Identification of Cys94 as the distal ligand to the Fe(III) heme in the transcriptional regulator RcoM-2 from Burkholderia xenovorans.

The CO-responsive transcriptional regulator RcoM from Burkholderia xenovorans (BxRcoM) was recently identified as a Cys(thiolate)-ligated heme protein that undergoes a redox-mediated ligand switch; however, the Cys bound to the Fe(III) heme was not identified. To that end, we generated and purified three Cys-to-Ser variants of BxRcoM-2--C94S, C127S, and C130S--and examined their spectroscopic properties in order to identify the native Cys(thiolate) ligand. Electronic absorption, resonance Raman, and electron paramagnetic resonance (EPR) spectroscopies demonstrate that the C127S and C130S variants, like wild-type BxRcoM-2, bind a six-coordinate low-spin Fe(III) heme using a Cys/His ligation motif. In contrast, electronic absorption and resonance Raman spectra of the C94S variant are most consistent with a mixture of five-coordinate high-spin and six-coordinate low-spin Fe(III) heme, neither of which are ligated by a Cys(thiolate) ligand. The EPR spectrum of C94S is dominated by a large, axial high-spin Fe(III) signal, confirming that the native ligation motif is not maintained in this variant. Together, these data reveal that Cys(94) is the distal Fe(III) heme ligand in BxRcoM-2; by sequence alignment, Cys(94) is also implicated as the distal Fe(III) heme ligand in BxRcoM-1, another homologue found in the same organism.

Burkholderia xenovorans RcoM(Bx)-1, a transcriptional regulator system for sensing low and persistent levels of carbon monoxide.

The single-component RcoM transcription factor couples an N-terminally bound heme cofactor with a C-terminal "LytTR" DNA-binding domain. Here the RcoM(Bx)-1 protein from Burkholderia xenovorans LB400 was heterologously expressed and then purified in a form with minimal bound CO (~10%) and was found to stably bind this effector with a nanomolar affinity. DNase I protection assays demonstrated that the CO-associated form binds with a micromolar affinity to two ~60-bp DNA regions, each comprised of a novel set of three direct-repeat binding sites spaced 21 bp apart on center. Binding to each region was independent, while binding to the triplet binding sites within a region was cooperative, depended upon spacing and sequence, and was marked by phased DNase I hyperactivity and protection patterns consistent with considerable changes in the DNA conformation of the nucleoprotein complex. Each protected binding site spanned a conserved motif (5'-TTnnnG-3') that was present, in triplicate, in putative RcoM-binding regions of more than a dozen organisms. In vivo screens confirmed the functional importance of the conserved "TTnnnG" motif residues and their triplet arrangement and were also used to determine an improved binding motif [5'-CnnC(C/A)(G/A)TTCAnG-3'] that more closely corresponds to canonical LytTR domain/DNA-binding sites. A low-affinity but CO-dependent binding of RcoM(Bx)-1 to a variety of DNA probes was demonstrated in vitro. We posit that for the RcoM(Bx)-1 protein, the high CO affinity combined with multiple low-affinity DNA-binding events constitutes a transcriptional "accumulating switch" that senses low but persistent CO levels.

Complete genome sequence of Rhodospirillum rubrum type strain (S1).

Rhodospirillum rubrum (Esmarch 1887) Molisch 1907 is the type species of the genus Rhodospirillum, which is the type genus of the family Rhodospirillaceae in the class Alphaproteobacteria. The species is of special interest because it is an anoxygenic phototroph that produces extracellular elemental sulfur (instead of oxygen) while harvesting light. It contains one of the most simple photosynthetic systems currently known, lacking light harvesting complex 2. Strain S1(T) can grow on carbon monoxide as sole energy source. With currently over 1,750 PubMed entries, R. rubrum is one of the most intensively studied microbial species, in particular for physiological and genetic studies. Next to R. centenum strain SW, the genome sequence of strain S1(T) is only the second genome of a member of the genus Rhodospirillum to be published, but the first type strain genome from the genus. The 4,352,825 bp long chromosome and 53,732 bp plasmid with a total of 3,850 protein-coding and 83 RNA genes were sequenced as part of the DOE Joint Genome Institute Program DOEM 2002.

