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Background The Type VI secretion system (T6SS) has been identified in several different bacteria, including the plant pathogenPantoea ananatis. Previous in silico analyses described three different T6SS loci present in the pathogenic strain of P. ananatis LMG 20103. This initial investigation has been extended to include an additional seven sequenced strains of P. ananatis together with 39 strains from different ecological niches. Comparative and phylogenetic analyses were used to investigate the distribution, evolution, intra-strain variability and operon structure of the T6SS in the sequenced strains. Results Three different T6SS loci were identified in P. ananatis strain LMG 20103 and designated PA T6SS 1-3. PA T6SS-1 was present in all sequenced strains of P. ananatis and in all 39 additional strains examined in this study. In addition, PA T6SS-1 included all 13 core T6SS genes required for synthesis of a functional T6SS. The plasmid-borne PA T6SS-2 also included all 13 core T6SS genes but was restricted to only 33% (15/46) of the strains examined. In addition, PA T6SS-2 was restricted to strains of P. ananatis isolated from symptomatic plant material. This finding raises the possibility of an association between PA T6SS-2 and either pathogenicity or host specificity. The third cluster PA T6SS-3 was present in all strains analyzed in this study but lacked 11 of the 13 core T6SS genes suggesting it may not encoded a functional T6SS. Inter-strain variability was also associated with hcp and vgrG islands, which are associated with the T6SS and encode a variable number of proteins usually of unknown function. These proteins may play a role in the fitness of different strains in a variety of ecological niches or as candidate T6SS effectors. Phylogenetic analysis indicated that PA T6SS-1 and PA T6SS-2 are evolutionarily distinct. Conclusion Our analysis indicates that the three T6SSs of P. ananatis appear to have been independently acquired and may play different roles relating to pathogenicity, host range determination and/or niche adaptation. Future work will be directed toward understanding the roles that these T6SSs play in the biology of P. ananatis. [ABSTRACT FROM AUTHOR]