Biological gas channels for NH3 and CO2: evidence that Rh (Rhesus) proteins are CO2 channels.

Physiological evidence from our laboratory indicates that Amt/Mep proteins are gas channels for NH3, the first biological gas channels to be described. This view has now been confirmed by structural evidence and is displacing the previous belief that Amt/Mep proteins were active transporters for the NH4+ ion. Still disputed is the physiological substrate for Rh proteins, the only known homologues of Amt/Mep proteins. Many think they are mammalian ammonium (NH4+ or NH3) transporters. Following Monod's famous dictum, "Anything found to be true of E. coli must also be true of elephants" [Perspect. Biol. Med. 47(1) (2004) 47], we explored the substrate for Rh proteins in the unicellular green alga Chlamydomonas reinhardtii. C. reinhardtii is one of the simplest organisms to have Rh proteins and it also has Amt proteins. Physiological studies in this microbe indicate that the substrate for Rh proteins is CO2 and confirm that the substrate for Amt proteins is NH3. Both are readily hydrated gases. Knowing that transport of CO2 is the ancestral function of Rh proteins supports the inference from hematological research that a newly evolving role of the human Rh30 proteins, RhCcEe and RhD, is to help maintain the flexible, flattened shape of the red cell.

Phosphoaspartates in bacterial signal transduction.

Bacteria use a strategy referred to as two-component signal transduction to sense a variety of stimuli and initiate an appropriate response. Signal processing begins with proteins referred to as histidine kinases. In most cases, these are membrane-bound receptors that respond to environmental cues. Histidine kinases use ATP as a phosphodonor to phosphorylate a conserved histidine residue. Subsequent transfer of the phosphoryl group to a conserved aspartyl residue in the cognate response regulator results in an appropriate output. Recent structural studies of activated (phosphorylated) response regulators and their aspartate-bearing regulatory domains have provided insight into the links between the chemistry and biology of these ubiquitous regulatory proteins. Chemical aspects of their function appear to generalize broadly to enzymes that adopt a phosphoaspartate intermediate.

Evidence that fungal MEP proteins mediate diffusion of the uncharged species NH(3) across the cytoplasmic membrane.

Methylammonium and ammonium (MEP) permeases of Saccharomyces cerevisiae belong to a ubiquitous family of cytoplasmic membrane proteins that transport only ammonium (NH(4)(+) + NH(3)). Transport and accumulation of the ammonium analog [(14)C]methylammonium, a weak base, led to the proposal that members of this family were capable of energy-dependent concentration of the ammonium ion, NH(4)(+). In bacteria, however, ATP-dependent conversion of methylammonium to gamma-N-methylglutamine by glutamine synthetase precludes its use in assessing concentrative transport across the cytoplasmic membrane. We have confirmed that methylammonium is not metabolized in the yeast S. cerevisiae and have shown that it is little metabolized in the filamentous fungus Neurospora crassa. However, its accumulation depends on the energy-dependent acidification of vacuoles. A Deltavph1 mutant of S. cerevisiae and a Deltavma1 mutant, which lack vacuolar H(+)-ATPase activity, had large (fivefold or greater) defects in the accumulation of methylammonium, with little accompanying defect in the initial rate of transport. A vma-1 mutant of N. crassa largely metabolized methylammonium to methylglutamine. Thus, in fungi as in bacteria, subsequent energy-dependent utilization of methylammonium precludes its use in assessing active transport across the cytoplasmic membrane. The requirement for a proton gradient to sequester the charged species CH(3)NH(3)(+) in acidic vacuoles provides evidence that the substrate for MEP proteins is the uncharged species CH(3)NH(2). By inference, their natural substrate is NH(3), a gas. We postulate that MEP proteins facilitate diffusion of NH(3) across the cytoplasmic membrane and speculate that human Rhesus proteins, which lie in the same domain family as MEP proteins, facilitate diffusion of CO(2).

BeF(3)(-) acts as a phosphate analog in proteins phosphorylated on aspartate: structure of a BeF(3)(-) complex with phosphoserine phosphatase.

