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Characterization of CetA and CetB, a bipartite energy taxis system in Campylobacter jejuni.

Elliott KT, Dirita VJ - Mol. Microbiol. (2008)

Bottom Line: The PAS domain (a sensory domain named after three proteins Per, ARNT and Sim, where it was first identified) is thought to interact directly with the Aer HAMP domain to transmit this signal to the highly conserved domain (HCD) found in chemotaxis receptors.CetA has two transmembrane helices in a helical hairpin while CetB is a peripheral membrane protein tightly associated with the membrane.This study provides a foundation for further characterization of signal transduction mechanisms within CetA/CetB.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Michigan, Ann Arbor, MI 48109, USA.

ABSTRACT
The energy taxis receptor Aer, in Escherichia coli, senses changes in the redox state of the electron transport system via an flavin adenine dinucleotide cofactor bound to a PAS domain. The PAS domain (a sensory domain named after three proteins Per, ARNT and Sim, where it was first identified) is thought to interact directly with the Aer HAMP domain to transmit this signal to the highly conserved domain (HCD) found in chemotaxis receptors. An apparent energy taxis system in Campylobacter jejuni is composed of two proteins, CetA and CetB, that have the domains of Aer divided between them. CetB has a PAS domain, while CetA has a predicted transmembrane region, HAMP domain and the HCD. In this study, we examined the expression of cetA and cetB and the biochemical properties of the proteins they encode. cetA and cetB are co-transcribed independently of the flagellar regulon. CetA has two transmembrane helices in a helical hairpin while CetB is a peripheral membrane protein tightly associated with the membrane. CetB levels are CetA dependent. Additionally, we demonstrated that both CetA and CetB participate in complexes, including a likely CetB dimer and a complex that may include both CetA and CetB. This study provides a foundation for further characterization of signal transduction mechanisms within CetA/CetB.

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location and activities of LacZ and PhoA fusions to CetA.A. Locations of PhoA and LacZ fusions to truncated or full-length CetA are indicated. LacZ or PhoA was fused C-terminally to the truncated or full-length protein.B. Alkaline phosphatase activity of PhoA fusions.C. β-Galactosidase activity of LacZ fusions. In both (B) and (C), ‘PhoA’ and ‘LacZ’ indicate the empty vectors pTrcphoA and pTrcLacZ respectively.
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fig04: location and activities of LacZ and PhoA fusions to CetA.A. Locations of PhoA and LacZ fusions to truncated or full-length CetA are indicated. LacZ or PhoA was fused C-terminally to the truncated or full-length protein.B. Alkaline phosphatase activity of PhoA fusions.C. β-Galactosidase activity of LacZ fusions. In both (B) and (C), ‘PhoA’ and ‘LacZ’ indicate the empty vectors pTrcphoA and pTrcLacZ respectively.

Mentions: We made phoA and lacZ fusions such that alkaline phosphatase or β-galactosidase would be fused C-terminally to full-length CetA or to CetA that was truncated at residue 5, 24, 50 or 140 (Fig. 4A). These fusions were expressed in an E. coli strain lacking lacZ and phoA and assayed for alkaline phosphatase and β-galactosidase activity. The only alkaline phosphatase fusion construct with significant activity was that at His-24 of CetA (Fig. 4B). The β-galactosidase fusion at this location (His-24) was also the fusion with the lowest β-galactosidase activity (Fig. 4C). This fusion did have β-galactosidase activity above background levels, however. Work in other laboratories has indicated that β-galactosidase fusions to periplasmic regions, which do not lead to translocation of the fusion but rather embed the protein in the membrane, can sometimes lead to degradation of the fusion and release of native β-galactosidase, giving rise to activity (Georgiou et al., 1988; Gott and Boos, 1988). These studies caution that use of β-galactosidase fusions must be complemented by an alternative topological probe, such as alkaline phosphatase, which is likely a more reliable indicator of subcellular localization. Together, our results indicate that His-24 is accessible to the periplasm, whereas all of the other fusion locations are found in the cytoplasm. These data support our prediction that CetA has two transmembrane helices in a helical hairpin.


