The Aer2 chemoreceptor from Vibrio vulnificus is a tri-PAS-heme oxygen sensor.

The marine pathogen Vibrio vulnificus senses and responds to environmental stimuli via two chemosensory systems and 42-53 chemoreceptors. Here, we present an analysis of the V. vulnificus Aer2 chemoreceptor, VvAer2, which is the first V. vulnificus chemoreceptor to be characterized. VvAer2 is related to the Aer2 receptors of other gammaproteobacteria, but uncharacteristically contains three PAS domains (PAS1-3), rather than one or two. Using an E. coli chemotaxis hijack assay, we determined that VvAer2, like other Aer2 receptors, senses and responds to O2 . All three VvAer2 PAS domains bound pentacoordinate b-type heme and exhibited similar O2 affinities. PAS2 and PAS3 both stabilized O2 via conserved Iß-Trp residues, but PAS1, which was easily oxidized in vitro, was unaffected by Iß-Trp replacement. Our results support a model in which PAS1 is largely dispensable for O2 -mediated signaling, whereas PAS2 modulates PAS3 signaling, and PAS3 signals to the downstream domains. Each PAS domain appeared to be positionally optimized, because PAS swapping caused altered signaling properties, and neither PAS1 nor PAS2 could replace PAS3. Our findings strengthen previous conclusions that Aer2 receptors are O2 sensors, but with distinct N-terminal domain arrangements that facilitate, modulate and tune responses based on environmental signals.

New Roles for HAMP Domains: the Tri-HAMP Region of Pseudomonas aeruginosa Aer2 Controls Receptor Signaling and Cellular Localization.

The Aer2 chemoreceptor from Pseudomonas aeruginosa is an O2 sensor involved in stress responses, virulence, and tuning the behavior of the chemotaxis (Che) system. Aer2 is the sole receptor of the Che2 system. It is soluble, but membrane associated, and forms complexes at the cell pole during stationary phase. The domain arrangement of Aer2 is unusual, with a PAS sensing domain sandwiched between five HAMP domains, followed by a C-terminal kinase-control output domain. The first three HAMP domains form a poly-HAMP chain N-terminal to the PAS sensing domain. HAMP domains are often located between signal input and output domains, where they transduce signals. Given that HAMP1 to 3 reside N-terminal to the input-output pathway, we undertook a systematic examination of their function in Aer2. We found that HAMP1 to 3 influence PAS signaling over a considerable distance, as the majority of HAMP1, 2 and 3 mutations, and deletions of helical phase stutters, led to nonresponsive signal-off or off-biased receptors. PAS signal-on lesions that mimic activated Aer2 also failed to override N-terminal HAMP signal-off replacements. This indicates that HAMP1 to 3 are critical coupling partners for PAS signaling and likely function as a cohesive unit and moveable scaffold to correctly orient and poise PAS dimers for O2-mediated signaling in Aer2. HAMP1 additionally controlled the clustering and polar localization of Aer2 in P. aeruginosa. Localization was not driven by HAMP1 charge, and HAMP1 signal-off mutants still localized. Employing HAMP as a clustering and localization determinant, as well as a facilitator of PAS signaling, are newly recognized roles for HAMP domains. IMPORTANCE P. aeruginosa is an opportunistic pathogen that interprets environmental stimuli via 26 chemoreceptors that signal through 4 distinct chemosensory systems. The second chemosensory system, Che2, contains a receptor named Aer2 that senses O2 and mediates stress responses and virulence and tunes chemotactic behavior. Aer2 is membrane associated, but soluble, and has three N-terminal HAMP domains (HAMP1 to 3) that reside outside the signal input-output pathway of Aer2. In this study, we determined that HAMP1 to 3 facilitate O2-dependent signaling from the PAS sensing domain and that HAMP1 controls the formation of Aer2-containing polar foci in P. aeruginosa. Both of these are newly recognized roles for HAMP domains that may be applicable to other non-signal-transducing HAMP domains and poly-HAMP chains.

