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1.
Taylor BL 《Molecular microbiology》2007,65(6):1415-1424
Aer, the Escherichia coli aerotaxis (oxygen-sensing) receptor, is representative of a small class of receptors that face the cytoplasm in bacteria. Instead of sensing oxygen directly, Aer detects redox changes in the electron transport system or cytoplasm when the bacteria enter or leave a hypoxic microniche. As a result, Aer sensing also enables bacteria to avoid environments where carbon deficiency, unfavourable reduction potential or other insults would limit energy production. An FAD-binding PAS domain is the sensor for Aer and a HAMP domain interacts with the PAS domain to form an input-output module for signal transduction. By analogy to the first solution structure of an isolated HAMP domain from Archaeoglobus, Aer HAMP is proposed to fold into a four-helix bundle that rotates between a signal-on and signal-off conformation. Aer is the first protein in which a PAS-HAMP input-output module has been investigated. The structure and signal transduction mechanism of Aer is providing important insights into signalling by PAS and HAMP domains. 相似文献
2.
Delineating PAS‐HAMP interaction surfaces and signalling‐associated changes in the aerotaxis receptor Aer
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The Escherichia coli aerotaxis receptor, Aer, monitors cellular oxygen and redox potential via FAD bound to a cytosolic PAS domain. Here, we show that Aer‐PAS controls aerotaxis through direct, lateral interactions with a HAMP domain. This contrasts with most chemoreceptors where signals propagate along the protein backbone from an N‐terminal sensor to HAMP. We mapped the interaction surfaces of the Aer PAS, HAMP and proximal signalling domains in the kinase‐off state by probing the solvent accessibility of 129 cysteine substitutions. Inaccessible PAS‐HAMP surfaces overlapped with a cluster of PAS kinase‐on lesions and with cysteine substitutions that crosslinked the PAS β ‐scaffold to the HAMP AS‐2 helix. A refined Aer PAS‐HAMP interaction model is presented. Compared to the kinase‐off state, the kinase‐on state increased the accessibility of HAMP residues (apparently relaxing PAS‐HAMP interactions), but decreased the accessibility of proximal signalling domain residues. These data are consistent with an alternating static‐dynamic model in which oxidized Aer‐PAS interacts directly with HAMP AS‐2, enforcing a static HAMP domain that in turn promotes a dynamic proximal signalling domain, resulting in a kinase‐off output. When PAS‐FAD is reduced, PAS interaction with HAMP is relaxed and a dynamic HAMP and static proximal signalling domain convey a kinase‐on output. 相似文献
3.
Genetic analysis of the HAMP domain of the Aer aerotaxis sensor localizes flavin adenine dinucleotide-binding determinants to the AS-2 helix 总被引:7,自引:4,他引:3
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HAMP domains are signal transduction domains typically located between the membrane anchor and cytoplasmic signaling domain of the proteins in which they occur. The prototypical structure consists of two helical amphipathic sequences (AS-1 and AS-2) connected by a region of undetermined structure. The Escherichia coli aerotaxis receptor, Aer, has a HAMP domain and a PAS domain with a flavin adenine dinucleotide (FAD) cofactor that senses the intracellular energy level. Previous studies reported mutations in the HAMP domain that abolished FAD binding to the PAS domain. In this study, using random and site-directed mutagenesis, we identified the distal helix, AS-2, as the component of the HAMP domain that stabilizes FAD binding. AS-2 in Aer is not amphipathic and is predicted to be buried. Mutations in the sequence coding for the contiguous proximal signaling domain altered signaling by Aer but did not affect FAD binding. The V264M residue replacement in this region resulted in an inverted response in which E. coli cells expressing the mutant Aer protein were repelled by oxygen. Bioinformatics analysis of aligned HAMP domains indicated that the proximal signaling domain is conserved in other HAMP domains that are not involved in chemotaxis or aerotaxis. Only one null mutation was found in the coding sequence for the HAMP AS-1 and connector regions, suggesting that these are not active signal transduction sites. We consider a model in which the signal from FAD is transmitted across a PAS-HAMP interface to AS-2 or the proximal signaling domain. 相似文献
4.
