Mycoplasma genitalium is an emerging sexually transmitted pathogen. This wall-less microorganism is among the smallest, self-replicating cell known. Its streamlined genome is an appealing model of a minimal cell. Behind this apparent simplicity, its cell membrane hides a complex cytoskeleton that shapes and polarizes the cell. In this way, cells show a differentiated tip structure, known as terminal organelle (TO), which is involved in key processes of its parasitic way of life. Moreover, TO is involved in gliding motility. This unique motility mechanism is related in many aspects of the biology of this microorganism, with especial relevance in pathogenesis. Nevertheless, the mechanics behind it is still poorly characterized. The general aim of the present work is to deepen the understanding of this mechanism by studying the specific contribution of different domains of proteins previously related with the TO or motility. TO is organized around an internal cytoskeletal structure. MG218 protein is one of the main proteins of the cytoskeleton and has a central role in the TO formation. Recently, a novel protein that shares the C-terminal sequence of MG218 has been identified. This new protein (MG218-s) is translated from a specific mRNA transcribed from a promoter located inside mg218 gene but the precise translation start remains undefined. To gain insight into the function of this protein, in the first chapter we have addressed the construction of a MG218-s null strain still expressing the MG218 full-length protein. In order to obtain this mutant, a new strategy to introduce point mutations in the M. genitalium genome has been designed. This strategy allowed the identification of the starting Met of the MG218-s protein and the generation of an MG218-s null mutant that helped to understand the role of this new protein. The second and third chapters are focused on the study of the MG200 protein, which is an important element of the motility machinery. MG200 and MG386 protein are the first elements to be related exclusively to motility in M. genitalium. These proteins share common features, being remarkable the presence of a well conserved Enriched in Aromatic and Glycine Residues domain (EAGR box). This domain is exclusively found in mycoplasmas, apparently always in proteins related with motility and/or with the terminal organelle architecture, as MG312. The analysis of the crystal structure of the MG200 EAGR box supported a role in protein-protein interactions, indicating that it can be a platform for interactions with other macromolecules. However, no apparent structural defects in the TO architecture have been shown by mutant cells bearing deletions in proteins containing EAGR boxes. To further understand the role of this domain, the second chapter analyzes the phenotype of mutants that express the MG200 protein bearing a deletion of the EAGR box domain. The observations made suggest that the EAGR boxes would play a relevant and specific role in motility. Interestingly, MG200 is a multi-domain protein that has homology to the Hsp40 co-chaperones. Transposon disruption of MG_200 locus led to adherent strains with a non-motile phenotype. A recent analysis of these mutants has evidenced the presence of an N-terminus MG200 fragment, which contains a J-domain and a glycine and phenylalanine rich region (G/F) characteristic of DnaJ proteins. In the third chapter, we have studied the contribution of the amino-terminal domains of MG200 in its general function.