Beyond the stress response: absence of RelA protein hampers the cell wall structuring and cell size in Liquorilactobacillus vini

The role of (p)ppGpp alarmone produced by RelA protein is well documented as a response to stress and nutritional deficiency in bacteria. However, little information is reported for Lactobacillus sensu lato, including the remaining species of old genus Lactobacillus and the derived species recently allocated in new genera. The present study aimed to characterize for the first time the effect of the inactivation of relA in a representative of the Liquorilactobacillus genus. Results obtained have revealed an unexpected role of RelA protein on the cell wall homeostasis and cell size. L.vini ΔrelA showed low growth and increased cell size, related to overexpression of genes responsible for DNA transcription and repair and down-expression of the fusA gene. The low growth also resulted in the loss of the flagellum in Δ relA , which may also be associated with the fragility of the cell wall, ease of lysis and resistance to beta-lactams of the Δ relA mutant. Cell wall homeostasis was deregulated mainly by the down-expression of the gene encoding alanine ligase and murB , which may have triggered the overexpression of the genes encoding PBPs proteins. Growth data suggest that the absence of the RelA protein promoted a disconnection between the cell cycle and cell division in L. vini , and a consequent reduction in the growth rate of the culture, cell wall fragility and beta-lactam resistance, expanding the RelA function in Liquorilactobacillus beyond the stringent response.