scholarly journals Methylation-Independent Aerotaxis Mediated by the Escherichia coli Aer Protein

2004 ◽  
Vol 186 (12) ◽  
pp. 3730-3737 ◽  
Author(s):  
Sergei I. Bibikov ◽  
Andrew C. Miller ◽  
Khoosheh K. Gosink ◽  
John S. Parkinson
Keyword(s):  
Escherichia Coli ◽  
Deleterious Effect ◽  
Behavioral Responses ◽  
Cellular Redox ◽  
E Coli ◽  
Signaling Domain ◽  
Chemotaxis Proteins ◽  
Homogeneous Chemical ◽  
Signaling Domains ◽  
Lines Of Evidence

ABSTRACT Aer is a membrane-associated protein that mediates aerotactic responses in Escherichia coli. Its C-terminal half closely resembles the signaling domains of methyl-accepting chemotaxis proteins (MCPs), which undergo reversible methylation at specific glutamic acid residues to adapt their signaling outputs to homogeneous chemical environments. MCP-mediated behaviors are dependent on two specific enzymes, CheR (methyltransferase) and CheB (methylesterase). The Aer signaling domain contains unorthodox methylation sites that do not conform to the consensus motif for CheR or CheB substrates, suggesting that Aer, unlike conventional MCPs, might be a methylation-independent transducer. Several lines of evidence supported this possibility. (i) The Aer protein was not detectably modified by either CheR or CheB. (ii) Amino acid replacements at the putative Aer methylation sites generally had no deleterious effect on Aer function. (iii) Aer promoted aerotactic migrations on semisolid media in strains that lacked all four of the E. coli MCPs. CheR and CheB function had no influence on the rate of aerotactic movements in those strains. Thus, Aer senses and signals efficiently in the absence of deamidation or methylation, methylation changes, methylation enzymes, and methyl-accepting chemotaxis proteins. We also found that chimeric transducers containing the PAS-HAMP sensing domain of Aer joined to the signaling domain and methylation sites of Tar, an orthodox MCP, exhibited both methylation-dependent and methylation-independent aerotactic behavior. The hybrid Aear transducers demonstrate that methylation independence does not emanate from the Aer signaling domain but rather may be due to transience of the cellular redox changes that are thought to trigger Aer-mediated behavioral responses.

scholarly journals Chemotaxis of Escherichia coli to Pyrimidines: a New Role for the Signal Transducer Tap

2007 ◽  
Vol 190 (3) ◽  
pp. 972-979 ◽  
Author(s):  
Xianxian Liu ◽  
Rebecca E. Parales
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Growth Conditions ◽  
Wild Type ◽  
E Coli ◽  
C Terminus ◽  
Signaling Domain ◽  
Chemotaxis Proteins ◽  
Periplasmic Domain

ABSTRACT Escherichia coli exhibits chemotactic responses to sugars, amino acids, and dipeptides, and the responses are mediated by methyl-accepting chemotaxis proteins (MCPs). Using capillary assays, we demonstrated that Escherichia coli RP437 is attracted to the pyrimidines thymine and uracil and the response was constitutively expressed under all tested growth conditions. All MCP mutants lacking the MCP Tap protein showed no response to pyrimidines, suggesting that Tap, which is known to mediate dipeptide chemotaxis, is required for pyrimidine chemotaxis. In order to confirm the role of Tap in pyrimidine chemotaxis, we constructed chimeric chemoreceptors (Tapsr and Tsrap), in which the periplasmic and cytoplasmic domains of Tap and Tsr were switched. When Tapsr and Tsrap were individually expressed in an E. coli strain lacking all four native MCPs, Tapsr mediated chemotaxis toward pyrimidines and dipeptides, but Tsrap did not complement the chemotaxis defect. The addition of the C-terminal 19 amino acids from Tsr to the C terminus of Tsrap resulted in a functional chemoreceptor that mediated chemotaxis to serine but not pyrimidines or dipeptides. These results indicate that the periplasmic domain of Tap is responsible for detecting pyrimidines and the Tsr signaling domain confers on Tapsr the ability to mediate efficient chemotaxis. A mutant lacking dipeptide binding protein (DBP) was wild type for pyrimidine taxis, indicating that DBP, which is the primary chemoreceptor for dipeptides, is not responsible for detecting pyrimidines. It is not yet known whether Tap detects pyrimidines directly or via an additional chemoreceptor protein.