Protein phosphoaspartate bonds play a variety of roles. In response regulator proteins of two-component signal transduction systems, phosphorylation of an aspartate residue is coupled to a change from an inactive to an active conformation. In phosphatases and mutases of the haloacid dehalogenase (HAD) superfamily, phosphoaspartate serves as an intermediate in phosphotransfer reactions, and in P-type ATPases, also members of the HAD family, it serves in the conversion of chemical energy to ion gradients. In each case, lability of the phosphoaspartate linkage has hampered a detailed study of the phosphorylated form. For response regulators, this difficulty was recently overcome with a phosphate analog, BeF(3)(-), which yields persistent complexes with the active site aspartate of their receiver domains. We now extend the application of this analog to a HAD superfamily member by solving at 1.5-A resolution the x-ray crystal structure of the complex of BeF(3)(-) with phosphoserine phosphatase (PSP) from Methanococcus jannaschii. The structure is comparable to that of a phosphoenzyme intermediate: BeF(3)(-) is bound to Asp-11 with the tetrahedral geometry of a phosphoryl group, is coordinated to Mg(2+), and is bound to residues surrounding the active site that are conserved in the HAD superfamily. Comparison of the active sites of BeF(3)(-) x PSP and BeF(3)(-) x CeY, a receiver domain/response regulator, reveals striking similarities that provide insights into the function not only of PSP but also of P-type ATPases. Our results indicate that use of BeF(3)(-) for structural studies of proteins that form phosphoaspartate linkages will extend well beyond response regulators.

Isolation of Escherichia coli mRNA and comparison of expression using mRNA and total RNA on DNA microarrays.

Bacterial messenger RNA (mRNA) is not coherently polyadenylated, whereas mRNA of Eukarya can be separated from stable RNAs by virtue of polyadenylated 3'-termini. We have developed a method to isolate Escherichia coli mRNA by polyadenylating it in crude cell extracts with E. coli poly(A) polymerase I and purifying it by oligo(dT) chromatography. Differences in lacZRNA levels were similar with purified mRNA and total RNA in dot blot hydridizations for cultures grown with or without gratuitous induction of the lactose operon. More broadly, changes in gene expression upon induction were similar when cDNAs primed from mRNA or total RNA with random hexanucleotides were hydridized to DNA microarrays for the E. coli genome. Comparable signal intensities were obtained with only 1% as much oligo(dT)-purified mRNA as total RNA, and hence in vitro poly(A) tailing appears to be selective for mRNA. These and additional studies of genome-wide expression with DNA microarrays provide evidence that in vitro poly(A) tailing works universally for E. coli mRNAs.

Crystal structure of an activated response regulator bound to its target.

The chemotactic regulator CheY controls the direction of flagellar rotation in Escherichia coli. We have determined the crystal structure of BeF3--activated CheY from E. coli in complex with an N-terminal peptide derived from its target, FliM. The structure reveals that the first seven residues of the peptide pack against the beta4-H4 loop and helix H4 of CheY in an extended conformation, whereas residues 8-15 form two turns of helix and pack against the H4-beta5-H5 face. The peptide binds the only region of CheY that undergoes noticeable conformational change upon activation and would most likely be sandwiched between activated CheY and the remainder of FliM to reverse the direction of flagellar rotation.

Nitrogen regulatory protein C-controlled genes of Escherichia coli: scavenging as a defense against nitrogen limitation.

Nitrogen regulatory protein C (NtrC) of enteric bacteria activates transcription of genes/operons whose products minimize the slowing of growth under nitrogen-limiting conditions. To reveal the NtrC regulon of Escherichia coli we compared mRNA levels in a mutant strain that overexpresses NtrC-activated genes [glnL(Up)] to those in a strain with an ntrC (glnG) null allele by using DNA microarrays. Both strains could be grown under conditions of nitrogen excess. Thus, we could avoid differences in gene expression caused by slow growth or nitrogen limitation per se. Rearranging the spot images from microarrays in genome order allowed us to detect all of the operons known to be under NtrC control and facilitated detection of a number of new ones. Many of these operons encode transport systems for nitrogen-containing compounds, including compounds recycled during cell-wall synthesis, and hence scavenging appears to be a primary response to nitrogen limitation. In all, approximately 2% of the E. coli genome appears to be under NtrC control, although transcription of some operons depends on the nitrogen assimilation control protein, which serves as an adapter between NtrC and final sigma(70)-dependent promoters.

