Preámbulo

The suitability of the selected five NTS as outlined in section 3.3. as an export and soluble partner in the fusion design was tested. To this aim, all five His-tagged candidates (AmiA, HyaA, PaoA, TorA and YcbK) were expressed in W3110 WT, MC4100 WT and MC4100 Tat-null strains in LB at 30°C induced for 2 h at 30°C (Table 2-3, condition 1). The cells were fractionated according to the PureFrac method (section 4.4, Appendix 4) and the expression and export profiles analysed by SDS-PAGE and Western blot. As shown in Figure 5-1A, the expression of the 5 NTS was measured through various degrees of success. YcbK was not expressed even though the plasmid sequence was confirmed before and after the experiment to rule out nucleic acid mutations. The growth profile of YcbK was similar to those of the other NTS in the WT strains with respect to that during the induction phase (Appendix 5), a lower OD600 was reached compared to the control strain (empty vector). This suggests that YcbK was

expressed but bearing in mind the results on analysis, was instable in the cytoplasm and so was degraded. YcbK is currently of unknown function, is not in an operon structure with other genes which could highlight its role or the necessity of partners or chaperones essential for expression, folding and stability (section 3.3.). Computational analysis using Virtual Footprint of the region upstream of the open reading frame of ycbK within the chromosome revealed a conserved -10 and -35 promoter region as well as RpoD, CRP, ArgR and Sigma70 binding sites, which strongly support that this gene is at least transcribed (Munch et al., 2005, Solovyev, 2011). The absence of YcbK expression in this study could be due to several reasons: (i) requirement of unknown chaperones and/or partners, (ii) requirement of an unknown metal ion which was not found in the supplied LB, (iii) instability brought by the aerobic conditions as some Tat substrates are involved in anaerobic growth. Furthermore, AmiA, HyaA and PaoA were expressed but most of the proteins were detected in the insoluble fractions demonstrating their instability irrespective of the strain. TorA was the only candidate to express in a significant amount and showed partial stability as demonstrated by the intensity of the detected bands in the soluble fractions (Figure 5-1A).

Chapter 5 - NTS

77 Regarding export, the three highly unstable candidates AmiA, HyaA and PaoA showed no sign of crossing the inner membrane. AmiA has been overexpressed previously but no evidence of the subcellular localisation have been sought using isolated fractions (Ize et al., 2003, Bernhardt and De Boer, 2003). HyaA is known to form a dimer with HyaB and this complex is believed to be Tat exported using the signal peptide of HyaA (Rodrigue et al., 1999), although no direct evidence have been published. Moreover, evidence of the chaperone HyaE binding the HyaA signal peptide have been highlighted by Dubini and Sargent (2003). However, no evidence of HyaE being required for HyaA export has been presented. It is plausible that the chaperone HyaE and/or the partner HyaB may need to be co-expressed in order for the complex to be exported by the Tat mechanism. In relation To PaoA, a more complex story has emerged from other authors. A cytoplasmic variant of PaoA has been expressed in the cytoplasm where it was found to interact with the PaoB and PaoC partners to form the PaoABC complex (section 3.3.) (Neumann et al., 2009, Correia et al., 2016). Lee et al. (2014) confirmed the formation of this complex but also proved that both partners are required for Tat export of the complex as “hitchhikers” thanks to the signal peptide of PaoA. The team also demonstrated the implication of the PaoD chaperone in improving the complex stability and its requirement to gain activity. In this case, PaoA would require co-expression of the partners PaoB and PaoC as well as potentially, the chaperone PaoD for Tat export. Due to the success of export reported by Lee et al. (2014), this highlighted that the PaoA experiment needed further optimization which was performed and reported in section 5.2.

The only successful candidate, with regards to export in the first experiment, was TorA, as a significant amount of proteins was detected in the periplasmic fraction of the WT strains. Conversely, no proteins were detected in the periplasm of the Tat-null strain (Figure 5-1A). Since the control markers LacI and MBP were both detected in their respective fractions, this confirms TorA was specifically exported by Tat and supports previous published data (Weiner et al., 1998, Jack et al., 2004). TorA, even without its chaperone TorD, is known to fold to a near native conformation although at a slower rate (Pommier et al., 1998, Ilbert et al., 2003).

Chapter 5 - NTS

78 The fractionation controls represented in Figure 5-1B indicate the purity of the isolated fractions, as confirmed by the absence of LacI and MBP contaminating proteins in the respective periplasmic and cytoplasmic fractions. Overall, only TorA was exported by Tat without any further optimization. However, the DNA sequence coding for this protein rendered the plasmid unstable which made TorA technically impractical. Therefore, none of the five selected candidates were fit to use as soluble partner as such and would require optimization.