Leptospira interrogans Aer2: an Unusual Membrane-Bound PAS-Heme Oxygen Sensor.

In this study, we provide the first characterization of a chemoreceptor from Leptospira interrogans, the cause of leptospirosis. This receptor is related to the Aer2 receptors that have been studied in other bacteria. In those organisms, Aer2 is a soluble receptor with one or two PAS-heme domains and signals in response to O2 binding. In contrast, L. interrogans Aer2 (LiAer2) is an unusual membrane-bound Aer2 with a periplasmic domain and three cytoplasmic PAS-heme domains. Each of the three PAS domains bound b-type heme via conserved Eη-His residues. They also bound O2 and CO with similar affinities to each other and other PAS-heme domains. However, all three PAS domains were uniquely hexacoordinate in the deoxy-heme state, whereas other Aer2-PAS domains are pentacoordinate. Similar to other Aer2 receptors, LiAer2 could hijack the E. coli chemotaxis pathway but only when it was expressed with an E. coli high-abundance chemoreceptor. Unexpectedly, the response was inverted relative to classic Aer2 receptors. That is, LiAer2 caused E. coli to tumble (it was signal-on) in the absence of O2 and to stop tumbling in its presence. Thus, an endogenous ligand in the deoxy-heme state was correlated with signal-on LiAer2, and its displacement for gas-binding turned signaling off. This response also occurred in a soluble version of LiAer2 lacking the periplasmic domain, transmembrane (TM) region, and first two PAS domains, meaning that PAS3 alone was sufficient for O2-mediated control. Future studies are needed to understand the unique signaling mechanisms of this unusual Aer2 receptor. IMPORTANCE Leptospira interrogans, the cause of the zoonotic infection leptospirosis, is found in soil and water contaminated with animal urine. L. interrogans survives in complex environments with the aid of 12 chemoreceptors, none of which has been explicitly studied. In this study, we characterized the first L. interrogans chemoreceptor, LiAer2, and reported its unique characteristics. LiAer2 is membrane-bound, has three cytoplasmic PAS-heme domains that each bound hexacoordinate b-type heme and O2 turned LiAer2 signaling off. An endogenous ligand in the deoxy-heme state was correlated with signal-on LiAer2 and its displacement for O2-binding turned signaling off. Our study corroborated previous findings that Aer2 receptors are O2 sensors, but also demonstrated that they do not all function the same way.

Oxygen-Induced Conformational Changes in the PAS-Heme Domain of the Pseudomonas aeruginosa Aer2 Receptor.

The Aer2 receptor from Pseudomonas aeruginosa has an O2-binding PAS-heme domain that stabilizes O2 via a Trp residue in the distal heme pocket. Trp rotates ∼90° to bond with the ligand and initiate signaling. Although the isolated PAS domain is monomeric, both in solution and in a cyanide-bound crystal structure, an unliganded structure forms a dimer. An overlay of the two structures suggests possible signaling motions but also predicts implausible clashes at the dimer interface when the ligand is bound. Moreover, in a full-length Aer2 dimer, PAS is sandwiched between multiple N- and C-terminal HAMP domains, which would feasibly restrict PAS motions. To explore the PAS dimer interface and signal-induced motions in full-length Aer2, we introduced Cys substitutions and used thiol-reactive probes to examine in vivo accessibility and residue proximities under both aerobic and anaerobic conditions. In vivo, PAS dimers were retained in full-length Aer2 in the presence and absence of O2, and the dimer interface was consistent with the isolated PAS dimer structure. O2-mediated changes were also consistent with structural predictions in which the PAS N-terminal caps move apart and the C-terminal DxT region moves closer together. The DxT motif links PAS to the C-terminal HAMP domains and was critical for PAS-HAMP signaling. Removing the N-terminal HAMP domains altered the distal PAS dimer interface and prevented signaling, even after signal-on lesions were introduced into PAS. The N-terminal HAMP domains thus facilitate the O2-dependent shift of PAS to the signal-on conformation, clarifying their role upstream of the PAS-sensing domain.

