RelA and SpoT of Gram-negative organisms critically regulate cellular levels of (p)ppGpp. Here, we have dissected the spoT gene function of the cholera pathogen Vibrio cholerae by extensive genetic analysis. Unlike Escherichia coli, V. choleraeΔrelAΔspoT cells accumulated (p)ppGpp upon fatty acid or glucose starvation. The result strongly suggests RelA-SpoT-independent (p)ppGpp synthesis in V. cholerae. By repeated subculturing of a V. choleraeΔrelAΔspoT mutant, a suppressor strain with (p)ppGpp0 phenotype was isolated. Bioinformatics analysis of V. cholerae whole genome sequence allowed identification of a hypothetical gene (VC1224), which codes for a small protein (∼29 kDa) with a (p)ppGpp synthetase domain and the gene is highly conserved in vibrios; hence it has been named relV. Using E. coliΔrelA or ΔrelAΔspoT mutant we showed that relV indeed codes for a novel (p)ppGpp synthetase. Further analysis indicated that relV gene of the suppressor strain carries a point mutation at nucleotide position 676 of its coding region (ΔrelAΔspoT relV676), which seems to be responsible for the (p)ppGpp0 phenotype. Analysis of a V. choleraeΔrelAΔspoTΔrelV triple mutant confirmed that apart from canonical relA and spoT genes, relV is a novel gene in V. cholerae responsible for (p)ppGpp synthesis.