Characterization of CetA and CetB, a bipartite energy taxis system in Campylobacter jejuni.

Elliott KT, Dirita VJ - Mol. Microbiol. (2008)

location and activities of LacZ and PhoA fusions to CetA.A. Locations of PhoA and LacZ fusions to truncated or full-length CetA are indicated. LacZ or PhoA was fused C-terminally to the truncated or full-length protein.B. Alkaline phosphatase activity of PhoA fusions.C. β-Galactosidase activity of LacZ fusions. In both (B) and (C), ‘PhoA’ and ‘LacZ’ indicate the empty vectors pTrcphoA and pTrcLacZ respectively.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2628428&req=5

fig04: location and activities of LacZ and PhoA fusions to CetA.A. Locations of PhoA and LacZ fusions to truncated or full-length CetA are indicated. LacZ or PhoA was fused C-terminally to the truncated or full-length protein.B. Alkaline phosphatase activity of PhoA fusions.C. β-Galactosidase activity of LacZ fusions. In both (B) and (C), ‘PhoA’ and ‘LacZ’ indicate the empty vectors pTrcphoA and pTrcLacZ respectively.
Mentions: We made phoA and lacZ fusions such that alkaline phosphatase or β-galactosidase would be fused C-terminally to full-length CetA or to CetA that was truncated at residue 5, 24, 50 or 140 (Fig. 4A). These fusions were expressed in an E. coli strain lacking lacZ and phoA and assayed for alkaline phosphatase and β-galactosidase activity. The only alkaline phosphatase fusion construct with significant activity was that at His-24 of CetA (Fig. 4B). The β-galactosidase fusion at this location (His-24) was also the fusion with the lowest β-galactosidase activity (Fig. 4C). This fusion did have β-galactosidase activity above background levels, however. Work in other laboratories has indicated that β-galactosidase fusions to periplasmic regions, which do not lead to translocation of the fusion but rather embed the protein in the membrane, can sometimes lead to degradation of the fusion and release of native β-galactosidase, giving rise to activity (Georgiou et al., 1988; Gott and Boos, 1988). These studies caution that use of β-galactosidase fusions must be complemented by an alternative topological probe, such as alkaline phosphatase, which is likely a more reliable indicator of subcellular localization. Together, our results indicate that His-24 is accessible to the periplasm, whereas all of the other fusion locations are found in the cytoplasm. These data support our prediction that CetA has two transmembrane helices in a helical hairpin.

Bottom Line: The PAS domain (a sensory domain named after three proteins Per, ARNT and Sim, where it was first identified) is thought to interact directly with the Aer HAMP domain to transmit this signal to the highly conserved domain (HCD) found in chemotaxis receptors.CetA has two transmembrane helices in a helical hairpin while CetB is a peripheral membrane protein tightly associated with the membrane.This study provides a foundation for further characterization of signal transduction mechanisms within CetA/CetB.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Michigan, Ann Arbor, MI 48109, USA.

ABSTRACT
The energy taxis receptor Aer, in Escherichia coli, senses changes in the redox state of the electron transport system via an flavin adenine dinucleotide cofactor bound to a PAS domain. The PAS domain (a sensory domain named after three proteins Per, ARNT and Sim, where it was first identified) is thought to interact directly with the Aer HAMP domain to transmit this signal to the highly conserved domain (HCD) found in chemotaxis receptors. An apparent energy taxis system in Campylobacter jejuni is composed of two proteins, CetA and CetB, that have the domains of Aer divided between them. CetB has a PAS domain, while CetA has a predicted transmembrane region, HAMP domain and the HCD. In this study, we examined the expression of cetA and cetB and the biochemical properties of the proteins they encode. cetA and cetB are co-transcribed independently of the flagellar regulon. CetA has two transmembrane helices in a helical hairpin while CetB is a peripheral membrane protein tightly associated with the membrane. CetB levels are CetA dependent. Additionally, we demonstrated that both CetA and CetB participate in complexes, including a likely CetB dimer and a complex that may include both CetA and CetB. This study provides a foundation for further characterization of signal transduction mechanisms within CetA/CetB.

Show MeSH
Related in: MedlinePlus