RESUMO
Chemotaxis in Bacteria and Archaea depends on the presence of hexagonal polar arrays composed of membrane-bound chemoreceptors that interact with rings of baseplate signaling proteins. In the alphaproteobacterium Azospirillum brasilense, chemotaxis is controlled by two chemotaxis signaling systems (Che1 and Che4) that mix at the baseplates of two spatially distinct membrane-bound chemoreceptor arrays. The subcellular localization and organization of transmembrane chemoreceptors in chemotaxis signaling clusters have been well characterized but those of soluble chemoreceptors remain relatively underexplored. By combining mutagenesis, microscopy, and biochemical assays, we show that the cytoplasmic chemoreceptors AerC and Tlp4b function in chemotaxis and localize to and interact with membrane-bound chemoreceptors and chemotaxis signaling proteins from both polar arrays, indicating that soluble chemoreceptors are promiscuous. The interactions of AerC and Tlp4b with polar chemotaxis signaling clusters are not equivalent and suggest distinct functions. Tlp4b, but not AerC, modulates the abundance of chemoreceptors within the signaling clusters through an unknown mechanism. The AerC chemoreceptor, but not Tlp4b, is able to traffic in and out of chemotaxis signaling clusters depending on its level of expression. We also identify a role of the chemoreceptor composition of chemotaxis signaling clusters in regulating their polar subcellular organization. The organization of chemotaxis signaling proteins as large membrane-bound arrays underlies chemotaxis sensitivity. Our findings suggest that the composition of chemoreceptors may fine-tune chemotaxis signaling not only through their chemosensory specificity but also through their role in the organization of polar chemotaxis signaling clusters. IMPORTANCE Cytoplasmic chemoreceptors represent about 14% of all chemoreceptors encoded in bacterial and archaeal genomes, but little is known about how they interact with and function in large polar assemblies of membrane-bound chemotaxis signaling clusters. Here, we show that two soluble chemoreceptors with a role in chemotaxis are promiscuous and interact with two distinct membrane-bound chemotaxis signaling clusters that control all chemotaxis responses in Azospirillum brasilense. We also found that any change in the chemoreceptor composition of chemotaxis signaling clusters alters their polar organization, suggesting a dynamic interplay between the sensory specificity of chemotaxis signaling clusters and their polar membrane organization.
Assuntos
Azospirillum brasilense , Quimiotaxia , Quimiotaxia/fisiologia , Azospirillum brasilense/genética , Azospirillum brasilense/metabolismo , Proteínas de Bactérias/metabolismo , Células Quimiorreceptoras , Citoplasma/metabolismo , Proteínas Quimiotáticas Aceptoras de Metil/genéticaRESUMO
Bacterial chemoreceptors control the activity of the associated CheA kinase in response to chemical gradients and, consequently, regulate the swimming behavior of the cell. However, such control is not direct but requires the participation of the essential coupling protein CheW, which is structurally homologous to the carboxy-terminal domain of the kinase. The actual role of this small coupling protein is somehow intriguing. It has been demonstrated that it is absolutely essential for chemoreceptor control of the kinase, in spite of the occurrence of direct contacts between chemoreceptors and CheA. In addition, CheW plays an essential role in the assembly of the large macromolecular arrays that combine chemoreceptors of different specificities, and it is therefore responsible for molecular interactions that provide such arrays with remarkable signaling properties. In this work, we analyze truncated CheW derivatives that are still able to control the kinase but have lost the ability to connect signaling units. We demonstrate that these two activities can work separately and speculate about the significance of the roles of these two different activities in the context of the chemoreceptor cluster.
Assuntos
Proteínas de Bactérias/ultraestrutura , Quimiotaxia , Proteínas de Escherichia coli/ultraestrutura , Escherichia coli/ultraestrutura , Histidina Quinase/ultraestrutura , Proteínas Quimiotáticas Aceptoras de Metil/ultraestrutura , Proteínas de Bactérias/genética , Sítios de Ligação , Quimiotaxia/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Histidina Quinase/genética , Proteínas Quimiotáticas Aceptoras de Metil/genética , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Transdução de Sinais/genética , Homologia Estrutural de ProteínaRESUMO
Chemoreceptors are dimeric proteins that contain a periplasmic or extracellular domain for ligand binding and an extremely well-conserved cytoplasmic domain for output response control. This latter domain consists in a long α-helical hairpin that forms a four-helix coiled-coil bundle in the dimer. Dimers associate into trimers of dimers in the crystal structure obtained for the cytoplasmic domain of the Escherichia coli serine chemoreceptor, Tsr. Further studies confirmed that this crystal structure reflects the basic unit within the in vivo organization of chemoreceptors. The trimers of dimers form large and stable chemoreceptor clusters in all the prokaryotes that have been studied. Here, we describe the use of TMEA, a trifunctional cross-linker that reacts with sulfhydryl groups, as a tool to study the geometry and dynamics of the interaction between receptors of the same or different types in living cells.