scholarly journals Ligand-Specific Activation of Escherichia coli Chemoreceptor Transmethylation

2004 ◽  
Vol 186 (22) ◽  
pp. 7556-7563 ◽  
Author(s):  
Frances M. Antommattei ◽  
Jennifer B. Munzner ◽  
Robert M. Weis
Keyword(s):  
Escherichia Coli ◽  
E Coli ◽  
Specific Effects ◽  
C Terminus ◽  
Chemotaxis Proteins ◽  
Chemosensory Pathways ◽  
Receptor Dimers ◽  
Signaling Domains ◽  
Receptor Dimer ◽  
Methyl Group Transfer

ABSTRACT Adaptation in the chemosensory pathways of bacteria like Escherichia coli is mediated by the enzyme-catalyzed methylation (and demethylation) of glutamate residues in the signaling domains of methyl-accepting chemotaxis proteins (MCPs). MCPs can be methylated in trans, where the methyltransferase (CheR) molecule catalyzing methyl group transfer is tethered to the C terminus of a neighboring receptor. Here, it was shown that E. coli cells exhibited adaptation to attractant stimuli mediated through either engineered or naturally occurring MCPs that were unable to tether CheR as long as another MCP capable of tethering CheR was also present, e.g., either the full-length aspartate or serine receptor (Tar or Tsr). Methylation of isolated membrane samples in which engineered tethering and substrate receptors were coexpressed demonstrated that the truncated substrate receptors (trTsr) were efficiently methylated in the presence of tethering receptors (Tar with methylation sites blocked) relative to samples in which none of the MCPs had tethering sites. The effects of ligand binding on methylation were investigated, and an increase in rate was produced only with serine (the ligand specific for the substrate receptor trTsr); no significant change in rate was produced by aspartate (the ligand specific for the tethering receptor Tar). Although the overall efficiency of methylation was lower, receptor-specific effects were also observed in trTar- and trTsr-containing samples, where neither Tar nor Tsr possessed the CheR binding site at the C terminus. Altogether, the results are consistent with a ligand-induced conformational change that is limited to the methylated receptor dimer and does not spread to adjacent receptor dimers.

scholarly journals Identification and Characterization of a New Lipoprotein, NlpI, in Escherichia coli K-12

1999 ◽  
Vol 181 (14) ◽  
pp. 4318-4325 ◽  
Author(s):  
Masaru Ohara ◽  
Henry C. Wu ◽  
Krishnan Sankaran ◽  
Paul D. Rick
Keyword(s):  
Escherichia Coli ◽  
Direct Consequence ◽  
Insertion Mutant ◽  
Polynucleotide Phosphorylase ◽  
Wild Type ◽  
E Coli ◽  
K 12 ◽  
Identification And Characterization ◽  
Lines Of Evidence

ABSTRACT We report here the identification of a new lipoprotein, NlpI, inEscherichia coli K-12. The NlpI structural gene (nlpI) is located between the genes pnp(polynucleotide phosphorylase) and deaD (RNA helicase) at 71 min on the E. coli chromosome. The nlpI gene encodes a putative polypeptide of approximately 34 kDa, and multiple lines of evidence clearly demonstrate that NlpI is indeed a lipoprotein. An nlpI::cm mutation rendered growth of the cells osmotically sensitive, and incubation of the insertion mutant at an elevated temperature resulted in the formation of filaments. The altered phenotype of the mutant was a direct consequence of the mutation in nlpI, since it was complemented by the wild-type nlpI gene alone. Overexpression of the unaltered nlpI gene in wild-type cells resulted in the loss of the rod morphology and the formation of single prolate ellipsoids and pairs of prolate ellipsoids joined by partial constrictions. NlpI may be important for an as-yet-undefined step in the overall process of cell division.

