The CadC proline cluster and its role for EF-P dependency

CadC belongs to the family of ToxR-like transcriptional activators (Watson et al., 1992). These proteins are characterized by a conserved modular composition consisting of a N- terminal cytoplasmic winged helix-turn-helix DNA-binding motif followed by a single transmembrane domain and a C-terminal periplasmic sensing domain (Egan et al., 2002, Hase & Mekalanos, 1998; Merrell & Camilli, 2000; Miller et al., 1987) (Fig. 17A). A cytoplasmic putative unstructured region between aa 104 and aa 158 interconnects the input and output domains. As shown before, impaired translation elongation of CadC in efp mutant strains is restricted to this part (Fig. 16B). This raises the question for a specific recognition sequence. Interestingly, a cluster of four prolines (Pro120-Pro121-Pro122-Ile123-Pro124) was found in this region (Fig. 17B). Three of the prolines are consecutive, the last one is separated by an isoleucine. When the unstructured region of E. coli and four additional species of the

Enterobacteriaceae were blasted, the proline cluster could be observed in all of these species (Fig. 17B).

Of the 20 canonical amino acids, proline is very special in that it has a secondary α-amino group. Accordingly, proline is a particularly poor acceptor during peptide-bond formation (Muto & Ito, 2008; Pavlov et al., 2009). Since EF-P has been shown to stimulate peptide- bond formation in vitro (Glick et al., 1979; Glick & Ganoza, 1975), it was hypothesized that EF-P might support translation by overcoming the detrimental effect of the consecutive prolines and releasing ribosomal stalling. To test this hypothesis, two in vivo approaches were used:

60 Figure 17: The proline cluster in the unstructured loop region of CadC.

A) CadC domains. CadC consists of a N-terminal helix-turn-helix (HTH) motif for DNA binding and a periplasmic domain for pH sensing. Domain models were based on the crystal structures of the periplasmic domain of E. coli CadC (PDB code: 3LY7) (Eichinger et al., 2011) and of the DNA-binding domain of E. coli PhoB (PDB code: 1QQI) (Blanco et al., 2002), which shows high similarity to CadC. TM (grey): transmembrane domain. Dark red: unstructured loop region.

B) Amino acid alignment of the unstructured region of CadC in some selected enterobacteria (E. coli, Escherichia fergusonii, Klebsiella pneumonia, Edwardsiella tarda and Salmonella enterica). Sequences were compared using Clustal Omega (Sievers et al., 2011). Proline clusters are marked by black boxes.

Firstly, each single proline of full length CadC was exchanged in plasmid p5C (pBBR-MCS5 PcadC-cadC) by alanine resulting in plasmids p5C-P120A, p5C-P121A, p5C-P122A and p5C-

124A. Additionally, the complete proline cluster was substituted in plasmid p5C-P/A. Strain JW4107 (∆efp) harboring plasmids p5C-P120A, p5C-P121A, p5C-P122A and p5C-124A, respectively, and the BW25113 wildtype were cultivated in LB medium and harvested at an OD600 of 1. After purification of the membrane vesicles, 150 µg were loaded onto a SDS gel and the CadC amount was compared. Strikingly, CadC with P/A substitutions expressed in the ∆efp mutant reached nearly fully (p5C-P/A) or partially (p5C-P120A, p5C-P121A, p5C- P122A) wildtype like protein amounts (Fig. 18A) confirming that the three consecutive prolines are crucial for EF-P dependency. By contrast, mutants harboring plasmid p5C-P124A

61 remained EF-P dependent. This result indicates that three prolines in a row are sufficient for the EF-P dependent ribosomal stalling.

Secondly, the CadC´-LacZ-translational fusion TL158 in plasmid p3LC-TL158 was employed to generate proline to alanine derivatives as well and to depict the translational rate as function of relative β-galactosidase activity. The β-galactosidase activities of the wildtype (efp+) and the mutant (efp-) were compared and indicated by an efp-/efp+ ratio. In accordance to Fig. 16B, the ∆efp mutant harboring plasmid p3LC -TL158 showed a highly decreased β- galactosidase activity (0.13) (Fig. 18B). By contrast, constructs p3LC-TL158-P121A and p3LC -TL158-P/A resulted in an EF-P independent phenotype with a ratio of 0.75 and 0.85, respectively. Comparable to the Western Blot results, mutants harboring p3LC-TL158-P120A (0.3) or p3LC-TL158-P122A (0.4) were partially EF-P independent, the mutant harboring p3LC-TL158-P124A remained fully EF-P dependent (0.13). These results confirm again the importance of three consecutive prolines for EF-P dependent translation. Furthermore, this hypothesis is supported by the fact that an interruption of consecution in p3CL-TL158-P121A led to a stronger EF-P independent phenotype compared to p3LC-TL158-P120A and p3LC- TL158-P122A (Fig. 18B).

To examine if the disruption of the proline cluster results not only in higher CadC amounts, but also in increased CadA activities in the ∆efp mutant, strain JW4095 (∆cadC) and JW4107 (∆efp) harboring plasmids p5C-P120A, p5C-P121A, p5C-P122A, p5C-124A and p5C-P/A were cultivated microaerobically in pH 5.8 buffered KE medium. Cells were harvested at their exponential growth phase and the β-galactosidase activities between efp+ and efp- cells were compared. Remarkably, plasmids p5C-P121A and p5C-P/A led to full complementation of the

∆efp phenotype and even to higher CadA activities in wildtype cells containing plasmid p5C.

∆efp cells harboring plasmids p5C-P120A and p5C-P122A achieved only 21% and 47% of the wildtype β-galactosidase activities, respectively (Fig. 18C). As expected, wildtype CadC (p5C) and the P124A exchange failed to show any remarkable CadA activity in the ∆efp

62 Figure 18: Disruption of the CadC proline cluster leads to EF-P independency.

A) CadC protein levels in various mutants. The protein levels of full-length CadC were determined by quantitative Western blotting using CadC-specific antibodies. A cadC deletion mutant was used as negative control, and wildtype CadC in the efp+ strain was assigned to 100%. The CadC signal intensity was analyzed by using the program ImageJ.

B) β-Galactosidase activities in wildtype BW25113 (efp+) and JW4107 (efp-) cells harboring translational CadC´-LacZ fusions (TL158) and the corresponding proline/alanine CadC substitutes.

C) The CadA phenotype in in wildtype BW25113 (efp+) and JW4107 (efp-) cells harboring p5C (full-length cadC) and the corresponding proline/alanine CadC substitutes.