Assuntos
Escherichia coli/metabolismo , Maleimidas/metabolismo , Proteínas Quimiotáticas Aceptoras de Metil/química , Proteínas Quimiotáticas Aceptoras de Metil/metabolismo , Membrana Celular/metabolismo , Reagentes de Ligações Cruzadas , Cristalografia por Raios X , Escherichia coli/química , Modelos Moleculares , Domínios Proteicos , Multimerização Proteica , Transdução de SinaisRESUMO
Bacterial chemoreceptors are dimeric membrane proteins that transmit signals from a periplasmic ligand-binding domain to the interior of the cells. The highly conserved cytoplasmic domain consists of a long hairpin that in the dimer forms a four-helix coiled-coil bundle. The central region of the bundle couples changes in helix packing that occur in the membrane proximal region to the signaling tip, controlling the activity of an associated histidine kinase. This subdomain contains certain glycine residues that are postulated to form a hinge in chemoreceptors from enteric bacteria and have been largely postulated to play a role in the coupling mechanism, and/or in the formation of higher-order chemoreceptor assemblies. In this work, we directly assessed the importance of the "glycine hinge" by obtaining nonfunctional replacements at each of its positions in the Escherichia coli serine receptor Tsr and characterizing them. Our results indicate that, rather than being essential for proper receptor-receptor interaction, the "glycine hinge" residues are involved in the ability of the receptor to switch between different signaling states. Mainly, the C-helix residue G439 has a key role in shifting the equilibrium toward a kinase-activating conformation. However, we found second-site mutations that restore the chemotactic proficiency of some of the "glycine hinge" mutants, suggesting that a complete hinge is not strictly essential. Rather, glycine residues seem to favor the coupling activity that relies on some other structural features of the central subdomain.
Assuntos
Escherichia coli K12/química , Proteínas Quimiotáticas Aceptoras de Metil/química , Transdução de Sinais , Substituição de Aminoácidos , Escherichia coli K12/genética , Proteínas Quimiotáticas Aceptoras de Metil/genética , Mutação de Sentido Incorreto , Estrutura Secundária de ProteínaRESUMO
Piscirickettsia salmonis is the etiological agent of piscirickettsiosis, which, as the main systemic disease in the Chilean salmon industry, causes significant economic losses. This bacterium can produce biofilm as a persistence and survival strategy in adverse conditions. In other bacteria, cheA is a key gene for modulating the onset of bacterial chemotaxis, as well as having a secondary role in biofilm production. Notwithstanding this association, the potential relationships between biofilm formation and genes involved in P. salmonis chemotaxis are poorly understood. This study aimed to determine P. salmonis cheA gene expression when grown in different culture media known to induce biofilm production. Piscirickettsia salmonis AUSTRAL-005 produced moderate/high biofilm levels after 144 h of incubation in the AUSTRAL-SRS and marine broths. In contrast, LF-89 biofilm production was weak/nonexistent in the aforementioned broths. Both assessed P. salmonis strains contained the cheYZA operon. Additionally, AUSTRAL-005 cheA transcripts increased in both culture media. In conclusion, these results suggest potential relationships between biofilm formation and genes related to chemotaxis in the fish pathogen P. salmonis.
