| Summary: | <p>Bacteria swim towards improving conditions by controlling flagellar activity via signals (CheY) sent from chemosensory protein clusters, which respond to changing stimuli. The best studied chemotactic bacterium, <em>E. coli</em>, has one transmembrane chemosensory protein cluster controlling flagellar behaviour. <em>R. sphaeroides</em> has two clusters, one transmembrane and one cytoplasmic. The roles of the two clusters in regulating swimming and chemosensory behaviour are explored here.</p> <p>Newly-developed software was used to measure the effect of deleting or mutating each chemotaxis protein on unstimulated swimming and on the chemosensory response to dynamic change. New behaviours were identified by using much larger sample sizes than previous studies. <em>R. sphaeroides</em> chemotaxis mutants were classified as (i) stoppy unresponsive; (ii) smooth unresponsive or (iii) stoppy inhibited compared to wildtype swimming and chemosensory behaviour. The data showed that the ability to stop during free-swimming is not necessarily connected to the ability to respond to a chemotaxis challenge. The data suggested a new model of connectivity between the two chemosensory pathways.</p> <p>CheY<sub>3</sub> and CheY<sub>4</sub> are phosphorylated by the transmembrane polar cluster in response to external chemoeffector concentrations. CheY<sub>6</sub>-P produced by the cytoplasmic cluster is a requirement for chemotaxis, whether or not the polar cluster is able to produce CheY<sub>6</sub>-P. CheY<sub>6</sub>-P stops the motor, whereas CheY<sub>3,4</sub>-P allow smooth swimming. When chemoeffector levels fall, the signals through CheY<sub>3,4</sub> fall, allowing CheY<sub>6</sub>-P to bind and stop the motor. As the polar cluster adapts to the fall by the action of the adaptation proteins CheB<sub>1</sub> and CheR<sub>2</sub>, the concentration of CheY<sub>3,4</sub>-P increases again, to compete with CheY<sub>6</sub>-P and allow periods of smooth swimming. Under aerobic conditions, the cytoplasmic cluster controls the basal stopping frequency and does not appear to respond to external chemoeffector changes.</p> <p>The role of the adaptation proteins in resetting the signalling state in <em>R. sphaeroides</em> is unclear, particularly the roles of the proteins associated with the cytoplasmic cluster, CheB<sub>2</sub> and CheR<sub>3</sub>. Tandem mass spectrometry was used to identify glutamate and glutamine (EQ) sites on the cytoplasmic <em>R. sphaeroides</em> chemoreceptor TlpT that are deamidated and methylated by the <em>R. sphaeroides</em> adaptation homologues. In <em>E. coli</em>, adaptation sites are usually EQ/EQ pairs. However the sites reported in TlpT vary at the first residue in the pair. Mutation of the putative EQ adaptation sites caused changes in adaptation, suggesting that CheY<sub>6</sub>-P levels are controlled and reset by CheB<sub>2</sub> and CheR<sub>3</sub> controlling the adaptation state of TlpT.</p>
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