Repik A Rebbapragada A Johnson MS Haznedar JO Zhulin IB Taylor BL 《Molecular microbiology》2000,36(4):806-816
PAS domains sense oxygen, redox potential and light, and are implicated in behaviour, circadian rhythmicity, development and metabolic regulation. Although PAS domains are widespread in archaea, bacteria and eukaryota, the mechanism of signal transduction has been elucidated only for the bacterial photo sensor PYP and oxygen sensor FixL. We investigated the signalling mechanism in the PAS domain of Aer, the redox potential sensor and aerotaxis transducer in Escherichia coli. Forty-two residues in Aer were substituted using cysteine-replacement mutagenesis. Eight mutations resulted in a null phenotype for aerotaxis, the behavioural response to oxygen. Four of them also led to the loss of the non-covalently bound FAD cofactor. Three mutant Aer proteins, N34C, F66C and N85C, transmitted a constant signal-on bias. One mutation, Y111C, inverted signalling by the transducer so that positive stimuli produced negative signals and vice versa. Residues critical for signalling were mapped onto a three-dimensional model of the Aer PAS domain, and an FAD-binding site and 'active site' for signal transduction are proposed. 相似文献
5.
Interactions between the PAS and HAMP domains of the Escherichia coli aerotaxis receptor Aer
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The Escherichia coli energy-sensing Aer protein initiates aerotaxis towards environments supporting optimal cellular energy. The Aer sensor is an N-terminal, FAD-binding, PAS domain. The PAS domain is linked by an F1 region to a membrane anchor, and in the C-terminal half of Aer, a HAMP domain links the membrane anchor to the signaling domain. The F1 region, membrane anchor, and HAMP domain are required for FAD binding. Presumably, alterations in the redox potential of FAD induce conformational changes in the PAS domain that are transmitted to the HAMP and C-terminal signaling domains. In this study we used random mutagenesis and intragenic pseudoreversion analysis to examine functional interactions between the HAMP domain and the N-terminal half of Aer. Missense mutations in the HAMP domain clustered in the AS-2 alpha-helix and abolished FAD binding to Aer, as previously reported. Three amino acid replacements in the Aer-PAS domain, S28G, A65V, and A99V, restored FAD binding and aerotaxis to the HAMP mutants. These suppressors are predicted to surround a cleft in the PAS domain that may bind FAD. On the other hand, suppression of an Aer-C253R HAMP mutant was specific to an N34D substitution with a predicted location on the PAS surface, suggesting that residues C253 and N34 interact or are in close proximity. No suppressor mutations were identified in the F1 region or membrane anchor. We propose that functional interactions between the PAS domain and the HAMP AS-2 helix are required for FAD binding and aerotactic signaling by Aer. 相似文献
6.
HAMP domains, found in many bacterial signal transduction proteins, generally transmit an intramolecular signal between an extracellular sensory domain and an intracellular signaling domain. Studies of HAMP domains in proteins where both the input and output signals occur intracellularly are limited to those of the Aer energy taxis receptor of Escherichia coli, which has both a HAMP domain and a sensory PAS domain. Campylobacter jejuni has an energy taxis system consisting of the domains of Aer divided between two proteins, CetA (HAMP domain containing) and CetB (PAS domain containing). In this study, we found that the CetA HAMP domain differs significantly from that of Aer in the predicted secondary structure. Using similarity searches, we identified 55 pairs of HAMP/PAS proteins encoded by adjacent genes in a diverse group of microorganisms. We propose that these HAMP/PAS pairs form a new family of bipartite energy taxis receptors. Within these proteins, we identified nine residues in the HAMP domain and proximal signaling domain that are highly conserved, at least three of which are required for CetA function. Additionally, we demonstrated that CetA contributes to the invasion of human epithelial cells by C. jejuni, while CetB does not. This finding supports the hypothesis that members of HAMP/PAS pairs possess the capacity to act independently of each other in cellular traits other than energy taxis. 相似文献
7.
