Comparison with data from other organisms

The interactions detected in this study were compared to interactions between the Che proteins from other organisms. Of particular interest for comparison was the mammalian-two hybrid dataset from Pyrococcus horikoshii (Usui et al., 2005),

be-cause this is the only dataset from an archaeal organism. The Che proteins tested in this study were CheB, CheC1, CheC2, CheD, CheR, CheW, CheY, DUF439 (see chap-ter 7), and one MCP. Unfortunately, the only inchap-teraction found with these proteins

Table 6.5:Physical and functional interactions between Che proteins described in litera-ture.

Interaction Species

CheW-MCP HP¹, EC^2,3, TD⁴, CJ³⁹

CheR-MCP ST⁵

CheV-MCP CJ³⁹

CheA-CheW HP¹, EC^2,6,3, TD⁴, CJ³⁹

CheA-CheB EC⁷

CheA-CheX TD⁴

CheA-CheZ EC^8,9

CheA-CheY HP¹, EC10,11,12,13,3,7, TM¹⁴, TD⁴, CJ³⁹

CheC-CheD PH¹⁵, TM¹⁶, BS¹⁷

CheC-CheA BS¹⁸

CheC-MCP BS¹⁸

CheD-MCP BS¹⁸, TM¹⁶

CheY-CheZ EC^19,20,21

CheY-FliM EC^22,23

CheR methylates MCP EC²⁴, BS²⁵, HS³⁸ CheB demethylates MCP EC²⁴, BS²⁶, HS³⁸ CheB deamidates MCP EC²⁷, HS^28,38 CheD deamidates MCP BS²⁹, TM¹⁶ CheD demethylates MCP TM¹⁶

CheA phosphorylates CheY EC³⁰, BS³¹, HS³² CheA phosphorylates CheB EC^33,30, BS³⁴, HS(hom) CheA phosphorylates CheV BS³⁴

CheX dephosphorylates CheY BB³⁵ CheZ dephosphorylates CheY EC³⁶

CheC dephosphorylates CheY BS³⁷, HS(hom) FliY dephosphorylated CheY BS³⁷

HP:H. pylori, EC:E. coli, TD:T. denticola, ST:S. typhimurium, BS:B. subtilis, HSH. salinarum, BB:

B. burgdorferi. References: ¹Rainet al.(2001),²Boukhvalovaet al.(2002),³ Schusteret al.(1993),

4Sim et al. (2005), ⁵Djordjevic and Stock (1998), ⁶ Gegner and Dahlquist (1991), ⁷Yamamoto et al.(2005),⁸Wang and Matsumura(1996),⁹ Kottet al.(2004),¹⁰Shukla and Matsumura(1995),

11McEvoyet al. (1998),¹² Welch et al.(1998),¹³Gouetet al.(2001),¹⁴Parket al.(2004),¹⁵ Usui et al.(2005),¹⁶Chaoet al.(2006),¹⁷Rosario and Ordal(1996),¹⁸ Kirbyet al.(2001),¹⁹Zhaoet al.

(2002),²⁰Sourjik and Berg(2004),²¹ Blat and Eisenbach(1996),²²Sourjik and Berg(2002a),²³Lee et al. (2001), ²⁴ Sherris and Parkinson (1981), ²⁵Kirsch et al. (1993b), ²⁶Kirsch et al. (1993a), ²⁷ Kehry et al. (1983), ²⁸Koch (2005), ²⁹Kristich and Ordal (2002),³⁰ Hesset al. (1988), ³¹Bischoff et al. (1993),³²Rudolph et al. (1995), ³³ Stewartet al. (1990),³⁴Karatan et al.(2001),³⁵Motaleb et al. (2005),³⁶ Silversmithet al.(2003),³⁷Szurmantet al.(2004),³⁸Kochet al.(2008),³⁹Parrish et al.(2007). HS(hom) means that this reaction was not experimentally verified inH. salinarum, but concluded to occur by homology.

was CheC1-CheD, so that a detailed comparison with the H. salinarum dataset is not possible. Generally, the interaction detection in that study was rather low; the mam-malian two-hybrid system might not be the optimal method to analyse interactions of proteins from a hyperthermophilic archaeon.

In the two large-scale studies carried out in E. coli by Butland et al. (2005) and Arifuzzamanet al.(2006), only interactions between CheW and two MCPs were iden-tified, and several proteins not expected to be related to chemotaxis fished with some Che proteins. There was, however, no overlap with the unexpected proteins fished in the present study.