Assuntos
Quimiotaxia/genética , Regulação Bacteriana da Expressão Gênica/genética , Óperon/genética , Piscirickettsia/genética , Animais , Biofilmes/crescimento & desenvolvimento , Linhagem Celular , Quimiotaxia/fisiologia , Meios de Cultura/química , Doenças dos Peixes/microbiologia , Peixes/microbiologia , Genes Bacterianos/genética , Proteínas Quimiotáticas Aceptoras de Metil/genética , Proteínas Quimiotáticas Aceptoras de Metil/fisiologia , Microscopia Eletrônica de Varredura , Piscirickettsia/crescimento & desenvolvimento , Piscirickettsia/patogenicidade , Infecções por Piscirickettsiaceae/microbiologia , Virulência/genética , Virulência/fisiologiaRESUMO
Bacterial chemoreceptors sense environmental stimuli and govern cell movement by transmitting the information to the flagellar motors. The highly conserved cytoplasmic domain of chemoreceptors consists in an alpha-helical hairpin that forms in the homodimer a coiled-coil four-helix bundle. Several classes of chemoreceptors that differ in the length of the coiled-coil structure were characterized. Many bacterial species code for chemoreceptors that belong to different classes, but how these receptors are organized and function in the same cell remains an open question. E. coli cells normally code for single class chemoreceptors that form extended arrays based on trimers of dimers interconnected by the coupling protein CheW and the kinase CheA. This structure promotes effective coupling between the different receptors in the modulation of the kinase activity. In this work, we engineered functional derivatives of the Tsr chemoreceptor of E. coli that mimic receptors whose cytoplasmic domain is longer by two heptads. We found that these long Tsr receptors did not efficiently mix with the native receptors and appeared to function independently. Our results suggest that the assembly of membrane-bound receptors of different specificities into mixed clusters is dictated by the length-class to which the receptors belong, ensuring cooperative function only between receptors of the same class.
Assuntos
Proteínas de Bactérias/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/fisiologia , Proteínas de Membrana/metabolismo , Transdução de Sinais , Estresse Fisiológico , Proteínas de Bactérias/genética , Escherichia coli/genética , Proteínas de Escherichia coli , Histidina Quinase , Proteínas de Membrana/genética , Proteínas Quimiotáticas Aceptoras de MetilRESUMO
Chemical signals sensed on the periplasmic side of bacterial cells by transmembrane chemoreceptors are transmitted to the flagellar motors via the histidine kinase CheA, which controls the phosphorylation level of the effector protein CheY. Chemoreceptor arrays comprise remarkably stable supramolecular structures in which thousands of chemoreceptors are networked through interactions between their cytoplasmic tips, CheA, and the small coupling protein CheW. To explore the conformational changes that occur within this protein assembly during signalling, we used in vivo cross-linking methods to detect close interactions between the coupling protein CheW and the serine receptor Tsr in intact Escherichia coli cells. We identified two signal-sensitive contacts between CheW and the cytoplasmic tip of Tsr. Our results suggest that ligand binding triggers changes in the receptor that alter its signalling contacts with CheW (and/or CheA).
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cisteína/metabolismo , Histidina Quinase , Proteínas de Membrana/metabolismo , Proteínas Quimiotáticas Aceptoras de Metil , Modelos Moleculares , Ligação Proteica , Estrutura Secundária de Proteína , Transdução de SinaisRESUMO
Chemoreceptors transmit signals from the environment to the flagellar motors via a histidine kinase that controls the phosphorylation level of the effector protein CheY. The cytoplasmic domain of chemoreceptors is strongly conserved and consists of a long alpha-helical hairpin that forms, in the dimer, a coiled-coil four-helix bundle. Changes in this domain during evolution are characterized by the presence of seven-residue insertions/deletions located symmetrically with respect to the hairpin turn, suggesting that specific interactions between the helices that form the hairpin are required for function. We assessed the impact of seven-residue deletions on the signalling ability and higher-order organization of the serine chemoreceptor from Escherichia coli. Our results indicate that symmetry alterations between the two branches of the cytoplasmic hairpin seriously compromise chemoreceptor function. Shorter functional versions of Tsr with symmetrical deletions form mixed trimers of dimers when coexpressed with Tar, the aspartate receptor of E. coli. However, Tar function in those cells is impaired, suggesting that the length difference between receptors introduces non-functional distortions into the chemoreceptor cluster. This observation is reinforced by the analysis of coexpression of Tar with chemoreceptors from Rhodobacter sphaeroides that naturally belong to a shorter-length class.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Deleção de Sequência , Proteínas de Bactérias/genética , Dimerização , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana/genética , Proteínas Quimiotáticas Aceptoras de Metil , Estrutura Terciária de Proteína , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismoRESUMO
Chemotactic behavior in bacteria relies on the sensing ability of large chemoreceptor clusters that are usually located at the cell pole. In Escherichia coli, chemoreceptors exhibit higher-order interactions within those clusters based on a trimer-of-dimers organization. This architecture is conserved in a variety of other bacteria and archaea, implying that receptors in many microorganisms form trimer-of-dimer signaling teams. To gain further insight into the assembly and dynamic behavior of receptor trimers of dimers, we used in vivo cross-linking targeted to cysteine residues at various positions that define six different levels along the cytoplasmic signaling domains of the aspartate and serine chemoreceptors, Tar and Tsr, respectively. We found that the cytoplasmic domains of these receptors are close to each other near the trimer contact region at the cytoplasmic tip and lie farther apart as the receptor dimers approach the cytoplasmic membrane. Tar and Tsr reporter sites within the same or closely adjacent levels readily formed mixed cross-links, whereas reporters located different distances from the tip did not. These findings indicate that there are no significant vertical displacements of one dimer with respect to the others within the trimer unit. Attractant stimuli had no discernible effect on the cross-linking efficiency of any of the reporters tested, but a strong osmotic stimulus reproducibly enhanced cross-linking at most of the reporter sites, indicating that individual dimers may move closer together under this condition.