Ligand responses of Vfr, the virulence factor regulator from Pseudomonas aeruginosa.

Vfr, a transcription factor homologous to the Escherichia coli cyclic AMP (cAMP) receptor protein (CRP), regulates many aspects of virulence in Pseudomonas aeruginosa. Vfr, like CRP, binds to cAMP and then recognizes its target DNA and activates transcription. Here we report that Vfr has important functional differences from CRP in terms of ligand sensing and response. First, Vfr has a significantly higher cAMP affinity than does CRP, which might explain the mysteriously unidirectional functional complementation between the two proteins (S. E. H. West et al., J. Bacteriol. 176:7532-7542, 1994). Second, Vfr is activated by both cAMP and cGMP, while CRP is specific to cAMP. Mutagenic analyses show that Thr133 (analogous to Ser128 of CRP) is the key residue for both of these distinct Vfr properties. On the other hand, substitutions that cause cAMP-independent activity in Vfr are similar to those seen in CRP, suggesting that a common cAMP activation mechanism is present. In the course of these analyses, we found a remarkable class of Vfr variants that have completely reversed the regulatory logic of the protein: they are active in DNA binding without cAMP and are strongly inhibited by cAMP. The physiological impact of Vfr's ligand sensing and response is discussed, as is a plausible basis for the fundamental change in protein allostery in the novel group of Vfr variants.

The poor growth of Rhodospirillum rubrum mutants lacking RubisCO is due to the accumulation of ribulose-1,5-bisphosphate.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) catalyzes the first step of CO(2) fixation in the Calvin-Benson-Bassham (CBB) cycle. Besides its function in fixing CO(2) to support photoautotrophic growth, the CBB cycle is also important under photoheterotrophic growth conditions in purple nonsulfur photosynthetic bacteria. It has been assumed that the poor photoheterotrophic growth of RubisCO-deficient strains was due to the accumulation of excess intracellular reductant, which implied that the CBB cycle is important for maintaining the redox balance under these conditions. However, we present analyses of cbbM mutants in Rhodospirillum rubrum that indicate that toxicity is the result of an elevated intracellular pool of ribulose-1,5-bisphosphate (RuBP). There is a redox effect on growth, but it is apparently an indirect effect on the accumulation of RuBP, perhaps by the regulation of the activities of enzymes involved in RuBP regeneration. Our studies also show that the CBB cycle is not essential for R. rubrum to grow under photoheterotrophic conditions and that its role in controlling the redox balance needs to be further elucidated. Finally, we also show that CbbR is a positive transcriptional regulator of the cbb operon (cbbEFPT) in R. rubrum, as seen with related organisms, and define the transcriptional organization of the cbb genes.

Sustaining N2-dependent growth in the presence of CO.

Low levels of carbon monoxide inhibit the N(2)-dependent growth of Rhodospirillum rubrum unless the ∼100-residue CowN protein is expressed. Expression requires the CO-responsive regulator RcoM and is maximal in cells grown in the presence of CO and a poor nitrogen source, consistent with the role of CowN in N(2) fixation.

Elimination of Rubisco alters the regulation of nitrogenase activity and increases hydrogen production in Rhodospirillum rubrum.

Nitrogenase not only reduces atmospheric nitrogen to ammonia, but also reduces protons to hydrogen (H(2)). The nitrogenase system is the primary means of H(2) production under photosynthetic and nitrogen-limiting conditions in many photosynthetic bacteria, including Rhodospirillum rubrum. The efficiency of this biological H(2) production largely depends on the nitrogenase enzyme and the availability of ATP and electrons in the cell. Previous studies showed that blockage of the CO(2) fixation pathway in R. rubrum induced nitrogenase activity even in the presence of ammonium, presumably to remove excess reductant in the cell. We report here the re-characterization of cbbM mutants in R. rubrum to study the effect of Rubisco on H(2) production. Our newly constructed cbbM mutants grew poorly in malate medium under anaerobic conditions. However, the introduction of constitutively active NifA (NifA*), the transcriptional activator of the nitrogen fixation (nif) genes, allows cbbM mutants to dissipate the excess reductant through the nitrogenase system and improves their growth. Interestingly, we found that the deletion of cbbM alters the posttranslational regulation of nitrogenase activity, resulting in partially active nitrogenase in the presence of ammonium. The combination of mutations in nifA, draT and cbbM greatly increased H(2) production of R. rubrum, especially in the presence of excess of ammonium. Furthermore, these mutants are able to produce H(2) over a much longer time frame than the wild type, increasing the potential of these recombinant strains for the biological production of H(2).