scholarly journals Cellular Stoichiometry of Methyl-Accepting Chemotaxis Proteins inSinorhizobium meliloti

2017 ◽  
Vol 200 (6) ◽  
Author(s):  
Hardik M. Zatakia ◽  
Timofey D. Arapov ◽  
Veronika M. Meier ◽  
Birgit E. Scharf
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Sinorhizobium Meliloti ◽  
High Abundance ◽  
Detailed Knowledge ◽  
Class Iii ◽  
Content Type ◽  
E Coli ◽  
Chemotaxis Proteins ◽  
Carboxy Terminal

ABSTRACTThe chemosensory system inSinorhizobium melilotihas several important deviations from the widely studied enterobacterial paradigm. To better understand the differences between the two systems and how they are optimally tuned, we determined the cellular stoichiometry of the methyl-accepting chemotaxis proteins (MCPs) and the histidine kinase CheA inS. meliloti. Quantitative immunoblotting was used to determine the total amount of MCPs and CheA per cell inS. meliloti. The MCPs are present in the cell in high abundance (McpV), low abundance (IcpA, McpU, McpX, and McpW), and very low abundance (McpY and McpZ), whereas McpT was below the detection limit. The approximate cellular ratio of these three receptor groups is 300:30:1. The chemoreceptor-to-CheA ratio is 23.5:1, highly similar to that seen inBacillus subtilis(23:1) and about 10 times higher than that inEscherichia coli(3.4:1). Different fromE. coli, the high-abundance receptors inS. melilotiare lacking the carboxy-terminal NWETF pentapeptide that binds the CheR methyltransferase and CheB methylesterase. Using transcriptionallacZfusions, we showed that chemoreceptors are positively controlled by the master regulators of motility, VisNR and Rem. In addition, FlbT, a class IIA transcriptional regulator of flagellins, also positively regulates the expression of most chemoreceptors except for McpT and McpY, identifying chemoreceptors as class III genes. Taken together, these results demonstrate that the chemosensory complex and the adaptation system inS. melilotideviates significantly from the established enterobacterial paradigm but shares some similarities withB. subtilis.IMPORTANCEThe symbiotic soil bacteriumSinorhizobium melilotiis of great agricultural importance because of its nitrogen-fixing properties, which enhances growth of its plant symbiont, alfalfa. Chemotaxis provides a competitive advantage for bacteria to sense their environment and interact with their eukaryotic hosts. For a better understanding of the role of chemotaxis in these processes, detailed knowledge on the regulation and composition of the chemosensory machinery is essential. Here, we show that chemoreceptor gene expression inS. melilotiis controlled through the main transcriptional regulators of motility. Chemoreceptor abundance is much lower inS. melilotithan inEscherichia coliandBacillus subtilis. Moreover, the chemoreceptor-to-kinase CheA ratio is different from that ofE. colibut similar to that ofB. subtilis.

scholarly journals Cluster II che Genes from Pseudomonas aeruginosa Are Required for an Optimal Chemotactic Response

2002 ◽  
Vol 184 (16) ◽  
pp. 4374-4383 ◽  
Author(s):  
Abel Ferrández ◽  
Andrew C. Hawkins ◽  
Douglas T. Summerfield ◽  
Caroline S. Harwood
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Gene Clusters ◽  
Soft Agar ◽  
Chemotactic Response ◽  
Wild Type ◽  
E Coli ◽  
Chemotaxis Proteins ◽  
Complete Set ◽  
K 12

