We recently showed thatLactobacillus sakei, a natural meat-borne lactic acid bacterium, can colonize thegastrointestinal tracts (GIT) of axenic mice but that this colonization in the intestinal environment selectsL.sakeimutants showing modified colony morphology (small and rough) and cell shape, most probably resultingfrom the accumulation of various mutations that confer a selective advantage for persistence in the GIT. In thepresent study, we analyzed such clones, issued from three differentL. sakeistrains, in order to determine whichfunctions were modified in the mutants. In the elongated filamentous cells of the rough clones, transmissionelectron microscopy (TEM) analysis showed a septation defect and dotted and slanted black bands, suggestingthe presence of a helical structure around the cells. Comparison of the cytoplasmic and cell wall/membraneproteomes of the meat isolateL. sakei23K and of one of its rough derivatives revealed a modified expressionfor 38 spots. The expression of six oxidoreductases, several stress proteins, and four ABC transporters wasstrongly reduced in the GIT-adapted strain, while the actin-like MreB protein responsible for cell shaping wasupregulated. In addition, the expression of several enzymes involved in carbohydrate metabolism was modified,which may correlate with the observation of modified growth of mutants on various carbon sources. Theseresults suggest that the modifications leading to a better adaptation to the GIT are pleiotropic and arecharacterized in a rough mutant by a different stress status, a cell wall modification, and modified use of energysources, leading to an improved fitness for the colonization of the GIT.