Mutagenesis and functional characterization of the four domains of GlnD, a bifunctional nitrogen sensor protein.

GlnD is a bifunctional uridylyltransferase/uridylyl-removing enzyme (UTase/UR) and is believed to be the primary sensor of nitrogen status in the cell by sensing the level of glutamine in enteric bacteria. It plays an important role in nitrogen assimilation and metabolism by reversibly regulating the modification of P(II) protein; P(II) in turn regulates a variety of other proteins. GlnD appears to have four distinct domains: an N-terminal nucleotidyltransferase (NT) domain; a central HD domain, named after conserved histidine and aspartate residues; and two C-terminal ACT domains, named after three of the allosterically regulated enzymes in which this domain is found. Here we report the functional analysis of these domains of GlnD from Escherichia coli and Rhodospirillum rubrum. We confirm the assignment of UTase activity to the NT domain and show that the UR activity is a property specifically of the HD domain: substitutions in this domain eliminated UR activity, and a truncated protein lacking the NT domain displayed UR activity. The deletion of C-terminal ACT domains had little effect on UR activity itself but eliminated the ability of glutamine to stimulate that activity, suggesting a role for glutamine sensing by these domains. The deletion of C-terminal ACT domains also dramatically decreased UTase activity under all conditions tested, but some of these effects are due to the competition of UTase activity with unregulated UR activity in these variants.

Cyclic di-GMP allosterically inhibits the CRP-like protein (Clp) of Xanthomonas axonopodis pv. citri.

The protein Clp from Xanthomonas axonopodis pv. citri regulates pathogenesis and is a member of the CRP (cyclic AMP receptor protein) superfamily. We show that unlike the DNA-binding activity of other members of this family, the DNA-binding activity of Clp is allosterically inhibited by its effector and that cyclic di-GMP serves as that effector at physiological concentrations.

Effect of perturbation of ATP level on the activity and regulation of nitrogenase in Rhodospirillum rubrum.

Nitrogenase activity in Rhodospirillum rubrum and in some other photosynthetic bacteria is regulated in part by the availability of light. This regulation is through a posttranslational modification system that is itself regulated by P(II) homologs in the cell. P(II) is one of the most broadly distributed regulatory proteins in nature and directly or indirectly senses nitrogen and carbon signals in the cell. However, its possible role in responding to light availability remains unclear. Because P(II) binds ATP, we tested the hypothesis that removal of light would affect P(II) by changing intracellular ATP levels, and this in turn would affect the regulation of nitrogenase activity. This in vivo test involved a variety of different methods for the measurement of ATP, as well as the deliberate perturbation of intracellular ATP levels by chemical and genetic means. To our surprise, we found fairly normal levels of nitrogenase activity and posttranslational regulation of nitrogenase even under conditions of drastically reduced ATP levels. This indicates that low ATP levels have no more than a modest impact on the P(II)-mediated regulation of NifA activity and on the posttranslational regulation of nitrogenase activity. The relatively high nitrogenase activity also shows that the ATP-dependent electron flux from dinitrogenase reductase to dinitrogenase is also surprisingly insensitive to a depleted ATP level. These in vivo results disprove the simple model of ATP as the key energy signal to P(II) under these conditions. We currently suppose that the ratio of ADP/ATP might be the relevant signal, as suggested by a number of recent in vitro analyses.

Identification and functional characterization of NifA variants that are independent of GlnB activation in the photosynthetic bacterium Rhodospirillum rubrum.