HAMP domains are sensory transduction modules that connect input and output domains in diverse signaling proteins from archaea, bacteria, and lower eukaryotes. Here, we employed in vivo disulfide cross-linking to explore the structure of the HAMP domain in the Escherichia coli aerotaxis receptor Aer. Using an Aer HAMP model based on the structure of Archaeoglobus fulgidus Af1503-HAMP, the closest residue pairs at the interface of the HAMP AS-1 and AS-2' helices were determined and then replaced with cysteines and cross-linked in vivo. Except for a unique discontinuity in AS-2, the data suggest that the Aer HAMP domain forms a parallel four-helix bundle that is similar to the structure of Af1503. The HAMP discontinuity was associated with a segment of AS-2 that was recently shown to interact with the Aer-PAS sensing domain. The four-helix HAMP bundle and its discontinuity were maintained in both the kinase-on and kinase-off states of Aer, although differences in the rates of disulfide formation also indicated the existence of different HAMP conformations in the kinase-on and kinase-off states. In particular, the kinase-on state was accompanied by significantly increased disulfide formation rates at the distal end of the HAMP four-helix bundle. This indicates that HAMP signaling may be associated with a tilting of the AS-1 and AS-2' helices, which may be the signal that is transmitted to the kinase control region of Aer. 相似文献
8.
PAS domain of the Aer redox sensor requires C-terminal residues for native-fold formation and flavin adenine dinucleotide binding
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The Aer protein in Escherichia coli is a membrane-bound, FAD-containing aerotaxis and energy sensor that putatively monitors the redox state of the electron transport system. Binding of FAD to Aer requires the N-terminal PAS domain and residues in the F1 region and C-terminal HAMP domain. The PAS domains of other PAS proteins are soluble in water. To investigate properties of the PAS domain, we subcloned segments of the aer gene from E. coli that encode the PAS domain with and without His6 tags and expressed the PAS peptides in E. coli. The 20-kDa His6-Aer2-166 PAS-F1 fragment was purified as an 800-kDa complex by gel filtration chromatography, and the associating protein was identified by N-terminal sequencing as the chaperone protein GroEL. None of the N-terminal fragments of Aer found in the soluble fraction was released from GroEL, suggesting that these peptides do not fold correctly in an aqueous environment and require a motif external to the PAS domain for proper folding. Consistent with this model, peptide fragments that included the membrane binding region and part (Aer2-231) or all (Aer2-285) of the HAMP domain inserted into the membrane, indicating that they were released by GroEL. Aer2-285, but not Aer2-231, bound FAD, confirming the requirement for the HAMP domain in stabilizing FAD binding. The results raise an interesting possibility that residues outside the PAS domain that are required for FAD binding are essential for formation of the PAS native fold. 相似文献
9.
Structure-function relationships in the HAMP and proximal signaling domains of the aerotaxis receptor Aer 总被引:2,自引:1,他引:1
Aer, the Escherichia coli aerotaxis receptor, faces the cytoplasm, where the PAS (Per-ARNT-Sim)-flavin adenine dinucleotide (FAD) domain senses redox changes in the electron transport system or cytoplasm. PAS-FAD interacts with a HAMP (histidine kinase, adenylyl cyclase, methyl-accepting protein, and phosphatase) domain to form an input-output module for Aer signaling. In this study, the structure of the Aer HAMP and proximal signaling domains was probed to elucidate structure-function relationships important for signaling. Aer residues 210 to 290 were individually replaced with cysteine and then cross-linked in vivo. The results confirmed that the Aer HAMP domain is composed of two α-helices separated by a structured loop. The proximal signaling domain consisted of two α-helices separated by a short undetermined structure. The Af1503 HAMP domain from Archaeoglobus fulgidus was recently shown to be a four-helix bundle. To test whether the Af1503 HAMP domain is a prototype for the Aer HAMP domain, the latter was modeled using coordinates from Af1503. Several findings supported the hypothesis that Aer has a four-helix HAMP structure: (i) cross-linking independently identified the same residues at the dimer interface that were predicted by the model, (ii) the rate of cross-linking for residue pairs was inversely proportional to the β-carbon distances measured on the model, and (iii) clockwise lesions that were not contiguous in the linear Aer sequence were clustered in one region in the folded HAMP model, defining a potential site of PAS-HAMP interaction during signaling. In silico modeling of mutant Aer proteins indicated that the four-helix HAMP structure was important for Aer stability or maturation. The significance of the HAMP and proximal signaling domain structure for signal transduction is discussed. 相似文献
10.
