Protocolo de Participación Efectiva de las Víctimas del conflicto armado

Given the lack of data, the current models of the flagellar motor and its sensory control implement adaptation in a very simplified form that only considers methylation by CheR and demethylation by CheB. In the model, CheR methylates transducers at a constant rate and CheB-P demethylates transducers at a rate that is proportional to the CheB- P concentration. However, the data presented here clearly show that CheD, CheC1 and CheC3 are indeed involved in chemo and phototaxis ofH. salinarum and more specifically in adaptation as suggested by their homologies to the B. subtilis proteins. In order to include these proteins in the model, further data are required that either directly or indirectly point to their function.

Using the MpcT antibody, Western Blots of the membrane fractions of theche mutants could yield a first indication of the transducer methylation states in these mutants. In B. subtilis such analyses revealed large deviations in the methylation states of the trans- ducers from cheD and cheC mutants (Rosario et al., 1995; Kirby et al., 2001; Kristich and Ordal, 2002). It would be interesting to compare the effects of the∆cheC1, ∆cheC3 mutation and the∆cheC2 mutation that was recently generated by Elisabeth Weidinger (2007). Mass spectrometric analysis of transducer methylation states in mutants has proved to yield a deeper insight into the functions of H. salinarum CheR and especially CheB. So far, the data point to CheB as the transducer Gln deamidase inH. salinarum, but the analysis has not been done with the ∆cheD mutant and the cytosolic fraction containing the soluble Htrs was excluded from the analysis.

homologs would be in vitro phosphorylation assays. In these assays CheY is phospho- rylated with 32P and the loss of the label over time is followed in autoradiograhps. A comparison of the kinetics of label loss in absence and presence of the putative phos- phatases allows to conclude phosphatase activity. In addition, the phosphatase activities of the CheC homologs could be assayed in presence of CheD to see if CheD activates phosphatase activities of the CheC homologs. Such an approach was successfully applied with the Thermotoga maritima proteins that were heterologously expressed in E. coli (Chao et al., 2006). The thermophilic nature of these proteins facilitates their separation from the mesophilic E. coli proteins. However, in case of H. salinarum the situation is different. Heterologous expression of functional halophilic proteins in E. coli is not trivial. CheA and CheY had to be purified from inclusion bodies followed by refolding. Another even more serious problem is that the half-life of H. salinarum CheY-P seems to be extraordinarily short in the first place. Dephosphorylation of CheA by CheY could only be shown by loss of the label on CheA, the phosphorylated CheY species was not seen on autoradiographs (Rudolph et al., 1995). Given that the CheY preparation was not contaminated with one of the CheC homologs, this means that it will not be possible to study dephosphorylation the classical way and at the same time raises the question whether a CheY-P phosphatase is required in H. salinarum at all.

In this study, the effects of specific single deletions of chemotaxis genes were investi- gated. Surprisingly, in some of the mutants phototaxis and chemotaxis were affected to different degrees e.g. deletion of cheC3 had severe effects on phototaxis but compara- bly mild effects on chemotaxis. This observation suggests that alternative Htr-mediated signaling pathways exist. In the case of phototaxis, it was possible to link the effects to the receptors/transducers SRI/HtrI and SRII/HtrII, respectively by investigating the effect of blue light and orange light stimuli. By contrast, the swarm plate assay that was used to study chemotaxis in general does not allow such differentiations. To address the question whether the deletions have differential effects on different Htrs involved in chemotaxis, assays have to be applied that allow the specific stimulation of these Htrs and at the same time allow the analysis of the cells´ response. The analysis is restricted to those Htrs with assigned function (see Fig. 2.13). In the capillary assay chemotactic efficiency is determined by counting the number of cells that swim into capillaries filled with specific chemicals. The tethered cell assay is a microscopic assay in which the cells are tethered via their flagella to the coverslip of a laminar-flow chamber. Depending on the rotational mode of the flagellar motor the cell bodies rotate either CW or CCW. The response of the cells is analyzed by inspection of changes of the rotational direction caused by a laminar flow of chemicals that passes the cells. One advantage of the assay is that it allows a dissection of excitation and adaptation efficiency. In addition, the response to attractant stimuli e.g. by addition of attractant and repellent stimuli e.g. by subsequent removal of the attractant can be analyzed in one setup.