Deciphering the Che2 chemosensory pathway and the roles of individual Che2 proteins from Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that senses and responds to its environment via four chemosensory systems. Oxygen activates the Che2 chemosensory system by binding to the PAS-heme domain of the Aer2 receptor. Ostensibly, the output of Che2 occurs via its response regulator CheY2, but controversy persists over CheY2's exact role. In this study, we show that CheY2 does not interact with the flagellar motor and that the Che2 system does not transfer phosphoryl groups to the chemotaxis (Che) system. We show that CheY2 instead provides feedback control of Aer2 adaptation. In the presence of O2 , Aer2 signaling increases the autophosphorylation of the histidine kinase CheA2, followed by CheY2-mediated dephosphorylation. CheY2 does not stably retain phosphate and may not signal the output of the Che2 system. Rather, CheY2 activity enhances the direct interaction of CheY2 with the adaptation protein CheD (a role often facilitated by CheC, which P. aeruginosa lacks). In the absence of O2 , Aer2 does not signal, and CheY2/CheD interactions attenuate. This frees CheD to augment CheR2-mediated methylation of Aer2, which enhances Aer2 signaling. CheD does not interact with CheR2, but most likely interacts with Aer2 via conserved CheD-binding motifs to make Aer2 a better methylation substrate.

Gas Sensing and Signaling in the PAS-Heme Domain of the Pseudomonas aeruginosa Aer2 Receptor.

The Aer2 chemoreceptor from Pseudomonas aeruginosa contains a PAS sensing domain that coordinates b-type heme and signals in response to the binding of O2, CO, or NO. PAS-heme structures suggest that Aer2 uniquely coordinates heme via a His residue on a 310 helix (H234 on Eη), stabilizes O2 binding via a Trp residue (W283), and signals via both W283 and an adjacent Leu residue (L264). Ligand binding may displace L264 and reorient W283 for hydrogen bonding to the ligand. Here, we clarified the mechanisms by which Aer2-PAS binds heme, regulates ligand binding, and initiates conformational signaling. H234 coordinated heme, but additional hydrophobic residues in the heme cleft were also critical for stable heme binding. O2 appeared to be the native Aer2 ligand (dissociation constant [Kd ] of 16 µM). With one exception, mutants that bound O2 could signal, whereas many mutants that bound CO could not. W283 stabilized O2 binding but not CO binding, and it was required for signal initiation; W283 mutants that could not stabilize O2 were rapidly oxidized to Fe(III). W283F was the only Trp mutant that bound O2 with wild-type affinity. The size and nature of residue 264 was important for gas binding and signaling: L264W blocked O2 binding, L264A and L264G caused O2-mediated oxidation, and L264K formed a hexacoordinate heme. Our data suggest that when O2 binds to Aer2, L264 moves concomitantly with W283 to initiate the conformational signal. The signal then propagates from the PAS domain to regulate the C-terminal HAMP and kinase control domains, ultimately modulating a cellular response.IMPORTANCEPseudomonas aeruginosa is a ubiquitous environmental bacterium and opportunistic pathogen that infects multiple body sites, including the lungs of cystic fibrosis patients. P. aeruginosa senses and responds to its environment via four chemosensory systems. Three of these systems regulate biofilm formation, twitching motility, and chemotaxis. The role of the fourth system, Che2, is unclear but has been implicated in virulence. The Che2 system contains a chemoreceptor called Aer2, which contains a PAS sensing domain that binds heme and senses oxygen. Here, we show that Aer2 uses unprecedented mechanisms to bind O2 and initiate signaling. These studies provide both the first functional corroboration of the Aer2-PAS signaling mechanism previously proposed from structure as well as a signaling model for Aer2-PAS receptors.