ABSTRACT Pseudomonas aeruginosa, a γ-proteobacterium, is motile by means of a single polar flagellum and is chemotactic to a variety of organic compounds and phosphate. P. aeruginosa has multiple homologues of Escherichia coli chemotaxis genes that are organized into five gene clusters. Previously, it was demonstrated that genes in cluster I and cluster V are essential for chemotaxis. A third cluster (cluster II) contains a complete set of che genes, as well as two genes, mcpA and mcpB, encoding methyl-accepting chemotaxis proteins. Mutations were constructed in several of the cluster II che genes and in the mcp genes to examine their possible contributions to P. aeruginosa chemotaxis. A cheB2 mutant was partially impaired in chemotaxis in soft-agar swarm plate assays. Providing cheB2 in trans complemented this defect. Further, overexpression of CheB2 restored chemotaxis to a completely nonchemotactic, cluster I, cheB-deficient strain to near wild-type levels. An mcpA mutant was defective in chemotaxis in media that were low in magnesium. The defect could be relieved by the addition of magnesium to the swarm plate medium. An mcpB mutant was defective in chemotaxis when assayed in dilute rich soft-agar swarm medium or in minimal-medium swarm plates containing any 1 of 60 chemoattractants. The mutant phenotype could be complemented by the addition of mcpB in trans. Overexpression of either McpA or McpB in P. aeruginosa or Escherichia coli resulted in impairment of chemotaxis, and these cells had smooth-swimming phenotypes when observed under the microscope. Expression of P. aeruginosa cheA2, cheB2, or cheW2 in E. coli K-12 completely disrupted wild-type chemotaxis, while expression of cheY2 had no effect. These results indicate that che cluster II genes are expressed in P. aeruginosa and are required for an optimal chemotactic response.

scholarly journals A zipped-helix cap potentiates HAMP domain control of chemoreceptor signaling

2018 ◽  
Vol 115 (15) ◽  
pp. E3519-E3528 ◽  
Author(s):  
Caralyn E. Flack ◽  
John S. Parkinson
Keyword(s):  
Escherichia Coli ◽  
Leucine Zipper ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Structural Motif ◽  
Heptad Repeat ◽  
Helix Bundle ◽  
Signaling Domain ◽  
Chemotaxis Proteins

Environmental awareness is an essential attribute for all organisms. The chemotaxis system of Escherichia coli provides a powerful experimental model for the investigation of stimulus detection and signaling mechanisms at the molecular level. These bacteria sense chemical gradients with transmembrane proteins [methyl-accepting chemotaxis proteins (MCPs)] that have an extracellular ligand-binding domain and intracellular histidine kinases, adenylate cyclases, methyl-accepting proteins, and phosphatases (HAMP) and signaling domains that govern locomotor behavior. HAMP domains are versatile input–output elements that operate in a variety of bacterial signaling proteins, including the sensor kinases of two-component regulatory systems. The MCP HAMP domain receives stimulus information and in turn modulates output signaling activity. This study describes mutants of the Escherichia coli serine chemoreceptor, Tsr, that identify a heptad-repeat structural motif (LLF) at the membrane-proximal end of the receptor signaling domain that is critical for HAMP output control. The homodimeric Tsr signaling domain is an extended, antiparallel, four-helix bundle that controls the activity of an associated kinase. The N terminus of each subunit adjoins the HAMP domain; the LLF residues lie at the C terminus of the methylation-helix bundle. We found, by using in vivo Förster resonance energy transfer kinase assays, that most amino acid replacements at any of the LLF residues abrogate chemotactic responses to serine and lock Tsr output in a kinase-active state, impervious to HAMP-mediated down-regulation. We present evidence that the LLF residues may function like a leucine zipper to promote stable association of the C-terminal signaling helices, thereby creating a metastable helix-packing platform for the N-terminal signaling helices that facilitates conformational control by the HAMP domains in MCP-family chemoreceptors.

scholarly journals CheR- and CheB-Dependent Chemosensory Adaptation System of Rhodobacter sphaeroides

2001 ◽  
Vol 183 (24) ◽  
pp. 7135-7144 ◽  
Author(s):  
Angela C. Martin ◽  
George H. Wadhams ◽  
Deepan S. H. Shah ◽  
Steven L. Porter ◽  
Jeevani C. Mantotta ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Rhodobacter Sphaeroides ◽  
High Abundance ◽  
E Coli ◽  
Polar Localization ◽  
Receptor Adaptation ◽  
Cell Assays ◽  
Chemotaxis Proteins ◽  
Conserved Region