For B. subtilis, the existence of a methylation-independent adaptation system that works at low stimulus concentrations was proposed (Kirsch et al., 1993a; Zimmer et al., 2002). In order to study if a methylation-independent adaptation system exists inH. sali- narum, the response of the che mutants to a variety of stimulus strengths has to be analyzed. It may be that some of the H. salinarum che mutants are able to respond

be non-methylatable in a strain devoid of these transducers and still observed methanol release upon their stimulation. Our studies on transducer methylation sites revealed that in the HtrII variant one methylation site was left that was not mutated, which would explain the methanol release. However, our data indicate that in case of the HtrI variant, all methylation sites were eliminated and thus methanol release must origin from other transducers.

The elucidation of the function of theR. sphaeroides PpfA protein may allow to predict the function ofH. salinarum ParA1. The phenotype of theH. salinarum ∆parA1 mutant and the location ofparA1 near theche gene cluster indicates that ParA1 like PpfA may be involved in targeting of soluble transducers to cytoplasmic clusters. Two aspects have to be studied to prove this hypothesis. First of all, the existence of cytoplasmic transducer clusters in H. salinarum has to be shown. In case cytoplasmic transducer clusters exist, the growth-stage-dependent localization and number of these clusters in wild type and the parA1 mutant has to be compared. One possibility is to generate fusions of soluble Htrs e.g. of Car with GFP and study the localization of these Htrs with fluorescence microscopy. As mentioned in section 3.1 this technique is not yet established for H. salinarum mostly due to inhibition of GFP fluorescence under halophilic growth conditions. However, in the meantime a salt-tolerant GFP variant was used in another halophilic organism, Haloferax volcanii (Reuter and Maupin-Furlow, 2004). This GFP variant may also work in H. salinarum. Alternative methods are immunogold labeling, immunofluorescence labeling or cryo EM. Unfortunately, all of these methods require at some stage that the cells are treated with low salt buffer, which usually destroys the cells. The second aspect is to study more specifically and in more detail the influence of the parA1 deletion on cytoplasmic versus TM transducers. This can be done with the aforementioned capillary and tethered cell assays.

In this study, the CW biases of the mutants were determined by counting the number of cells that were found with their flagellar bundle rotating in either direction. This allows a conclusion about the relative durations of CW and CCW swimming phases, but it does not tell anything about the absolute durations of CW and CCW swimming phases. Deviations from the wild type 50% CW ratio as such are already an interesting phenomenon, but the study of absolute distributions of CW and CCW swimming phases could reveal further deviations from wild type behavior even in the∆cheC1mutant that

showed a 50% CW bias. A B. subtilis cheB mutant has a close to wild type CW bias. However, analysis of the durations of CW and CCW swimming intervals revealed that the wild type bias is due to an almost equivalent decrease of both CW and CCW interval durations (Saulmon et al., 2004). Analysis of absolute CW and CCW durations requires the analysis of the behavior of single cells which could either be done by tethering cells or by single cell tracking. As mentioned in section 3.4.7 tethering of H. salinarum cells led to misinterpretations of the rotational bias. Therefore, single cell tracking seems to be the more suitable method.

Another interesting question is what a decreased percentage of responding cells exactly means. It could either mean that a certain percentage of cells never responds while the other cells respond or it could mean that in all cells the signaling efficiency is decreased. In B. subtilis it was found in at least two cases that in certain mutants subpopulations with distinct characteristics exist (Kirsch et al., 1993b; Kirby et al., 2001). To study such phenomena single cells have to be studied either in tethered cell assays or by single cell tracking.

CSPD Roche Diagnostics, Mannheim Germany

DIG-11-dUTP Roche Diagnostics, Mannheim Germany

DNA ladder 1 kb Invitrogen, Karlsruhe, Germany

DNA ladder 100 bp Peqlab, Erlangen, Germany

Neutralized bacteriological peptone L34 Oxoid, Basingstoke, Hampshire, UK Paraffin wax white, pastilles, Riedel de Haen, Seelze, Germany congealing point 50 - 61°C

PEG600 Sigma Aldrich, St. Louis, USA

Tris Riedel de Haen, Seelze, Germany

Vaseline white Riedel de Haen, Seelze, Germany