The activity of NifA, the transcriptional activator of the nitrogen fixation (nif) gene, is tightly regulated in response to ammonium and oxygen. However, the mechanisms for the regulation of NifA activity are quite different among various nitrogen-fixing bacteria. Unlike the well-studied NifL-NifA regulatory systems in Klebsiella pneumoniae and Azotobacter vinelandii, in Rhodospirillum rubrum NifA is activated by a direct protein-protein interaction with the uridylylated form of GlnB, which in turn causes a conformational change in NifA. We report the identification of several substitutions in the N-terminal GAF domain of R. rubrum NifA that allow NifA to be activated in the absence of GlnB. Presumably these substitutions cause conformational changes in NifA necessary for activation, without interaction with GlnB. We also found that wild-type NifA can be activated in a GlnB-independent manner under certain growth conditions, suggesting that some other effector(s) can also activate NifA. An attempt to use Tn5 mutagenesis to obtain mutants that altered the pool of these presumptive effector(s) failed, though much rarer spontaneous mutations in nifA were detected. This suggests that the necessary alteration of the pool of effector(s) for NifA activation cannot be obtained by knockout mutations.

The transcription regulator RcoM-2 from Burkholderia xenovorans is a cysteine-ligated hemoprotein that undergoes a redox-mediated ligand switch.

Spectroscopic characterization of the newly discovered heme-PAS domain sensor protein BxRcoM-2 reveals that this protein undergoes redox-dependent ligand switching and CO- and NO-induced ligand displacement. The aerobic bacterium Burkholderia xenovorans expresses two homologous heme-containing proteins that promote CO-dependent transcription in vivo. These regulators of CO metabolism, BxRcoM-1 and BxRcoM-2, are gas-responsive heme-PAS domain proteins like mammalian neuronal PAS domain protein 2 (NPAS2) and the direct oxygen sensor from Escherichia coli ( EcDos). BxRcoM-2 was studied using electronic absorption, MCD, resonance Raman, and EPR spectroscopies. In the Fe(III) oxidation state, the heme is low-spin and six-coordinate with a cysteine(thiolate) as one of the two ligands. The sixth ligand is a histidine (His (74)), which is present in all states of the protein that were studied. Reduction to the Fe(II) oxidation state results in replacement of the cysteine(thiolate) with a neutral thioether ligand, Met (104). CO and NO bind to the Fe(II) BxRcoM-2 heme opposite the histidine ligand. Thus, BxRcoM-2 employs coordination state changes similar to those known for CO-sensing CooA, with redox-dependent loss of a cysteine(thiolate) ligand and displacement of a relatively weakly bound axial ligand by the effector gas molecule. Like EcDos, the weakly bound axial ligand that is displaced is methionine.

Two-state allosteric modeling suggests protein equilibrium as an integral component for cyclic AMP (cAMP) specificity in the cAMP receptor protein of Escherichia coli.

Activation of the cAMP receptor protein (CRP) from Escherichia coli is highly specific to its allosteric ligand, cAMP. Ligands such as adenosine and cGMP, which are structurally similar to cAMP, fail to activate wild-type CRP. However, several cAMP-independent CRP variants (termed CRP*) exist that can be further activated by both adenosine and cGMP, as well as by cAMP. This has remained a puzzle because the substitutions in many of these CRP* variants lie far from the cAMP-binding pocket (>10 A) and therefore should not directly affect that pocket. Here we show a surprising similarity in the altered ligand specificity of four CRP* variants with a single substitution in D53S, G141K, A144T, or L148K, and we propose a common basis for this phenomenon. The increased active protein population caused by an equilibrium shift in these variants is hypothesized to preferentially stabilize ligand binding. This explanation is completely consistent with the cAMP specificity in the activation of wild-type CRP. The model also predicts that wild-type CRP should be activated even by the lower-affinity ligand, adenosine, which we experimentally confirmed. The study demonstrates that protein equilibrium is an integral factor for ligand specificity in an allosteric protein, in addition to the direct effects of ligand pocket residues.

RcoM: a new single-component transcriptional regulator of CO metabolism in bacteria.

