Chemotaxis in Escherichia coli: construction and properties of lambda tsr transducing phage

Callahan, Ann M.; Frazier, Barbara L.; Parkinson, John S.

doi:10.1128/jb.169.3.1246-1253.1987

Cited by 27 publications

(15 citation statements)

References 30 publications

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“…To study the antimicrobial properties of nanostructured hydrogel webs containing silver, E. coli K12 strain RP437 35,36 was used. To visualize the biofilms with fluorescence microscopy, E. coli RP437 was labeled with constitutively expressed DsRed-Express fluorescent protein by transformation of plasmid pRSH103.…”

Section: Methodsmentioning

confidence: 99%

Antimicrobial Properties of Nanostructured Hydrogel Webs Containing Silver

et al. 2009

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The need exists for biomaterials that prevent biofilm formation and associated infections. In this report, we have studied the synthesis, processing, and antimicrobial behavior of new silver-containing thermoplastic hydrogel nanofibrous webs. Thermoplastic hydrogels were synthesized from multiblock PEG-POSS polyurethanes (PEG: poly(ethylene gylcol); POSS: polyhedral oligosilsesquioxane) and electrospun into nanofibrous webs (diameter ∼150 nm), with or without AgNO 3 . The nanofibrous hydrogels exhibited unusual shrinkage during water uptake, yielding a uniquely dense structure compared to hydrogels prepared from cast films. Antimicrobial activity was examined using exposure to Escherichia coli with daily refreshment of medium and inoculation to a controlled cell density. Nanofibrous hydrogels without silver featured the most rapid and most extensive biofilm formation, while the silver-containing nanofibrous hydrogel featured outstanding biofilm resistance, with biofilm formation taking hold only after 14 days of incubation in daily refreshed bacterial cultures. We envision application of the unique antimicrobial hydrogels as wound dressings that combine sustained bactericidal properties and lack of volumetric swelling during water uptake.

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Section: Methodsmentioning

confidence: 99%

Antimicrobial Properties of Nanostructured Hydrogel Webs Containing Silver

et al. 2009

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“…BL21(DE3) is deficient in the OmpT and Lon proteases and contains a prophage that carries the T7 RNA polymerase gene. Isogenic strains UU1117 (aer [7]), RP5882 (tsr [10]) and BT3312 (aer tsr [36]) are derivatives of the E. coli K-12 RP437 strain (32).…”

Section: Methodsmentioning

confidence: 99%

PAS Domain of the Aer Redox Sensor Requires C-Terminal Residues for Native-Fold Formation and Flavin Adenine Dinucleotide Binding

Herrmann

Johnson

et al. 2004

J Bacteriol

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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 His 6 tags and expressed the PAS peptides in E. coli. The 20-kDa His 6 -Aer 2-166 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 (Aer 2-231 ) or all (Aer 2-285 ) of the HAMP domain inserted into the membrane, indicating that they were released by GroEL. Aer 2-285 , but not Aer 2-231 , 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.PAS domains are sensory input domains and protein-protein interaction sites that have been identified recently in a large family of sensory proteins from all kingdoms of life (34,44,50,51; Simple Modular Architecture Research Tool [28,38; http: //smart.embl-heidelberg.de/]). The stimuli detected by PAS domains are diverse and in bacteria include light, oxygen, redox potential, and voltage (for reviews, see references 16, 21, and 44). PAS domains face the cytoplasm, unlike other sensory input domains that project into the periplasm or onto the external surface of a cell (44). These domains have a characteristic three-dimensional fold (20, 33) and bind a variety of cofactors, including 4-hydroxycinnamic acid, FMN, FAD, and heme (7,8,13,20,22,35,37). In eukaryotes, PAS domains have been shown to direct circadian rhythms (18), hypoxia responses (39), ion channel function (30), and development (40) (for a review, see reference 44). Considering the prominence of PAS domains in sensory proteins, surprisingly little is known about their role in signal transduction.We are interested in PAS sensing and signaling in Aer, an FAD-containing aerotaxis receptor in Escherichia coli (6,7,35,36). Aerotaxis is a rapid behavioral response to an oxygen gradient that guides cells to a region where the oxygen concentration is optimal for growth of the species (42,43,45). The Aer protein has an N-terminal PAS domain, an F1 segment, a 38-amino-acid hydrophobic sequence, a HAMP domain, and a C-terminal signaling domain (6, 36) (Fig. 1). The HAMP domain and high...