Assuntos
Proteínas de Bactérias/química , Células Quimiorreceptoras/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Proteínas de Membrana/química , Multimerização Proteica , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Células Quimiorreceptoras/metabolismo , Reagentes de Ligações Cruzadas/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Quimiotáticas Aceptoras de Metil , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Receptores de Superfície CelularRESUMO
About half of the human population is infected with Helicobacter pylori, a bacterium causing gastritis, peptic ulcer and progression to gastric cancer. Chemotaxis and flagellar motility are required for colonization and persistence of H. pylori in the gastric mucus layer. It is not completely clear which chemical gradients are used by H. pylori to maintain its position. TlpA, a chemotaxis receptor for arginine/ bicarbonate, has been identified. This study aimed to find out whether tlpA gene expression is required for the chemotactic response to arginine/bicarbonate. Wild-type motile H. pylori ATCC 700392 and H. pylori ATCC 43504, a strain having an interrupted tlpA gene, were used. Also, a tlpA-knockout mutant of H. pylori 700392 (H. pylori 700-tlpA::cat) was produced by homologous recombination. Expression of tlpA was assessed by a Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) assay. Chemotaxis was measured as a Relative Chemotaxis Response (RCR) by a modified capillary assay. H. pylori 700392 presented chemotaxis to arginine and sodium bicarbonate. H. pylori 700-tlpA::cat showed neither tlpA gene expression nor chemotaxis towards arginine and bicarbonate. Besides confirming that TlpA is a chemotactic receptor for arginine/bicarbonate in H. pylori, this study showed that tlpA gene expression is required for arginine/bicarbonate chemotaxis.
Assuntos
Arginina/farmacologia , Proteínas de Bactérias/genética , Bicarbonatos/farmacologia , Quimiotaxia/genética , Helicobacter pylori/genética , Proteínas de Membrana/genética , Expressão Gênica , Helicobacter pylori/efeitos dos fármacos , Proteínas Quimiotáticas Aceptoras de Metil , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Ureia/metabolismoRESUMO
The six copies of the response regulator CheY from Rhodobacter sphaeroides bind to the switch protein FliM. Phosphorylation by acetyl phosphate (AcP) was detected by tryptophan fluorescence quenching in three of the four CheYs that contain this residue. Autophosphorylation with Ac(32)P was observed in five CheY proteins. We also show that all of the cheY genes are expressed simultaneously; therefore, in vivo all of the CheY proteins could bind to FliM to control the chemotactic response. Consequently, we hypothesize that in this complex chemotactic system, the binding of some CheY proteins to FliM, does not necessarily imply switching of the flagellar motor.