Genomic analysis suggested the existence of a CO-sensing bacterial transcriptional regulator that couples an N-terminal PAS fold domain to a C-terminal DNA-binding LytTR domain. UV/visible-light spectral analyses of heterologously expressed, purified full-length proteins indicated that they contained a hexacoordinated b-type heme moiety that avidly binds CO and NO. Studies of protein variants strongly suggested that the PAS domain residues His74 and Met104 serve as the heme Fe(II) axial ligands, with displacement of Met104 upon binding of the gaseous effectors. Two RcoM (regulator of CO metabolism) homologs were shown to function in vivo as CO sensors capable of regulating an aerobic CO oxidation (cox) regulon. The genetic linkage of rcoM with both aerobic (cox) and anaerobic (coo) CO oxidation systems suggests that in different organisms RcoM proteins may control either regulon type.

Mechanism of the CO-sensing heme protein CooA: new insights from the truncated heme domain and UVRR spectroscopy.

The bacterial CO-sensing heme protein CooA activates expression of genes whose products perform CO-metabolism by binding its target DNA in response to CO binding. The required conformational change has been proposed to result from CO-induced displacement of the heme and of the adjacent C-helix, which connects the sensory and DNA-binding domains. Support for this proposal comes from UV Resonance Raman (UVRR) spectroscopy, which reveals a more hydrophobic environment for the C-helix residue Trp110 when CO binds. In addition, we find a tyrosine UVRR response, which is attributable to weakening of a Tyr55-Glu83 H-bond that anchors the proximal side of the heme. Both Trp and Tyr responses are augmented in the heme domain when the DNA-binding domain has been removed, apparently reflecting loss of the inter-domain restraint. This augmentation is abolished by a Glu83Gln substitution, which weakens the anchoring H-bond. The CO recombination rate following photolysis of the CO adduct is similar for truncated and full-length protein, though truncation does increase the rate of CO association in the absence of photolysis; together these data indicate that truncation causes a faster dissociation of the endogenous Pro2 ligand. These findings are discussed in the light of structural evidence that the N-terminal tail, once released from the heme, selects the proper orientation of the DNA-binding domain, via docking interactions.

Specificity and regulation of interaction between the PII and AmtB1 proteins in Rhodospirillum rubrum.

The nitrogen regulatory protein P(II) and the ammonia gas channel AmtB are both found in most prokaryotes. Interaction between these two proteins has been observed in several organisms and may regulate the activities of both proteins. The regulation of their interaction is only partially understood, and we show that in Rhodospirillum rubrum one P(II) homolog, GlnJ, has higher affinity for an AmtB(1)-containing membrane than the other two P(II) homologs, GlnB and GlnK. This interaction strongly favors the nonuridylylated form of GlnJ and is disrupted by high levels of 2-ketoglutarate (2-KG) in the absence of ATP or low levels of 2-KG in the presence of ATP. ADP inhibits the destabilization of the GlnJ-AmtB(1) complex in the presence of ATP and 2-KG, supporting a role for P(II) as an energy sensor measuring the ratio of ATP to ADP. In the presence of saturating levels of ATP, the estimated K(d) of 2-KG for GlnJ bound to AmtB(1) is 340 microM, which is higher than that required for uridylylation of GlnJ in vitro, about 5 microM. This supports a model where multiple 2-KG and ATP molecules must bind a P(II) trimer to stimulate release of P(II) from AmtB(1), in contrast to the lower 2-KG requirement for productive uridylylation of P(II) by GlnD.

Structure-based hypothesis on the activation of the CO-sensing transcription factor CooA.

The CooA family of proteins are prokaryotic CO-sensing transcription factors that regulate the expression of genes involved in the utilization of CO as an energy source. They are homodimeric proteins that contain two hemes. Each monomer contains an N-terminal heme-binding domain and a C-terminal DNA-binding domain. Binding of CO to the heme leads to activation by a large reorientation of the DNA-binding domain such that the DNA-binding domain is in position for specific DNA recognition. The crystal structure of CooA from Rhodospirillum rubrum [RrCooA; Lanzilotta et al. (2000), Nature Struct. Biol. 7, 876-880] in the inactive CO-free off-state shows that the N-terminal Pro residue of monomer A coordinates the heme of monomer B and vice versa. It now appears that the CO replaces the Pro ligand and that this change is coupled to the activation process. However, precisely how the replacement of the Pro ligand by CO results in structural changes some 25 A from the CO-binding site remains unknown. Here, the structure of a CooA variant from the thermophilic bacterium Carboxydothermus hydrogenoformans (ChCooA) is reported in which one monomer is fully in the on-state. The N-terminal region that is displaced by CO binding is now positioned between the heme-binding and DNA-binding domains, which requires movement of the N-terminus by approximately 20 A and thus serves as a bridge between the two domains that helps to orient the DNA-binding domain in position for DNA binding.