Functional analysis of three topoisomerases that regulate DNA supercoiling levels in Chlamydia.

Chlamydia is a medically important bacterium that infects eukaryotic cells. Temporal expression of chlamydial genes during the intracellular infection is proposed to be regulated by changes in DNA supercoiling levels. To understand how chlamydial supercoiling levels are regulated, we purified and analyzed three putative Chlamydia trachomatis topoisomerases. As predicted by sequence homology, CT189/190 are the two subunits of DNA gyrase, whereas CT643 is a topoisomerase I. CT660/661 have been predicted to form a second DNA gyrase, but the reconstitute holoenzyme decatenated and relaxed DNA, indicating that the proteins are subunits of topoisomerase IV. Promoter analysis showed that each topoisomerase is transcribed from its own operon by the major chlamydial RNA polymerase. Surprisingly, all three topoisomerase promoters had higher activity from a more supercoiled DNA template. This supercoiling-responsivesness is consistent with negative feedback control of topoisomerase I and topoisomerase IV expression, which is typical of other bacteria. However, activation of the chlamydial gyrase promoter by increased supercoiling is unorthodox compared with the relaxation-induced transcription of gyrase in other bacteria. We present a model in which supercoiling levels during the intracellular chlamydial developmental cycle are regulated by unusual positive feedback control of the gyrase promoter and the temporal expression of three topoisomerases.

Biochemical and cellular characterization of Helicobacter pylori RecA, a protein with high-level constitutive expression.

Helicobacter pylori is a bacterial pathogen colonizing half of the world's human population. It has been implicated in a number of gastric diseases, from asymptomatic gastritis to cancer. It is characterized by an amazing genetic variability that results from high mutation rates and efficient DNA homologous recombination and transformation systems. Here, we report the characterization of H. pylori RecA (HpRecA), a protein shown to be involved in DNA repair, transformation, and mouse colonization. The biochemical characterization of the purified recombinase reveals activities similar to those of Escherichia coli RecA (EcRecA). We show that in H. pylori, HpRecA is present in about 80,000 copies per cell during exponential growth and decreases to about 50,000 copies in stationary phase. The amount of HpRecA remains unchanged after induction of DNA lesions, suggesting that HpRecA is always expressed at a high level in order to repair DNA damage or facilitate recombination. We performed HpRecA localization analysis by adding a Flag tag to the protein, revealing two different patterns of localization. During exponential growth, RecA-Flag presents a diffuse pattern, overlapping with the DAPI (4',6-diamidino-2-phenylindole) staining of DNA, whereas during stationary phase, the protein is present in more defined areas devoid of DAPI staining. These localizations are not affected by inactivation of competence or DNA recombination genes. Neither UV irradiation nor gamma irradiation modified HpRecA localization, suggesting the existence of a constitutive DNA damage adaptation system.

Genetic dissection of Helicobacter pylori AddAB role in homologous recombination.

Helicobacter pylori infects the stomach of about half of the world's human population, frequently causing chronic inflammation at the origin of several gastric pathologies. One of the most remarkable characteristics of the species is its remarkable genomic plasticity in which homologous recombination (HR) plays a critical role. Here, we analyzed the role of the H. pylori homologue of the AddAB recombination protein. Bioinformatics analysis of the proteins unveils the similarities and differences of the H. pylori AddAB complex with respect to the RecBCD and AddAB complexes from Escherichia coli and Bacillus subtilis, respectively. Helicobacter pylori mutants lacking functional addB or/and addA show the same level of sensitivity to DNA-damaging agents such as UV or irradiation and of deficiency in intrachromosomal RecA-dependent HR. Epistasis analyses of both DNA repair and HR phenotypes, using double and triple recombination mutants, demonstrate that, in H. pylori, AddAB and RecOR complexes define two separate presynaptic pathways with little functional overlap. However, neither of these complexes participates in the RecA-dependent process of transformation of these naturally competent bacteria.