Helen L. Packer

Identification of a chemotaxis operon with two cheY genes in Rhodobacter sphaeroides

A large chemotaxis operon was identified in Rhodobacter sphaeroides WS8‐N using a probe based on the 3′ terminal portion of the Rhizobium meliloti cheA gene. Two genes homologous to the enteric cheY were identified in an operon also containing cheA, cheW, and cheR homologues. The deduced protein sequences of che gene products were aligned with those from Escherichia coli and shown to be highly conserved. A mutant with an interrupted copy of cheA showed normal patterns of swimming, unlike the equivalent mutants in E. coli which are smooth swimming. Tethered cheA mutant cells showed normal responses to changes in organic acids, but increased, inverted responses to sugars. The unusual behaviour of the cheA mutant and the identification of two homologues of cheY suggests that R. sphaeroides has at least two pathways controlling motor activity. To identify functional similarity between the newly identified R. sphaeroides Che pathway and the methyl‐accepting chemotaxis protein (MCP)‐dependent pathway in enteric bacteria, the R. sphaeroides cheW gene was expressed in a cheW mutant strain of E. coli and found to complement, causing a partial return to a swarming phenotype. In addition, expression of the R. sphaeroides gene in wild‐type E. coli resulted in the same increased tumbling and reduced swarming as seen when the native gene is over‐expressed in E. coli. The identification of che homologues in R. sphaeroides and complementation by cheW suggests the presence of MCPs in an organism previously considered to use only MCP‐independent sensing. The MCP‐dependent pathway, appears conserved. In R. sphaeroides this pathway may mediate responses to sugars, while responses to organic acids may in involve a second system, possibly using the second CheY protein identified in this study.

Tactic Responses to Oxygen in the Phototrophic Bacterium Rhodobacter sphaeroides WS8N

The temporal and spatial behavior of a number of mutants of the photosynthetic, facultative anaerobe Rhodobacter sphaeroides to both step changes and to gradients of oxygen was analyzed. Wild-type cells, grown under a range of conditions, showed microaerophilic behavior, accumulating in a 1.3-mm band about 1.3 mm from the meniscus of capillaries. Evidence suggests this is the result of two signaling pathways. The strength of any response depended on the growth and incubation conditions. Deletion of either the complete chemosensory operons 1 and 2 plus the response regulator genes cheY(4) and cheY(5) or cheA(2) alone led to the loss of all aerotactic responses, although the cells still swam normally. The Prr system of R. sphaeroides responds to electron flow through the alternative high-affinity cytochrome oxidase, cbb(3), controlling expression of a wide range of metabolic pathways. Mutants with deletions of either the complete Prr operon or the histidine kinase, PrrB, accumulated up to the meniscus but still formed a thick band 1.3 mm from the aerobic interface. This indicates that the negative aerotactic response to high oxygen levels depends on PrrB, but the mutant cells still retain the positive response. Tethered PrrB(-) cells also showed no response to a step-down in oxygen concentration, although those with deletions of the whole operon showed some response. In gradients of oxygen where the concentration was reduced at 0.4 micro M/s, tethered wild-type cells showed two different phases of response, with an increase in stopping frequency when the oxygen concentration fell from 80 to 50% dissolved oxygen and a decrease in stopping at 50 to 20% dissolved oxygen, with cells returning to their normal stopping frequency in 0% oxygen. PrrB and CheA(2) mutants showed no response, while PrrCBA mutants still showed some response.

The behavioural response of anaerobic Rhodobacter sphaeroides to temporal stimuli.

The behavioural response of Rhodobacter sphaeroides to temporal changes in the concentration of chemoeffectors, and to stimuli affecting electron transport, was analysed using tethered cells. Populations of photosynthetically grown tethered cells of R. sphaeroides showed a transient response, a stop followed by adaptation, to a stepwise reduction in the concentration of chemoattractants (such as organic acids or sugars) and terminal electron acceptors. A step-down response was also measured in free swimming cells to a reduction in light intensity. As this response appears to apply to all effectors this suggests that there is a sensory pathway in anaerobically grown R. sphaeroides which responds primarily to a reduction in a stimulus. R. sphaeroides therefore responds when moving down a concentration gradient. This is the inverse of the classical Escherichia coli-Salmonella typhimurium model of bacterial sensory behaviour in which bacteria respond primarily when there is an increase in an attractant concentration, i.e. when moving up a gradient. R. sphaeroides does show a chemokinetic response to an increase in concentration of a limited number of compounds but this response is sustained and accompanied by an increase in the rate of flagellar rotation and therefore not simply equivalent to the transient increase in smooth swimming measured in E. coli on addition of an attractant.