Phosphorylation-induced signal propagation in the response regulator ntrC.

The bacterial enhancer-binding protein NtrC is a well-studied response regulator in a two-component regulatory system. The amino (N)-terminal receiver domain of NtrC modulates the function of its adjacent output domain, which activates transcription by the sigma(54) holoenzyme. When a specific aspartate residue in the receiver domain of NtrC is phosphorylated, the dimeric protein forms an oligomer that is capable of ATP hydrolysis and transcriptional activation. A chemical protein cleavage method was used to investigate signal propagation from the phosphorylated receiver domain of NtrC, which acts positively, to its central output domain. The iron chelate reagent Fe-BABE was conjugated onto unique cysteines introduced into the N-terminal domain of NtrC, and the conjugated proteins were subjected to Fe-dependent cleavage with or without prior phosphorylation. Phosphorylation-dependent cleavage, which requires proximity and an appropriate orientation of the peptide backbone to the tethered Fe-EDTA, was particularly prominent with conjugated NtrC(D86C), in which the unique cysteine lies near the top of alpha-helix 4. Cleavage occurred outside the receiver domain itself and on the partner subunit of the derivatized monomer in an NtrC dimer. The results are commensurate with the hypothesis that alpha-helix 4 of the phosphorylated receiver domain of NtrC interacts with the beginning of the central domain for signal propagation. They imply that the phosphorylation-dependent interdomain and intermolecular interactions between the receiver domain of one subunit and the output domain of its partner subunit in an NtrC dimer precede-and may give rise to-the oligomerization needed for transcriptional activation.

NMR structure of activated CheY.

The CheY protein is the response regulator in bacterial chemotaxis. Phosphorylation of a conserved aspartyl residue induces structural changes that convert the protein from an inactive to an active state. The short half-life of the aspartyl-phosphate has precluded detailed structural analysis of the active protein. Persistent activation of Escherichia coli CheY was achieved by complexation with beryllofluoride (BeF(3)(-)) and the structure determined by NMR spectroscopy to a backbone r.m.s.d. of 0.58(+/-0.08) A. Formation of a hydrogen bond between the Thr87 OH group and an active site acceptor, presumably Asp57.BeF(3)(-), stabilizes a coupled rearrangement of highly conserved residues, Thr87 and Tyr106, along with displacement of beta4 and H4, to yield the active state. The coupled rearrangement may be a more general mechanism for activation of receiver domains.

Beryllofluoride mimics phosphorylation of NtrC and other bacterial response regulators.

Two-component systems, sensor kinase-response regulator pairs, dominate bacterial signal transduction. Regulation is exerted by phosphorylation of an Asp in receiver domains of response regulators. Lability of the acyl phosphate linkage has limited structure determination for the active, phosphorylated forms of receiver domains. As assessed by both functional and structural criteria, beryllofluoride yields an excellent analogue of aspartyl phosphate in response regulator NtrC, a bacterial enhancer-binding protein. Beryllofluoride also appears to activate the chemotaxis, sporulation, osmosensing, and nitrate/nitrite response regulators CheY, Spo0F, OmpR, and NarL, respectively. NMR spectroscopic studies indicate that beryllofluoride will facilitate both biochemical and structural characterization of the active forms of receiver domains.

"Switch I" mutant forms of the bacterial enhancer-binding protein NtrC that perturb the response to DNA.

NtrC (nitrogen regulatory protein C) is a bacterial enhancer-binding protein of 469 residues that activates transcription by sigma(54)-holoenzyme. A region of its transcriptional activation (central) domain that is highly conserved among homologous activators of sigma(54)-holoenzyme-residues 206-220-is essential for interaction with this RNA polymerase: it is required for contact with the polymerase and/or for coupling the energy from ATP hydrolysis to a change in the conformation of the polymerase that allows it to form transcriptionally productive open complexes. Several mutant NtrC proteins with amino acid substitutions in this region, including NtrC(A216V) and NtrC(G219K), have normal ATPase activity but fail in transcriptional activation. We now report that other mutant forms carrying amino acid substitutions at these same positions, NtrC(A216C) and NtrC(G219C), are capable of activating transcription when they are not bound to a DNA template (non-DNA-binding derivatives with an altered helix-turn-helix DNA-binding motif at the C terminus of the protein) but are unable to do so when they are bound to a DNA template, whether or not it carries a specific enhancer. Enhancer DNA remains a positive allosteric effector of ATP hydrolysis, as it is for wild-type NtrC but, surprisingly, appears to have become a negative allosteric effector for some aspect of interaction with sigma(54)-holoenzyme. The conserved region in which these amino acid substitutions occur (206-220) is equivalent to the Switch I region of a large group of purine nucleotide-binding proteins. Interesting analogies can be drawn between the Switch I region of NtrC and that of p21(ras).