ABSTRACT Rhodobacter sphaeroides has multiple homologues of most of the Escherichia coli chemotaxis genes, organized in three major operons and other, unlinked, loci. These includecheA 1 andcheR 1 (cheOp1) and cheA 2 ,cheR2 , andcheB1 (che Op2). In-frame deletions of thesecheR and cheB homologues were constructed and the chemosensory behaviour of the resultant mutants examined on swarm plates and in tethered cell assays. Under the conditions tested, CheR2 and CheB1 were essential for normal chemotaxis, whereas CheR1 was not.cheR 2 andcheB 1 , but notcheR 1 , were also able to complement the equivalent E. coli mutants. However, none of the proteins were required for the correct polar localization of the chemoreceptor McpG in R. sphaeroides. In E. coli, CheR binds to the NWETF motif on the high-abundance receptors, allowing methylation of both high- and low-abundance receptors. This motif is not contained on any R. sphaeroides chemoreceptors thus far identified, although 2 of the 13 putative chemoreceptors, McpA and TlpT, do have similar sequences. This suggests that CheR2 either interacts with the NWETF motif of E. coli methyl-accepting chemotaxis proteins (MCPs), even though its native motif may be slightly different, or with another conserved region of the MCPs. Methanol release measurements show that R. sphaeroides has an adaptation system that is different from that of Bacillus subtilis and E. coli, with methanol release measurable on the addition of attractant but not on its removal. Intriguingly, CheA2, but not CheA1, is able to phosphorylate CheB1, suggesting that signaling through CheA1 cannot initiate feedback receptor adaptation via CheB1-P.

scholarly journals Heterologous Expression ofPseudomonas putidaMethyl-Accepting Chemotaxis Proteins YieldsEscherichia coliChemotactic to Aromatic Compounds

2018 ◽  
Author(s):  
Clémence Roggo ◽  
Estelle Emilie Clerc ◽  
Noushin Hadadi ◽  
Nicolas Carraro ◽  
Roman Stocker ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Putida ◽  
Aromatic Compounds ◽  
Environmental Concern ◽  
Bacterial Chemotaxis ◽  
Gene Encoding ◽  
E Coli ◽  
Cell Accumulation ◽  
Chemotaxis Proteins ◽  
Chemical Availability

ABSTRACTEscherichia coli, commonly used in chemotaxis studies, is attracted mostly by amino acids, sugars and peptides. We envisioned modifying chemotaxis specificity ofE. coliby expressing heterologous chemoreceptors fromPseudomonas putidaenabling attraction either to toluene or benzoate. ThemcpTgene encoding the type 40H methyl-accepting chemoreceptor for toluene fromPseudomonas putidaMT53 and thepcaYgene for the type 40H receptor for benzoate and related molecules fromP. putidaF1 were expressed from thetrgpromoter on a plasmid in motile wild-typeE. coliMG1655.E. colicells expressing McpT accumulated in chemoattraction assays to sources with 60–200 μM toluene; less strongly than the response to 100 μM serine, but statistically significantly stronger than to sources without any added attractant. An McpT-mCherry fusion protein was detectably expressed inE. coliand yielding weak but distinguishable membrane and polar foci in 1% of cells.E. coliexpressing PcaY showed weak attraction to 0.1–1 mM benzoate but 50–70% of cells localized the PcaY-mCherry fusion to their membrane. We conclude that implementing heterologous receptors in theE. colichemotaxis network is possible and, upon improvement of the compatibility of the type 40H chemoreceptors, may bear interest for biosensing.IMPORTANCEBacterial chemotaxis might be harnessed for the development of rapid biosensors, in which chemical availability is deduced from cell accumulation to chemoattractants over time. Chemotaxis ofEscherichia colihas been well-studied, but the bacterium is not attracted to chemicals of environmental concern, such as aromatic solvents. We show here that heterologous chemoreceptors for aromatic compounds fromPseudomonas putidaat least partly functionally complement theE. colichemotaxis network, yielding cells attracted to toluene or benzoate. Complementation was still inferior to native chemoattractants like serine, but our study demonstrates the potential for obtaining selective sensing for aromatic compounds inE. coli.