In Escherichia coli, the aerotaxis receptor Aer is an atypical receptor because it senses intracellular redox potential. The Aer sensor is a cytoplasmic, N-terminal PAS domain that is tethered to the membrane by a 47-residue F1 linker. Here we investigated the function, topology, and orientation of F1 by employing random mutagenesis, cysteine scanning, and disulfide cross-linking. No native residue was obligatory for function, most deleterious substitutions had radically different side chain properties, and all F1 mutants but one were functionally rescued by the chemoreceptor Tar. Cross-linking studies were consistent with the predicted α-helical structure in the N-terminal F1 region and demonstrated trigonal interactions among the F1 linkers from three Aer monomers, presumably within trimer-of-dimer units, as well as binary interactions between subunits. Using heterodimer analyses, we also demonstrated the importance of arginine residues near the membrane interface, which may properly anchor the Aer protein in the membrane. By incorporating these data into a homology model of Aer, we developed a model for the orientation of the Aer F1 and PAS regions in an Aer lattice that is compatible with the known dimensions of the chemoreceptor lattice. We propose that the F1 region facilitates the orientation of PAS and HAMP domains during folding and thereby promotes the stability of the PAS and HAMP domains in Aer. 相似文献
11.
Asharie J. Campbell Kylie J. Watts Mark S. Johnson Barry L. Taylor 《Molecular microbiology》2010,77(3):575-586
The Aer receptor monitors internal energy (redox) levels in Escherichia coli with an FAD‐containing PAS domain. Here, we randomly mutagenized the region encoding residues 14–119 of the PAS domain and found 72 aerotaxis‐defective mutants, 24 of which were gain‐of‐function, signal‐on mutants. The mutations were mapped onto an Aer homology model based on the structure of the PAS–FAD domain in NifL from Azotobacter vinlandii. Signal‐on lesions clustered in the FAD binding pocket, the β‐scaffolding and in the N‐cap loop. We suggest that the signal‐on lesions mimic the ‘signal‐on’ state of the PAS domain, and therefore may be markers for the signal‐in and signal‐out regions of this domain. We propose that the reduction of FAD rearranges the FAD binding pocket in a way that repositions the β‐scaffolding and the N‐cap loop. The resulting conformational changes are likely to be conveyed directly to the HAMP domain, and on to the kinase control module. In support of this hypothesis, we demonstrated disulphide band formation between cysteines substituted at residues N98C or I114C in the PAS β‐scaffold and residue Q248C in the HAMP AS‐2 helix. 相似文献
12.
The energy taxis receptor Aer, in Escherichia coli , senses changes in the redox state of the electron transport system via an flavin adenine dinucleotide cofactor bound to a PAS domain. The PAS domain (a sensory domain named after three proteins P er, A RNT and S im, where it was first identified) is thought to interact directly with the Aer HAMP domain to transmit this signal to the highly conserved domain (HCD) found in chemotaxis receptors. An apparent energy taxis system in Campylobacter jejuni is composed of two proteins, CetA and CetB, that have the domains of Aer divided between them. CetB has a PAS domain, while CetA has a predicted transmembrane region, HAMP domain and the HCD. In this study, we examined the expression of cetA and cetB and the biochemical properties of the proteins they encode. cetA and cetB are co-transcribed independently of the flagellar regulon. CetA has two transmembrane helices in a helical hairpin while CetB is a peripheral membrane protein tightly associated with the membrane. CetB levels are CetA dependent. Additionally, we demonstrated that both CetA and CetB participate in complexes, including a likely CetB dimer and a complex that may include both CetA and CetB. This study provides a foundation for further characterization of signal transduction mechanisms within CetA/CetB. 相似文献
13.