Solution structure of the DNA-binding domain of NtrC with three alanine substitutions.

The structure of the 20 kDa C-terminal DNA-binding domain of NtrC from Salmonella typhimurium (residues Asp380-Glu469) with alanine replacing Arg456, Asn457, and Arg461, was determined by NMR spectroscopy. NtrC is a homodimeric enhancer-binding protein that activates the transcription of genes whose products are required for nitrogen metabolism. The 91-residue C-terminal domain contains the determinants necessary for dimerization and DNA-binding of the full length protein. The mutant protein does not bind to DNA but retains many characteristics of the wild-type protein, and the mutant domain expresses at high yield (20 mg/l) in minimal medium. Three-dimensional (1)H/(13)C/(15)N triple-resonance, (1)H-(13)C-(13)C-(1)H correlation and (15)N-separated nuclear Overhauser effect (NOE) spectroscopy experiments were used to make backbone and side-chain (1)H,(15)N, and (13)C assignments. The structures were calculated using a total of 1580 intra and inter-monomer distance and hydrogen bond restraints (88 hydrogen bonds; 44 hydrogen bond restraints), and 88 phi dihedral restraints for residues Asp400 through Glu469 in both monomers. A total of 54 ambiguous restraints (intra or inter-monomer) involving residues close to the 2-fold symmetry axis were also included. Each monomer consists of four helical segments. Helices A (Trp402-Leu414) and B (Leu421-His440) join with those of another monomer to form an antiparallel four-helix bundle. Helices C (Gln446-Leu451) and D (Ala456-Met468) of each monomer adopt a classic helix-turn-helix DNA-binding fold at either end of the protein. The backbone rms deviation for the 28 best of 40 starting structures is 0.6 (+/-0.2) A. Structural differences between the C-terminal domain of NtrC and the homologous Factor for Inversion Stimulation are discussed.

Mutations affecting motifs of unknown function in the central domain of nitrogen regulatory protein C.

The positive control function of the bacterial enhancer-binding protein NtrC resides in its central domain, which is highly conserved among activators of sigma54 holoenzyme. Previous studies of a small set of mutant forms specifically defective in transcriptional activation, called NtrC repressor [NtrC(Rep)] proteins, had enabled us to locate various functional determinants in the central domain. In this more comprehensive survey, the DNA encoding a major portion of the central domain was randomly mutagenized and mutated ntrC genes were introduced into the cell via multicopy expression plasmids. DNA sequencing of 95 isolates identified by a preliminary phenotypic screen revealed that the lesions in them caused 55 distinct single amino acid substitutions at 44 different positions. Assays of glnA transcription in vivo and in vitro yielded two conclusions. First, of the 41 mutant proteins that could be purified, 17 (1 known, 16 new) showed no detectable activity in either assay, thus qualifying them as true NtrC(Rep) proteins. These contained residue changes in six of the seven highly conserved regions in the central domain, including two never studied before. Second, some mutant proteins were inactive in vivo but were either marginally or fully active in vitro. Their surprising lack of activity in vivo may be accounted for by high levels of expression, which apparently decreased activation by these mutant proteins but not by wild-type NtrC (NtrCWT). Of particular interest were a subset of these proteins that exhibited greater transcriptional activation than NtrCWT at low concentrations. Their elevated activation capacities remain to be explained.

Sensing of nitrogen limitation by Bacillus subtilis: comparison to enteric bacteria.

