The Aer2 chemoreceptor from Pseudomonas aeruginosa contains a PAS sensing domain that coordinates b-type heme and signals in response to the binding of O 2 , CO, or NO. PAS-heme structures suggest that Aer2 uniquely coordinates heme via a His residue on a 3 10 helix (H234 on E), stabilizes O 2 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. O 2 appeared to be the native Aer2 ligand (dissociation constant [K d ] of 16 M). With one exception, mutants that bound O 2 could signal, whereas many mutants that bound CO could not. W283 stabilized O 2 binding but not CO binding, and it was required for signal initiation; W283 mutants that could not stabilize O 2 were rapidly oxidized to Fe(III). W283F was the only Trp mutant that bound O 2 with wildtype affinity. The size and nature of residue 264 was important for gas binding and signaling: L264W blocked O 2 binding, L264A and L264G caused O 2 -mediated oxidation, and L264K formed a hexacoordinate heme. Our data suggest that when O 2 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.IMPORTANCE Pseudomonas 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 O 2 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.KEYWORDS chemoreceptor, PAS domain, signal transduction, Pseudomonas aeruginosa, heme, oxygen P seudomonas aeruginosa is a common environmental bacterium and a significant cause of opportunistic human disease. It survives in complex environments with the aid of 26 chemoreceptors and four chemosensory systems that collectively sense environmental conditions and modify bacterial behavior. The roles of three of these chemosensory systems are known: one modulates type IV pili production and twitching motility (Pil-Chp system), another controls biofilm formation (Wsp system), and a third

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.

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.

Summary 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.

Bacterial chemosensory systems sense and respond to stimuli via chemoreceptors that activate phosphotransfer cascades and initiate cellular responses. Pseudomonas aeruginosa has four chemosensory systems, three of which regulate biofilm formation (the Wsp system), twitching motility (the Pil-Chp system), and flagellum-mediated chemotaxis (the Che system) (Kato et al., 2008; Sampedro et al., 2014). The role of P. aeruginosa's fourth chemosensory system, Che2, is not well understood, although it appears to be involved in stress responses and it impacts virulence (Garvis et al., 2009; Schuster et al., 2004). The P. aeruginosa chemotaxis system receives sensory input from 23 chemoreceptors, whereas the three additional chemosensory systems employ one receptor each (Ortega et al., 2017). The receptor for Che2 is Aer2 (McpB) and it is encoded within the che2 operon [(Hong et al., 2004), Figure 1a]. Che2 proteins (Y2, A2, W2, Aer2, R2, D, and B2) are expressed in stationary phase and form an Aer2-mediated complex near the cell pole that does not co-localize with chemotaxis proteins [(Guvener et al., 2006; Schuster et al., 2004; Yang and Briegel, 2020), Figure 1b]. The stimulus for Che2 is O 2 , which binds to the PAS-heme domain of Aer2 with the assistance of a Trp residue that rotates to bond with O 2 [(Airola et al., 2013;

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.

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, Li Aer2, and reported its unique characteristics. Li Aer2 is membrane-bound, has three cytoplasmic PAS-heme domains that each bound hexacoordinate b -type heme and O 2 turned Li Aer2 signaling off.

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 O 2 and mediates stress responses and virulence and tunes chemotactic behavior.