While toxins are described to be the major virulence factors in C. difficile infections, there is an increasing interest in the role of non-toxin factors in pathogenesis and virulence. In this study, we investigated the role of the two lcp genes, supposed to be non-redundant and known to be involved in the synthesis of the polysaccharide II by anchoring filaments to the peptidoglycan [1]. In this study, we constructed single and double mutants of lcp genes. Surprisingly, we were unsuccessful to obtain the double mutant whereas the two single mutants were easily obtained by an allelic exchange technique. This suggests that lcp genes have indeed redundant functions. To delete both lcp genes, we developed a conditional lethal mutant technique. The first step was to construct a strain containing a second copy of lcpB in a chosen region of the chromosome, expressed under the control of an anhydrotetracycline inducible promotor, pTet. Then, we replaced the ORFs by a resistance cassette in the native locus and so deleted both lcpA and lcpB. In this conditional mutant, we were able to modulate the expression of lcpB. Thanks to this tool and the production of highly specific anti-polysaccharide II antibodies, we highlighted the essentiality of the polysaccharide II anchoring. Using immunofluorescence microscopy, we showed that in single mutants, the polysaccharide II layer is abnormal in comparison to the wild-type strain. It is discontinued and holed in the lcpA mutant but smooth and inhomogeneous in the lcpB mutant. In the double mutant with low lcpB expression, we observed ellipsoid cells. Complementation with lcpA or lcpB restores the rod-shape morphology and the normal abundance of polysaccharide II. Additional results show a defect of the S-layer anchoring in the conditional mutant strain. In conclusion, our results show the critical role of polysaccharide II anchoring in growth, elongation, and correct surface set-up of C. difficile. Our technique provides new opportunities to study essential genes in C. difficile. [1] M. Chu et al., PLoS Pathog., vol. 12, no 10, oct. 2016.