Previous studies showed that Salmonella typhimurium apparently senses external nitrogen limitation as a decrease in the concentration of the internal glutamine pool. To determine whether the inverse relationship observed between doubling time and the glutamine pool size in enteric bacteria was also seen in phylogenetically distant organisms, we studied this correlation in Bacillus subtilis, a gram-positive, sporulating bacterium. We measured the sizes of the glutamine and glutamate pools for cells grown in batch culture on different nitrogen sources that yielded a range of doubling times, for cells grown in ammonia-limited continuous culture, and for mutant strains (glnA) in which the catalytic activity of glutamine synthetase was lowered. Although the glutamine pool size of B. subtilis clearly decreased under certain conditions of nitrogen limitation, particularly in continuous culture, the inverse relationship seen between glutamine pool size and doubling time in enteric bacteria was far less obvious in B. subtilis. To rule out the possibility that differences were due to the fact that B. subtilis has only a single pathway for ammonia assimilation, we disrupted the gene (gdh) that encodes the biosynthetic glutamate dehydrogenase in Salmonella. Studies of the S. typhimurium gdh strain in ammonia-limited continuous culture and of gdh glnA double-mutant strains indicated that decreases in the glutamine pool remained profound in strains with a single pathway for ammonia assimilation. Simple working hypotheses to account for the results with B. subtilis are that this organism refills an initially low glutamine pool by diminishing the utilization of glutamine for biosynthetic reactions and/or replenishes the pool by means of macromolecular degradation.

MgATP binding and hydrolysis determinants of NtrC, a bacterial enhancer-binding protein.

When phosphorylated, the dimeric form of nitrogen regulatory protein C (NtrC) of Salmonella typhimurium forms a larger oligomer(s) that can hydrolyze ATP and hence activate transcription by the sigma(54)-holoenzyme form of RNA polymerase. Studies of Mg-nucleoside triphosphate binding using a filter-binding assay indicated that phosphorylation is not required for nucleotide binding but probably controls nucleotide hydrolysis per se. Studies of binding by isothermal titration calorimetry indicated that the apparent K(d) of unphosphorylated NtrC for MgATPgammaS is 100 microM at 25 degrees C, and studies by filter binding indicated that the concentration of MgATP required for half-maximal binding is 130 microM at 37 degrees C. Filter-binding studies with mutant forms of NtrC defective in ATP hydrolysis implicated two regions of its central domain directly in nucleotide binding and three additional regions in hydrolysis. All five are highly conserved among activators of sigma(54)-holoenzyme. Regions implicated in binding are the Walker A motif and the region around residues G355 to R358, which may interact with the nucleotide base. Regions implicated in nucleotide hydrolysis are residues S207 and E208, which have been proposed to lie in a region analogous to the switch I effector region of p21(ras) and other purine nucleotide-binding proteins; residue R294, which may be a catalytic residue; and residue D239, which is the conserved aspartate in the putative Walker B motif. D239 appears to play a role in binding the divalent cation essential for nucleotide hydrolysis. Electron paramagnetic resonance analysis of Mn(2+) binding indicated that the central domain of NtrC does not bind divalent cation strongly in the absence of nucleotide.

Physical evidence for a phosphorylation-dependent conformational change in the enhancer-binding protein NtrC.

The bacterial enhancer-binding protein nitrogen regulatory protein C (NtrC) activates transcription by sigma54-containing RNA polymerase in a reaction that depends on ATP hydrolysis. Phosphorylation of an aspartate residue in the N-terminal receiver domain of NtrC induces oligomerization of the protein and activates the ATPase activity, which is a function of its central output domain. To study the role of the receiver domain of NtrC, which is known to act positively, we isolated mutant forms of the protein carrying single cysteine residues and derivatized them with a sulfhydryl-specific nitroxide reagent for electron paramagnetic resonance studies. Single cysteines were placed at four positions at which we had obtained constitutive amino acid substitutions, those that yield activity without phosphorylation. In only one case, derivatized C86 in alpha-helix 4 of the receiver domain, did the motion of the side chain become dramatically slower upon phosphorylation. Importantly, derivatized NtrCD86C (NtrCD86C*) activated transcription normally. Additional experiments indicated that the spectral change observed upon phosphorylation of NtrCD86C* was due to interdomain interactions rather than a conformational change within the N-terminal domain itself. These interactions did not appear to occur within a monomer. Although it is not clear whether the spectral change seen upon phosphorylation of NtrCD86C* is due to an interaction that occurs within a dimer of NtrC or requires the formation of higher-order oligomers, the change indicated that alpha-helix 4 of the receiver domain probably plays an important role in communication with the remainder of the protein.

