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<i>Rhodobacter</i> <i>sphaeroides</i> has two chemotaxis clusters, an <i>Escherichia coli</i>-like cluster with membrane-spanning chemoreceptors and a less-understood cytoplasmic cluster. The cytoplasmic CheA is split into CheA<sub>4</sub>, a kinase, and CheA<sub>3</sub>, a His-domain phosphorylated by CheA<sub>4</sub> and a phosphatase domain, which together phosphorylate and dephosphorylate motor-stopping CheY<sub>6</sub>. In bacterial two-hybrid analysis, one major cytoplasmic chemoreceptor, TlpT, interacted with CheA<sub>4</sub>, while the other, TlpC, interacted with CheA<sub>3</sub>. Both clusters have associated adaptation proteins. Deleting their methyltransferases and methylesterases singly and together removed chemotaxis, but with opposite effects. The cytoplasmic cluster signal overrode the membrane cluster signal. Methylation and demethylation of specific chemoreceptor glutamates controls adaptation. Tandem mass spectroscopy and bioinformatics identified four putative sites on TlpT, three glutamates and a glutamine. Mutating each glutamate to alanine resulted in smooth swimming and loss of chemotaxis, unlike similar mutations in <i>E. coli</i> chemoreceptors. Cells with two mutated glutamates were more stoppy than wild-type and responded and adapted to attractant addition, not removal. Mutating all four sites amplified the effect. Cells were non-motile, began smooth swimming on attractant addition, and rapidly adapted back to non-motile before attractant removal. We propose that TlpT responds and adapts to the cell's metabolic state, generating the steady-state concentration of motor-stopping CheY<sub>6</sub>~P. Membrane-cluster signalling produces a pulse of CheY<sub>3</sub>/CheY<sub>4</sub>~P that displaces CheY<sub>6</sub>~P and allows flagellar rotation and smooth swimming before both clusters adapt.

Original publication

DOI

10.3390/ijms20205095

Type

Journal article

Journal

International journal of molecular sciences

Publication Date

14/10/2019

Volume

20

Addresses

Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK.

Keywords

Cytoplasm, Cytosol, Rhodobacter sphaeroides, Bacterial Proteins, Escherichia coli Proteins, Adaptation, Physiological, Signal Transduction, Chemotaxis, Protein Processing, Post-Translational, Gene Deletion, Phosphorylation, Tandem Mass Spectrometry, Chemoreceptor Cells, Histidine Kinase, Methyl-Accepting Chemotaxis Proteins