7.2 Results and Discussion
7.2.5 Interpretation of deletion phenotypes
time-consuming. The two S9∆1 clones were investigated because they showed a slightly different phenotype in the phototaxis measurements (smooth-swimming vs.
some residual switching).
The numbers of cells observed swimming in each direction are shown in Table 7.1.
Wildtype cells show a distribution between forward and backward swimming of close to 50:50, as expected (Marwan et al., 1991; Rudolph and Oesterhelt, 1996). Cells of the deletion strain ∆1,∆2, and the double deletion ∆2-4 show a bias toward forward swimming of almost 100%. Again, this corresponds to the phenotype of cheY and cheA deletion strains (Rudolph and Oesterhelt, 1996; del Rosario et al., 2007). The slight discrepancy of both S9∆1 clones found in the cell tracking assay also showed up in this experiment, proving the reliability of the applied methods. Cells lacking OE2404R exhibit a rotational distribution of nearly 50:50, similar to wildtype.
is present, but not effective. The first of these two possibilities seems less likely, because the PPI data suggest a role for OE2401F and OE2402F between CheY and the flagellum, and not upstream of CheY. Additionally, the homology of the archaeal Che system to the bacterial one argues against the first hypothesis: Current understanding is that the Che system ofH. salinarum, with the ten known Che proteins, is complete up to CheY-P. Only for the part downstream of CheY-P have no homologs to bacterial proteins been found. However, it would be possible that the deletion of OE2401F and OE2402F do not affect the level of CheY-P directly but via a yet unknown side mechanism. In B. subtilis, for example, cells deleted for CheD exhibit a very tumbly phenotype, similar to CheA mutants (Rosario and Ordal, 1996). In H. salinarum, however, the deletion of no other Che protein than CheA and CheY actually leads to cells with smooth-swimming phenotype (compare Table 6.1). In all other cases some residual switching and swarm ring formation is present (Staudinger,2007), supporting the idea that the defect of the OE2401F and OE2402F deletion strains is located between CheY-P and the flagellum.
Besides the two above mentioned deletions, a smooth-swimming phenotype has also been observed for the CheY∗∗ strain (Staudinger, 2007; del Rosario et al., 2007). In this strain, the CheY protein carries two point mutations: the aspartate in position 10 is replaced by lysine, and the tyrosine in position 100 by tryptophane. InE. coli, a similarly modified CheY mimics CheY-P. Hence E. coli cells expressing this mutated CheY protein exhibit a tumbly phenotype (Scharf et al., 1998). In H. salinarum, the in the first moment unexpected phenotype of the CheY∗∗ strain (expected was an increased switching frequency) was explained by introducing asymmetry in the motor switch model (del Rosarioet al.,2007; Staudinger,2007). Since CheY∗∗ causes a smooth-swimming phenotype, the results obtained with ∆1, ∆2, ∆2-4 could also be explained with (drastically) elevated CheY-P concentrations. This could be due to missing CheY-P phosphatase activity or hyperactivation of CheA. This hypothesis seems rather unlikely because even in the absence of a phosphatase the level of CheY-P should be limited by the short half-life of CheY-P, which is in the range of few seconds (Rudolph et al., 1995). The idea of hyperactivation of CheA is not supported by the obtained PPI data. This could only be explained by some feedback mechanism via the additional Che proteins, but then, again, remains the question why the deletion of these additional Che proteins results in a less severe phenotype. For the whole speculation based on the phenotype of CheY∗∗ it should be noted that the effect of
the CheY doublemutation was just deduced from the E. coli protein – in H. salinarum it cannot be ruled out that the mutated protein is just non-functional instead of constitutively active (Staudinger,2007).
A further possibility to explain the behaviour of ∆1, ∆2, ∆2-4 is an influence of the deleted proteins on the switch factor fumarate, which might act independently of the Che system (see 6.1.1.5 for details). The first evidence for fumarate as switch factor was found in a straight-swimming mutant which could be reverted to wild-type behaviour by introducing fumarate into the cells (Marwanet al.,1990), demonstrating that a defect in fumarate signalling can cause a phenotype similar to the one observed for ∆1, ∆2, ∆2-4. However, the detected protein interactions with CheY provide strong evidence that the proteins examined in the current study play a role in the action of CheY and not exclusively in fumarate switching.
The role of OE2404R remained unclear. The ∆4 strains were not distinguishable from wildtype strains in the phototaxis measurement and with respect to the flagellar rotational bias but produced significantly smaller swarm rings. Reduced swarm ring size is generally considered as outcome of diminished chemotaxis capability (given that the motility in itself is not affected). Several alternative hypotheses can be envisioned to explain the differences in the phototaxis measurements (no difference to wt) and swarming (reduced when compared to wt). First, OE2404R might only be involved in chemotaxis and not phototaxis signalling. Second, this protein might be required for fast and effective adaptation. Third, OE2404R might be required for fine tuning of the response. The first hypothesis seems rather unlikely since no other evidence exists that chemical and light signals utilise separate pathways in H. salinarum. The other possibilities are related to the phototaxis assay: This assay monitors the reaction after one strong and sudden change in light intensity, but does not report the adaptation efficiency or the reaction to more subtle stimuli. Further experiments should be done to test these three hypotheses. Dose-response curves for the phototactic behaviour would be a promising approach to discriminate between hypothesis one and three.
Monitoring the cellular response after repeated phototactic stimulation could be used to test hypothesis two.
Finally, it should be mentioned that it is not exactly known what determines the swarming capability ofH. salinarum cells. The widely accepted explanation for swarm ring formation is the formation of chemical gradients due to consumption of nutrients and excretion of metabolic end- or by-products by the cells at the site of inoculation.
Cells capable of chemotaxis and motility sense these gradients and bias their movement from the site of inoculation to the periphery. However, straight-swimming mutants do not form swarm rings at all, although straight movement in any direction is the fastest way to reach the periphery. Thus switching seems to be crucial for movement in semi-solid agar. Theoretically random switching should also lead to spreading of the cells, similar to diffusion driven by Brownian motion with its randomly occurring turnarounds after collisions. To what extent biasing the switching events in response to chemical concentrations increases the swarm ring size has not been investigated for H. salinarum. For E. coli, Wolfe and Berg (1989) demonstrated that nonchemotactic cells can form swarm rings if they rotate their flagella both CW and CCW. The cells spread more effectively when tumbles were more frequent. This behaviour was explained by the observation that cells that do not tumble tend to get trapped in the agar. If these findings can be transfered toH. salinarum cells, which do not tumble at all but swim forward and backward, remains to be investigated.
Overall, it can be said that OE2404R is involved in taxis signal transduction in H. salinarum, but it either fulfils a non-essential function or it can be replaced by its homolog, OE2402F, with only minor constraints.