Structure of a transiently phosphorylated switch in bacterial signal transduction.

Receiver domains are the dominant molecular switches in bacterial signalling. Although several structures of non-phosphorylated receiver domains have been reported, a detailed structural understanding of the activation arising from phosphorylation has been impeded by the very short half-lives of the aspartylphosphate linkages. Here we present the first structure of a receiver domain in its active state, the phosphorylated receiver domain of the bacterial enhancer-binding protein NtrC (nitrogen regulatory protein C). Nuclear magnetic resonance spectra were taken during steady-state autophosphorylation/dephosphorylation, and three-dimensional spectra from multiple samples were combined. Phosphorylation induces a large conformational change involving a displacement of beta-strands 4 and 5 and alpha-helices 3 and 4 away from the active site, a register shift and an axial rotation in helix 4. This creates an exposed hydrophobic surface that is likely to transmit the signal to the transcriptional activation domain.

Physiological role for the GlnK protein of enteric bacteria: relief of NifL inhibition under nitrogen-limiting conditions.

In Klebsiella pneumoniae, NifA-dependent transcription of nitrogen fixation (nif) genes is inhibited by a flavoprotein, NifL, in the presence of molecular oxygen and/or combined nitrogen. We recently demonstrated that the general nitrogen regulator NtrC is required to relieve NifL inhibition under nitrogen (N)-limiting conditions. We provide evidence that the sole basis for the NtrC requirement is its role as an activator of transcription for glnK, which encodes a PII-like allosteric effector. Relief of NifL inhibition is a unique physiological function for GlnK in that the structurally related GlnB protein of enteric bacteria-apparently a paralogue of GlnK-cannot substitute. Unexpectedly, although covalent modification of GlnK by uridylylation normally occurs under N-limiting conditions, several lines of evidence indicate that uridylylation is not required for relief of NifL inhibition. When GlnK was synthesized constitutively from non-NtrC-dependent promoters, it was able to relieve NifL inhibition in the absence of uridylyltransferase, the product of the glnD gene, and under N excess conditions. Moreover, an altered form of GlnK, GlnKY51N, which cannot be uridylylated due to the absence of the requisite tyrosine, was still able to relieve NifL inhibition.

Ammonia acquisition in enteric bacteria: physiological role of the ammonium/methylammonium transport B (AmtB) protein.

Homologues of the amtB gene of enteric bacteria exist in all three domains of life. Although their products are required for transport of the ammonium analogue methylammonium in washed cells, only in Saccharomyces cerevisiae have they been shown to be necessary for growth at low NH4+ concentrations. We now demonstrate that an amtB strain of Escherichia coli also grows slowly at low NH4+ concentrations in batch culture, but only at pH values below 7. In addition, we find that the growth defect of an S. cerevisiae triple-mutant strain lacking the function of three homologues of the ammonium/methylammonium transport B (AmtB) protein [called methylammonium/ammonium permeases (MEP)] that was observed at pH 6.1 is relieved at pH 7.1. These results provide direct evidence that AmtB participates in acquisition of NH4+/NH3 in bacteria as well as eucarya. Because NH3 is the species limiting at low pH for a given total concentration of NH4+ + NH3, results with both organisms indicate that AmtB/MEP proteins function in acquisition of the uncharged form. We confirmed that accumulation of [14C]methylammonium depends on its conversion to gamma-N-methylglutamine, an energy-requiring reaction catalyzed by glutamine synthetase, and found that at pH 7, constitutive expression of AmtB did not relieve the growth defects of a mutant strain of Salmonella typhimurium that appears to require a high internal concentration of NH4+/NH3. Hence, contrary to previous views, we propose that AmtB/MEP proteins increase the rate of equilibration of the uncharged species, NH3, across the cytoplasmic membrane rather than actively transporting-that is, concentrating-the charged species, NH4+.