Hong CS Shitashiro M Kuroda A Ikeda T Takiguchi N Ohtake H Kato J 《FEMS microbiology letters》2004,231(2):247-252
It was previously shown that the chemotaxis gene cluster 1 (cheYZABW) was required for chemotaxis. In this study, the involvement of the same cluster in aerotaxis is described and two transducer genes for aerotaxis are identified. Aerotaxis assays of a number of deletion-insertion mutants of Pseudomonas aeruginosa PAO1 revealed that the chemotaxis gene cluster 1 and cheR are required for aerotaxis. Mutant strains which contained deletions in the methyl-accepting chemotaxis protein-like genes tlpC and tlpG showed decreased aerotaxis. A double mutant deficient in tlpC and tlpG was negative for aerotaxis. TlpC has 45% amino acid identity with the Escherichia coli aerotactic transducer Aer. The TlpG protein has a predicted C-terminal segment with 89% identity to the highly conserved domain of the E. coli serine chemoreceptor Tsr. A hydropathy plot of TlpG indicated that hydrophobic membrane-spanning regions are missing in TlpG. A PAS motif was found in the N-terminal domains of TlpC and TlpG. On this basis, the tlpC and tlpG genes were renamed aer and aer-2, respectively. No significant homology other than the PAS motif was detected in the N-terminal domains between Aer and Aer-2. 相似文献
14.
Zachary A. Maschmann Teck Khiang Chua Siddarth Chandrasekaran Hctor Ibez Brian R. Crane 《The Journal of biological chemistry》2022,298(12)
The Per–Arnt–Sim (PAS; named for the representative proteins: Period, Aryl hydrocarbon receptor nuclear translocator protein and Single-minded) domain of the dimeric Escherichia coli aerotaxis receptor Aer monitors cellular respiration through a redox-sensitive flavin adenine dinucleotide (FAD) cofactor. Conformational shifts in the PAS domain instigated by the oxidized FAD (FADOX)/FAD anionic semiquinone (FADASQ) redox couple traverse the HAMP (histidine kinases, adenylate cyclases, methyl-accepting chemotaxis proteins, and phosphatases) and kinase control domains of the Aer dimer to regulate CheA kinase activity. The PAS domain of Aer is unstable and has not been previously purified. Here, residue substitutions that rescue FAD binding in an FAD binding–deficient full-length Aer variant were used in combination to stabilize the Aer PAS domain. We solved the 2.4 Å resolution crystal structure of this variant, Aer-PAS-GVV, and revealed a PAS fold that contains distinct features associated with FAD-based redox sensing, such as a close contact between the Arg115 side chain and N5 of the isoalloxazine ring and interactions of the flavin with the side chains of His53 and Asn85 that are poised to convey conformational signals from the cofactor to the protein surface. In addition, we determined the FADox/FADASQ formal potentials of Aer-PAS-GVV and full-length Aer reconstituted into nanodiscs. The Aer redox couple is remarkably low at –289.6 ± 0.4 mV. In conclusion, we propose a model for Aer energy sensing based on the low potential of Aer-PAS–FADox/FADASQ couple and the inability of Aer-PAS to bind to the fully reduced FAD hydroquinone. 相似文献
15.
Escherichia coli chemoreceptors are type I membrane receptors that have a periplasmic sensing domain, a cytosolic signaling domain, and two transmembrane segments. The aerotaxis receptor, Aer, is different in that both its sensing and signaling regions are proposed to be cytosolic. This receptor has a 38-residue hydrophobic segment that is thought to form a membrane anchor. Most transmembrane prediction programs predict a single transmembrane-spanning segment, but such a topology is inconsistent with recent studies indicating that there is direct communication between the membrane flanking PAS and HAMP domains. We studied the overall topology and membrane boundaries of the Aer membrane anchor by a cysteine-scanning approach. The proximity of 48 cognate cysteine replacements in Aer dimers was determined in vivo by measuring the rate and extent of disulfide cross-linking after adding the oxidant copper phenanthroline, both at room temperature and to decrease lateral diffusion in the membrane, at 4 degrees C. Membrane boundaries were identified in membrane vesicles using 5-iodoacetamidofluorescein and methoxy polyethylene glycol 5000 (mPEG). To map periplasmic residues, accessible cysteines were blocked in whole cells by pretreatment with 4-acetamido-4'-maleimidylstilbene-2, 2' disulfonic acid before the cells were lysed in the presence of mPEG. The data were consistent with two membrane-spanning segments, separated by a short periplasmic loop. Although the membrane anchor contains a central proline residue that reaches the periplasm, its position was permissive to several amino acid and peptide replacements. 相似文献
16.