Swimming speed and chemokinetic response of Rhodobacter sphaeroides investigated by natural manipulation of the membrane potential

The ΔΨ of R. sphaeroides, grown under high light to reduce the levels of light‐harvesting bacteriochlorophyll, was naturally manipulated using light intensity. The relationship between ΔΨ and the swimming speed of free swimming populations of cells was investigated. After de‐energisation by incubation in the dark there was an apparent threshold of about −13 mV which had to be overcome before functional motor rotation could resume and at −45 mV the motor saturated. Further increases in ΔΨ over −45 mV did not increase the free swimming velocity. However, when a chemokinetic effector was added there was an increase in swimming speed, even though the ΔΨ was well above saturation, indicating that the chemokinetic response is independent of normal relationship between motor rotation and ΔΨ.

Behavioral Responses of Rhodobacter sphaeroides to Linear Gradients of the Nutrients Succinate and Acetate

ABSTRACT Rhodobacter sphaeroides cells were tethered by their flagella and subjected to increasing and decreasing nutrient gradients. Using motion analysis, changes in flagellar motor rotation were measured and the responses of the cells to the chemotactic gradients were determined. The steepness and concentration ranges of increasing and decreasing gradients were varied, and the bacterial responses were measured. This allowed the limits of gradients that would invoke changes in flagellar behavior to be determined and thus predicts the nature of gradients that would evoke chemotaxis in the environment. The sensory threshold was measured at 30 nM, and the response showed saturation at 150 μM. The study determined that cells detected and responded to changing concentration rates as low as 1 nM/s for acetate and 5 nM/s for succinate. The complex sensory system of R. sphaeroides responded to both increasing and decreasing concentration gradients of attractant with different sensitivities. In addition, transition phases involving changes in the motor speed and the smoothness of motor rotation were found.

The Rhodobacter sphaeroides flagellar motor is a variable-speed rotor.

The rotation rate of the unidirectional stop/start motor of Rhodobacter sphaeroides was investigated using computerised motion analysis of tethered cells. The R. sphaeroides motor was found to have a variable rotation rate compared to the virtually constant‐speed motor of wild‐type and CheR mutant (smooth swimming) Escherichia coli. In addition, the dynamics of the R. sphaeroides motor during stopping was analysed with no consistent correlation behaviour. The motor could go from full rotation to stop, or stop to full rotation within one video frame, i.e. 0.02 s, but it could also slow down into a stop or restart slowly, taking up to 0.25 s. The R. sphaeroides motor under chemokinetic stimulation was also analysed and was found to show increased torque generation and reduced variation in rotation rate.

The effect of pH on the growth and motility of Rhodobacter sphaeroides WS8 and the nature of the driving force of the flagellar motor

Rhodobacter sphaeroides WS8 grew, and swam vigorously, over the pH range 6 to 9. Sustained motility was, however, observed in populations of cells resuspended at pH values between 4.9 and 10.4, although the mean run speed was reduced at the extremes of pH. The ability of R. sphaeroides to swim in strong alkaline conditions prompted the question of whether motility at alkaline pH was powered by a sodium motive force, as has been found in the facultative alkalophilic Bacillus and Vibrio species, particularly as motility was found to be sensitive to the sodium channel inhibitor amiloride. The nature of the driving force of the flagellar motor was therefore investigated. It was found that R. sphaeroides was motile over the same pH range in the absence and presence of sodium ions. The protonophore CCCP was found to inhibit motility under all conditions, whereas monensin, an inhibitor of sodium pumps, had no effect upon motility in the presence or absence of sodium. It was concluded that the delta p is the driving force for the flagellar motor in R. sphaeroides at all values of pH. Amiloride, a specific inhibitor of the sodium-driven flagellar motor in alkalophilic Bacillus and Vibrio was shown to act non-specifically on the proton driven motor of R. sphaeroides, reducing the swimming speed of this organism in media with and without sodium to the same extent and over the complete pH range. Measurement of the delta p by using the electrochromic absorbance change of the carotenoid pigments to measure delta psi and 31P-NMR to measure delta pH showed that the maximum delta p was about -215 mV. At pH 10 the cells swam more slowly and the delta p was about -90 mV. These data suggest that the flagellar motor of R. sphaeroides is proton-driven under all conditions with a threshold for motor rotation below -90 mV and saturation at above -90 mV and below -215 mV.

Response kinetics of tethered bacteria to stepwise changes in nutrient concentration

We examined the changes in swimming behaviour of the bacterium Rhodobacter sphaeroides in response to stepwise changes in a nutrient (propionate), following the pre-stimulus motion, the initial response and the adaptation to the sustained concentration of the chemical. This was carried out by tethering motile cells by their flagella to glass slides and following the rotational behaviour of their cell bodies in response to the nutrient change. Computerised motion analysis was used to analyse the behaviour. Distributions of run and stop times were obtained from rotation data for tethered cells. Exponential and Weibull fits for these distributions, and variability in individual responses are discussed. In terms of parameters derived from the run and stop time distributions, we compare the responses to stepwise changes in the nutrient concentration and the long-term behaviour of 84 cells under 12 propionate concentration levels from 1 nM to 25 mM. We discuss traditional assumptions for the random walk approximation to bacterial swimming and compare them with the observed R. sphaeroides motile behaviour.