Another large-scale dataset was produced in H. pylori by Y2H analysis. This or-ganism has a rather simple chemotaxis system, with only four MCPs, CheA, CheW, CheY, and three CheVs (Tomb et al., 1997; O’Toole et al., 2000). In this study the interactions MCP-CheW-CheA-CheY were detected, as well as interactions with some unexpected proteins (no overlap to E. coli and H. salinarum unexpected proteins).

Further large-scale Y2H datasets exist forCampylobacter jejuni (Parrish et al., 2007) and Treponema pallidum (Rajagopalaet al., 2007). In C. jejuni, interactions between MCPs-CheV and MCPs-CheW-CheA-CheY were detected. Although this organism codes for CheR and CheB (Parkhill et al., 2000), no interactions for these proteins were reported. CheC and CheD are not present in C. jejuni. The T. pallidum dataset does not contain interactions between Che proteins.

Since large-scale studies did not deliver adequate data for comparison, the STRING (von Mering et al., 2007) and BIND (Alfarano et al., 2005) databases were queried for respective data from smaller studies carried out in any prokaryotic organism, and literature searching was done. Additionally, functional interactions (i. e. enzymatic reactions) between the Che and related proteins were collected from PubMed. Result-ing data (Table 6.5) was used to draw a general Che protein interaction network (Fig-ure 6.8). Most of the reported interactions were found in E. coli and the spirochaete T. denticola, only four physical interactions were reported for B. subtilis. This weak-ens the comparison with the H. salinarum network, which is with regard to the used proteins more closely related to the one from B. subtilis. For example, neither E. coli nor T. denticola code for a CheC or CheD protein.

The interactions of the core are generally in agreement between H. salinarum and other organism’s data. The H. salinarum dataset contains probably indirect interac-tions (CheY-CheW, CheY-MCP) because it was generated by AP-MS. In the

data-bases, no direct interaction between CheA and MCPs is deposited. In literature, it is reported that the CheA-MCP association generally depends on CheW (Gegneret al., 1992). A direct (weak) CheA-MCP interaction was suggested for inhibitory signalling (Ames and Parkinson, 1994) in E. coli. A direct interaction with CheA, however, is likely to occur in H. salinarum, because some transducers were fished with CheA, but not with either of the CheWs (see 6.2.2.3).

Interactions of CheR and CheB with Htrs could not be demonstrated inH. salinarum (except CheR-Htr12). In the databases, there is only one reference for a physical inter-action between CheR and a MCP, based on a crystal structure after co-crystallisation.

That means that these interactions seem to be hard to detect with PPI analysis meth-ods.

CheD plays an important role in theH. salinarum interaction network: it was found to interact with CheC2, CheC3, CheB, and OE2401F, OE2402F, and OE2404R (see chapter 7). Of these, only the interaction with CheC has been described before. In-stead of this, inB. subtilisan interaction of CheD with the MCPs was identified by Y2H analysis (Kirby et al., 2001). Such an interaction was not detected in H. salinarum.

This might be due to different functions of CheD in both organisms (see subsubsec-tion 6.1.1.3).

The interaction between CheC and CheD was earlier demonstrated inB. subtilis and interpreted as feedback loop to the transducers via CheD’s deamidase activity, which is decreased by CheC binding (Muff and Ordal, 2007). Furthermore, the interaction with CheD increased the CheY-P dephosphorylation activity of CheC 5-fold (Szurmant et al., 2004). CheY-P stabilises the CheC-CheD complex, thus closing the feedback circuit. The role of the CheC-CheD interaction in H. salinarum remains unclear. An effect on the activity of CheC is possible, whereas a feedback loop to the transducers in unlikely due to the lack of receptor deamidase activity of CheD.

Such a feedback loop could be formed by the CheD-CheB and CheC1-CheB in-teractions detected in this study. CheC1-CheB-CheD in H. salinarum might thus be analogue to CheD-CheC in B. subtilis.

In this study, an interaction between CheC2 and the proteins OE2402F and OE2404R was detected. These proteins were found to interact both with Che proteins (CheY, CheD, CheC2) and with the flagella-accessory proteins FlaCE and FlaD (these pro-teins are discussed in detail in chapter 7). Hence they might be constituents of or be associated with the archaeal flagellar motor switch. Provided that this hypothesis

holds true, the interaction with CheC2 might reflect a situation similar to B. subtilis:

In this organism, FliY, the main CheY-P phosphatase, is localised at the flagellar motor switch, and CheC, the second CheY-P phosphatase, at the signalling complex (Szurmant et al., 2004). A direct or indirect interaction of one of the CheCs with the signalling complex was, however, not identified in H. salinarum. Generally, phos-phatase localisation turned out to be a conserved and important principle in bacterial chemotaxis systems (Rao et al., 2005).