Loss- and gain-of-function mutations in the F1-HAMP region of the Escherichia coli aerotaxis transducer Aer
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The Escherichia coli Aer protein contains an N-terminal PAS domain that binds flavin adenine dinucleotide (FAD), senses aerotactic stimuli, and communicates with the output signaling domain. To explore the roles of the intervening F1 and HAMP segments in Aer signaling, we isolated plasmid-borne aerotaxis-defective mutations in a host strain lacking all chemoreceptors of the methyl-accepting chemotaxis protein (MCP) family. Under these conditions, Aer alone established the cell's run/tumble swimming pattern and modulated that behavior in response to oxygen gradients. We found two classes of Aer mutants: null and clockwise (CW) biased. Most mutant proteins exhibited the null phenotype: failure to elicit CW flagellar rotation, no aerosensing behavior in MCP-containing hosts, and no apparent FAD-binding ability. However, null mutants had low Aer expression levels caused by rapid degradation of apparently nonnative subunits. Their functional defects probably reflect the absence of a protein product. In contrast, CW-biased mutant proteins exhibited normal expression levels, wild-type FAD binding, and robust aerosensing behavior in MCP-containing hosts. The CW lesions evidently shift unstimulated Aer output to the CW signaling state but do not block the Aer input-output pathway. The distribution and properties of null and CW-biased mutations suggest that the Aer PAS domain may engage in two different interactions with HAMP and the HAMP-proximal signaling domain: one needed for Aer maturation and another for promoting CW output from the Aer signaling domain. Most aerotaxis-defective null mutations in these regions seemed to affect maturation only, indicating that these two interactions involve structurally distinct determinants. 相似文献
17.
Poly-HAMP domains are widespread in bacterial chemoreceptors, but previous studies have focused on receptors with single HAMP domains. The Pseudomonas aeruginosa chemoreceptor, Aer-2, has an unusual domain architecture consisting of a PAS-sensing domain sandwiched between three N-terminal and two C-terminal HAMP domains, followed by a conserved kinase control module. The structure of the N-terminal HAMP domains was recently solved, making Aer-2 the first protein with resolved poly-HAMP structure. The role of Aer-2 in P. aeruginosa is unclear, but here we show that Aer-2 can interact with the chemotaxis system of Escherichia coli to mediate repellent responses to oxygen, carbon monoxide and nitric oxide. Using this model system to investigate signalling and poly-HAMP function, we determined that the Aer-2 PAS domain binds penta-co-ordinated b-type haem and that reversible signalling requires four of the five HAMP domains. Deleting HAMP 2 and/or 3 resulted in a kinase-off phenotype, whereas deleting HAMP 4 and/or 5 resulted in a kinase-on phenotype. Overall, these data support a model in which ligand-bound Aer-2 PAS and HAMP 2 and 3 act together to relieve inhibition of the kinase control module by HAMP 4 and 5, resulting in the kinase-on state of the Aer-2 receptor. 相似文献
18.