Assuntos
Proteínas de Bactérias/metabolismo , Quimiotaxia/fisiologia , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana/metabolismo , Rhodobacter sphaeroides/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Proteínas Quimiotáticas Aceptoras de Metil , Dados de Sequência Molecular , Fosforilação , Rhodobacter sphaeroides/crescimento & desenvolvimentoRESUMO
The availability of the complete genome of the Gram-negative beta-proteobacterium Chromobacterium violaceum has increasingly impacted our understanding of this microorganism. This review focuses on the genomic organization and structural analysis of the deduced proteins of the chemosensory adaptation system of C. violaceum. C. violaceum has multiple homologues of most chemotaxis genes, organized mostly in clusters in the bacterial genome. We found at least 67 genes, distributed in 10 gene clusters, involved in the chemotaxis of C. violaceum. A close examination of the chemoreceptors methyl-accepting chemotaxis proteins (MCPs), and the deduced sequences of the members of the two-component signaling system revealed canonical motifs, described as essential for the function of the deduced proteins. The chemoreceptors found in C. violaceum include the complete repertoire of such genes described in bacteria, designated as tsr, tar, trg, and tap; 41 MCP loci were found in the C. violaceum genome. Also, the C. violaceum genome includes a large repertoire of the proteins of the chemosensory transducer system. Multiple homologues of bacterial chemotaxis genes, including CheA, CheB, CheD, CheR, CheV, CheY, CheZ, and CheW, were found in the C. violaceum genome.
Assuntos
Proteínas de Bactérias/genética , Quimiotaxia/genética , Chromobacterium/genética , Flagelos/genética , Genes Bacterianos/genética , Proteínas de Bactérias/fisiologia , Quimiotaxia/fisiologia , Chromobacterium/fisiologia , Flagelos/fisiologia , Genes Bacterianos/fisiologia , Genoma Bacteriano , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Proteínas Quimiotáticas Aceptoras de MetilRESUMO
Helicobacter pylori ATCC43504 responds chemotactically to aspartic acid and serine, but not to arginine, nor to sodium bicarbonate. In contrast, H. pylori ATCC700392 (strain 26695) shows chemotaxis to all four attractants. Open reading frame HP0099 from H. pylori 26695 is predicted to encode one of three methyl-accepting chemotaxis receptor proteins (MCPs). When Escherichia coli is transformed with a plasmid carrying HP0099 from strain 26695, the recombinants acquire chemotaxis to arginine, bicarbonate, and urea. In H. pylori 43504, the HP0099 gene is interrupted with a mini-IS605 insertion, which accounts for its inability to recognize arginine and bicarbonate as attractants. Together, these results argue that the H. pylori HP0099 gene encodes an MCP for arginine and bicarbonate.
Assuntos
Arginina/farmacologia , Proteínas de Bactérias , Quimiotaxia/efeitos dos fármacos , Helicobacter pylori/genética , Proteínas de Membrana/genética , Bicarbonato de Sódio/farmacologia , Sequência de Bases , Quimiotaxia/genética , Elementos de DNA Transponíveis , Helicobacter pylori/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Quimiotáticas Aceptoras de Metil , Dados de Sequência Molecular , Transdução de Sinais , Especificidade por Substrato , Ureia/farmacologiaRESUMO
We have cloned and sequenced a 2,262-bp chromosomal DNA fragment from the chemolithoautotrophic acidophilic bacterium Leptospirillum ferrooxidans. This DNA contained an open reading frame for a 577-amino-acid protein showing several characteristics of the bacterial chemoreceptors and, therefore, we named this gene lcrI for Leptospirillum chemotaxis receptor I. This is the first sequence reported for a gene from L. ferrooxidans encoding a protein. The lcrI gene showed both sigma 28-like and sigma 70-like putative promoters. The LcrI deduced protein contained two hydrophobic regions most likely corresponding to the two transmembrane regions present in all of the methyl-accepting chemotaxis proteins (MCPs) which make them fold with both periplasmic and cytoplasmic domains. We have proposed a cytoplasmic domain for LcrI, which also contains the highly conserved domain (HCD region), present in all of the chemotactic receptors, and two probable methylation sites. The in vitro expression of a DNA plasmid containing the 2,262-bp fragment showed the synthesis of a 58-kDa protein which was immunoprecipitated by antibodies against the Tar protein (an MCP from Escherichia coli), confirming some degree of antigenic conservation. In addition, this 58-kDa protein was expressed in E. coli, being associated with its cytoplasmic membrane fraction. It was not possible to determine a chemotactic receptor function for LcrI expressed in E. coli. This was most likely due to the fact that the periplasmic pH of E. coli, which differs by 3 to 4 pH units from that of acidophilic chemolithotrophs, does not allow the right conformation for the LcrI periplasmic domain.