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“…All strains used in this work were derived from RP437, an E. coli K-12 strain that is wild type for chemotaxis (28). The in vivo effects of Tsr fragments were studied in RP5700, which carries a complete deletion of the tsr CONSTITUTIVELY SIGNALING CHEMORECEPTOR FRAGMENTS 6341 locus [A(tsr)7028] but is otherwise chemotactic (7). RP3098…”

Section: Methodsmentioning

confidence: 99%

Constitutively signaling fragments of Tsr, the Escherichia coli serine chemoreceptor

1994

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Tsr, the serine chemoreceptor of Escherichia coli, has two signaling modes. One augments clockwise (CW) flagellar rotation, and the other augments counterclockwise (CCW) rotation. To identify the portion of the Tsr molecule responsible for these activities, we isolated soluble fragments of the Tsr cytoplasmic domain that could alter the flagellar rotation patterns of unstimulated wild-tpe cells. Residues 290 to 470 from wild-type Tsr generated a CW signal, whereas the same fragment with a single amino acid replacement (alanine 413 to valine) produced a CCW signal. The soluble components of the chemotaxis phosphorelay system needed for expression of these Tsr fragment signals were identified by epistasis analysis. Like full-length receptors, the fragments appeared to generate signals through interactions with the CheA autokinase and the CheW coupli factor. CheA was required for both signaling activities, whereas CheW was needed only for CW signaling. Purified Tsr fragments were also examined for effects on CheA autophosphorylation activity in vitro.Consistent with the in vivo findings, the CW fragment stimulated CheA, whereas the CCW f ent inhibited CheA. CheW was required for stimulation but not for inhibition. These findings demonstrate that a 180-residue segment of the Tsr cytoplasmic domain can produce two active signals. The CCW signal involves a direct contact between the receptor and the CheA kinase, whereas the CW signal requires participation of CheW as well. The correlation between the in vitro effects of Tsr signaling fragments on CheA activity and their in vivo behavioral effects lends convincing support to the phosphorelay model of chemotactic signaling.Methyl-accepting chemotaxis proteins (MCPs) mediate chemotactic responses in a variety of motile bacteria, ranging from archaebacteria to gram-positive and gram-negative eubacteria (reviewed in reference 11). These sensory transducers offer tractable models for molecular studies of transmembrane and intracellular signaling. Escherichia coli has four different MCPs that detect the attractants serine (Tsr), aspartate (Tar), dipeptides (Tap), and ribose and galactose (Trg). MCP molecules are about 550 amino acids in length and span the cytoplasmic membrane. They have a periplasmic ligand-binding domain that monitors attractant levels in the environment and a cytoplasmic signaling domain that controls rotation of the flagellar motors (14,21). Occupancy changes in the sensing domain modulate the output activity of the signaling domain to elicit chemotactic movements. For example, attractant increases promote smooth, forward swimming by reducing the probability of the episodes of clockwise (CW) flagellar rotation that cause turns or tumbling movements. Ensuing changes in MCP methylation state cancel excitatory stimulus signals to achieve sensory adaptation, thereby enabling the organism to detect further changes in its chemical environment (31).MCPs in E. coli appear to control swimming behavior by modulating the autophosphorylation activity of CheA, a cyto...

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Chemotaxis in Escherichia coli: construction and properties of lambda tsr transducing phage

Cited by 27 publications

References 30 publications

Antimicrobial Properties of Nanostructured Hydrogel Webs Containing Silver

Antimicrobial Properties of Nanostructured Hydrogel Webs Containing Silver

PAS Domain of the Aer Redox Sensor Requires C-Terminal Residues for Native-Fold Formation and Flavin Adenine Dinucleotide Binding

Constitutively signaling fragments of Tsr, the Escherichia coli serine chemoreceptor

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