Function of the N-terminal cap of the PAS domain in signaling by the aerotaxis receptor Aer
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Aer, the Escherichia coli receptor for behavioral responses to oxygen (aerotaxis), energy, and redox potential, contains a PAS sensory-input domain. Within the PAS superfamily, the N-terminal segment (N-cap) is poorly conserved and its role is not well understood. We investigated the role of the N-cap (residues 1 to 19) in the Aer PAS domain by missense and truncation mutagenesis. Aer-PAS N-cap truncations and an Aer-M21P substitution resulted in low cellular levels of the mutant proteins, suggesting that the N-terminal region was important for stabilizing the structure of the PAS domain. The junction of the N-cap and PAS core was critical for signaling in Aer. Mutations and truncations in the sequence encoding residues 15 to 21 introduced a range of phenotypes, including defects in FAD binding, constant tumbling motility, and an inverse response in which E. coli cells migrated away from oxygen concentrations to which they are normally attracted. The proximity of two N-cap regions in an Aer dimer was assessed in vivo by oxidatively cross-linking serial cysteine substitutions. Cross-linking of several cysteine replacements at 23 degrees C was attenuated at 10 degrees C, indicating contact was not at a stable dimer interface but required lateral mobility. We observed large multimers of Aer when we combined cross-linking of N-cap residues with a cysteine replacement that cross-links exclusively at the Aer dimer interface. This suggests that the PAS N-cap faces outwards in a dimer and that PAS-PAS contacts can occur between adjacent dimers. 相似文献
19.
Biofilm dispersion by Pseudomonas aeruginosa in response to environmental cues is dependent on the cytoplasmic BdlA protein harboring two sensory PAS domains and a chemoreceptor domain, TarH. The closest known and previously characterized BdlA homolog is the flavin adenine dinucleotide (FAD)-binding Aer, the redox potential sensor and aerotaxis transducer in Escherichia coli. Here, we made use of alanine replacement mutagenesis of the BdlA PAS domain residues previously demonstrated to be essential for aerotaxis in Aer to determine whether BdlA is a potential sensory protein. Five substitutions (D14A, N23A, W60A, I109A, and W182A) resulted in a null phenotype for dispersion. One protein, the BdlA protein with the G31A mutation (BdlA-G31A), transmitted a constant signal-on bias as it rendered P. aeruginosa biofilms hyperdispersive. The hyperdispersive phenotype correlated with increased interaction of BdlA-G31A with the phosphodiesterase DipA under biofilm growth conditions, resulting in increased phosphodiesterase activity and reduced biofilm biomass accumulation. We furthermore demonstrate that BdlA is a heme-binding protein. None of the BdlA protein variants analyzed led to a loss of the heme prosthetic group. The N-terminal PASa domain was identified as the heme-binding domain of BdlA, with BdlA-dependent nutrient-induced dispersion requiring the PASa domain. The findings suggest that BdlA plays a role in intracellular sensing of dispersion-inducing conditions and together with DipA forms a regulatory network that modulates an intracellular cyclic d-GMP (c-di-GMP) pool to enable dispersion. 相似文献
20.
HAMP domains, ~55 amino acid motifs first identified in histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases, operate as signal mediators in two-component signal transduction proteins. A bioinformatics study identified a coevolving signal-accepting network of 10 amino acids in membrane-delimited HAMP proteins. To probe the functionality of this network we used a HAMP containing mycobacterial adenylyl cyclase, Rv3645, as a reporter enzyme in which the membrane anchor was substituted by the Escherichia coli chemotaxis receptor for serine (Tsr receptor) and the HAMP domain alternately with that from the protein Af1503 of the archaeon Archaeoglobus fulgidus or the Tsr receptor. In a construct with the Tsr-HAMP, cyclase activity was inhibited by serine, whereas in a construct with the HAMP domain from A. fulgidus, enzyme activity was not responsive to serine. Amino acids of the signal-accepting network were mutually swapped between both HAMP domains, and serine signaling was examined. The data biochemically tentatively established the functionality of the signal-accepting network. Based on a two-state gearbox model of rotation in HAMP domain-mediated signal propagation, we characterized the interaction between permanent and transient core residues in a coiled coil HAMP structure. The data are compatible with HAMP rotation in signal propagation but do not exclude alternative models for HAMP signaling. Finally, we present data indicating that the connector, which links the α-helices of HAMP domains, plays an important structural role in HAMP function. 相似文献