A C-helix residue, Arg-123, has important roles in both the active and inactive forms of the cAMP receptor protein.

The cAMP receptor protein (CRP) of Escherichia coli exists in an equilibrium between active and inactive forms, and the effector, cAMP, shifts that equilibrium to the active form, thereby allowing DNA binding. For this equilibrium shift, a C-helix repositioning around the C-helix residues Thr-127 and Ser-128 has been reported as a critical local event along with proper beta4/beta5 positioning. Here we show that another C-helix residue, Arg-123, has a unique role in cAMP-dependent CRP activation in two different ways. First, Arg-123 is important for proper cAMP affinity, although it is not critical for the conformational change with saturating amounts of cAMP. Second, Arg-123 is optimal for stabilizing the inactive conformation of CRP when cAMP is absent, thereby allowing a maximal range of regulation by cAMP. However, Arg-123 does not appear to be critical for a functional response to cAMP, as has been proposed previously (Berman, H. M., Ten Eyck, L. F., Goodsell, D. S., Haste, N. M., Korney, A., and Taylor, S. S. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 45-50). Based on mutagenic evidence, we also propose the basis for the stabilization of the inactive form to be through a salt interaction between Asp-68 and Arg-123.

DNA binding by an imidazole-sensing CooA variant is dependent on the heme redox state.

CooA is a transcription factor from Rhodospirillum rubrum that is regulated by the binding of the small molecule effector, CO, to a heme moiety in the protein. The heme in CooA is axially ligated by two endogenous donors in the Fe(III) and Fe(II) states of the protein, and CO binding to the Fe(II) state results in replacement of the distal ligand. Reduction of the heme in the absence of CO results in a ligand switch on the proximal side, in which a cysteine thiolate in the Fe(III) state is replaced by a histidine in the Fe(II) state. Recently, a variant, termed RW CooA, was designed to respond to a new effector; Fe(II) RW CooA shows high specificity and induced DNA-binding activity in the presence of imidazole. Spectroscopic characterization of the imidazole adducts of RW CooA revealed that, unlike CO, imidazole binds to both Fe(III) RW CooA and Fe(II) RW CooA. The spectral characteristics are consistent with normal function of the redox-mediated ligand switch; Fe(III)-imidazole RW CooA bears a thiolate ligand and Fe(II)-imidazole RW CooA bears a neutral donor ligand. Since the effector binds to both redox states, RW CooA was used to probe the role of the redox-mediated ligand switch in the CooA activation mechanism. Functional studies of Fe(III)-imidazole and Fe(II)-imidazole ligated RW CooA demonstrate that only the Fe(II)-imidazole form is active for DNA binding. Thus, the ligand switch is essential for the activating conformational change and may prevent aberrant activation of CooA by other neutral diatomic molecules.

Roles of the heme and heme ligands in the activation of CooA, the CO-sensing transcriptional activator.

CooA of Rhodospirillum rubrum is a CO-sensing, heme-containing transcriptional activator that regulates the expression of the genes responsible for CO oxidation. We randomized the codons for residues 75-77 of CooA which include two proximal heme ligands, screened for both CO-dependent and CO-independent variants, and characterized in vivo and in vitro properties of selected CooA variants. The analysis showed that small residues at position 75 are critical and that, as previously suspected, His77 is absolutely necessary for CO responsiveness of CooA. Many hemeless variants altered at those residues were found to be constitutively active. We propose that proximal heme pocket residues of wild-type CooA have important role in stabilizing both active and inactive heme positions for its CO-sensing function.