scholarly journals Heterologous Expression ofPseudomonas putidaMethyl-Accepting Chemotaxis Proteins YieldsEscherichia coliCells Chemotactic to Aromatic Compounds

2018 ◽  
Vol 84 (18) ◽  
Author(s):  
Clémence Roggo ◽  
Estelle Emilie Clerc ◽  
Noushin Hadadi ◽  
Nicolas Carraro ◽  
Roman Stocker ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Putida ◽  
Aromatic Compounds ◽  
Environmental Concern ◽  
Bacterial Chemotaxis ◽  
Gene Encoding ◽  
Content Type ◽  
E Coli ◽  
Cell Accumulation ◽  
Chemotaxis Proteins

ABSTRACTEscherichia coli, commonly used in chemotaxis studies, is attracted mostly by amino acids, sugars, and peptides. We envisioned modifying the chemotaxis specificity ofE. coliby expressing heterologous chemoreceptors fromPseudomonas putidaenabling attraction either to toluene or benzoate. ThemcpTgene encoding the type 40-helical bundle (40H) methyl-accepting chemoreceptor for toluene fromPseudomonas putidaMT53 and thepcaYgene for the type 40H receptor for benzoate and related molecules fromP. putidaF1 were expressed from thetrgpromoter on a plasmid in motile wild-typeE. coliMG1655.E. colicells expressing McpT accumulated in chemoattraction assays to sources with 60 to 200 μM toluene, although less strongly than the response to 100 μM serine, but statistically significantly stronger than that to sources without any added attractant. An McpT-mCherry fusion protein was detectably expressed inE. coliand yielded weak but distinguishable membranes and polar foci in 1% of cells.E. colicells expressing PcaY showed weak attraction to 0.1 to 1 mM benzoate, but 50 to 70% of cells localized the PcaY-mCherry fusion to their membrane. We conclude that implementing heterologous receptors in theE. colichemotaxis network is possible and, upon improvement of the compatibility of the type 40H chemoreceptors, may bear interest for biosensing.IMPORTANCEBacterial chemotaxis might be harnessed for the development of rapid biosensors, in which chemical availability is deduced from cell accumulation to chemoattractants over time. Chemotaxis ofEscherichia colihas been well studied, but the bacterium is not attracted to chemicals of environmental concern, such as aromatic solvents. We show here that heterologous chemoreceptors for aromatic compounds fromPseudomonas putidaat least partly functionally complement theE. colichemotaxis network, yielding cells attracted to toluene or benzoate. Complementation was still inferior to native chemoattractants, like serine, but our study demonstrates the potential for obtaining selective sensing for aromatic compounds inE. coli.

scholarly journals Detecting selective sweeps in naturally occurring Escherichia coli.

Genetics ◽  
1994 ◽  
Vol 138 (4) ◽  
pp. 993-1003 ◽  
Author(s):  
D S Guttman ◽  
D E Dykhuizen
Keyword(s):  
Escherichia Coli ◽  
Frame Of Reference ◽  
Selective Sweeps ◽  
Genetic Hitchhiking ◽  
E Coli ◽  
Naturally Occurring ◽  
Selective Neutrality ◽  
Natural Isolates ◽  
Evolutionary Event ◽  
Lines Of Evidence

Abstract The nucleotide sequences of the gapA and pabB genes (separated by approximately 32.5 kb) were determined in 12 natural isolates of Escherichia coli. Three analyses were performed on the data. First, the levels of polymorphism at the loci were compared within and between E. coli and Salmonella strains relative to their degrees of constraint. Second, the gapA and pabB loci were analyzed by the Hudson-Kreitman-Aguadé (HKA) test for selective neutrality. Four additional dispersed genes (crr, putP, trp and gnd) were added to the analysis to provide the necessary frame of reference. Finally, the gene genealogies of gapA and pabB were examined for topological consistency within and between the loci. These lines of evidence indicate that some evolutionary event has recently purged the variability in the region surrounding the gapA and pabB loci in E. coli. This can best be explained by the spread of a selected allele through the global E. coli population by directional selection and the resulting loss in variability in the surrounding regions due to genetic hitchhiking.

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