorylation of H6'HepK-32P (Fig. 10), we saw no diminution of H6'HepK-dependent phosphorylation of H6DevR (Fig. 10B, lanes 1 and 2), perhaps because the latter reaction takes place much more rapidly. On the basis of the observed effect of ADP in vitro, we conjecture that the ratio of ATP to ADP in vivo may regulate the level of phosphorylated H6'HepK.
Because a chromosomal hepK::gfp fusion was expressed too weakly for us to localize the resulting fluorescence, we expressed hepK::gfp in a pDU1-based plasmid. Unlike expression of a glnA fusion in a pDU1-based plasmid, which was localized in all cells of N2-fixing filaments (63) as expected on the basis of biochemical data (64, 65), hepK was expressed primarily in proheterocysts (Fig. 12). This result appears consistent with the fact that it is in developing heterocysts that synthesis of heterocyst envelope polysaccharide, a process that is regulated by HepK (10), takes place.
As noted above, no human, nematode, or fruit fly sequences that presumptively encode two-component phosphorelay proteins have been identified. Because two-component phosphorelay systems are specifically involved with the establishment of virulence in some bacteria and fungi, sometimes via regulation of polysaccharides (66, 67), drugs targeted to such systems may prove highly specific for microbial pathogens (68, 69). Therefore, study of phosphorelay regulation of polysaccharide synthesis in Anabaena, a nonpathogen, may have relevance for drug development.
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FOOTNOTES |
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Permanent address: College of Life Sciences, Anhui Normal University, Wuhu, Anhui 241000, The People's Republic of China.
|| To whom correspondence should be addressed: MSU-DOE Plant Research Laboratory, Michigan State University, E. Lansing, MI 48824. Tel.: 517-353-2049; Fax: 517-353-9168; E-mail: wolk{at}msu.edu.
1 R. Zhou and C. P. Wolk, unpublished data.
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ACKNOWLEDGMENTS |
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REFERENCES |
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