Type VI secretion system (T6SS) is described as a macromolecular secretion machine that is utilized for bacterial competition. The gene clusters encoding T6SS are composed of core
tss
genes and
tag
genes. However, the clusters differ greatly in different pathogens due to the great changes accumulated during the long-term evolution. In this work, we identified a novel hypothetical periplasmic protein designated as AsaA which is encoded by the first gene of the T6SS cluster in the genus
Acinetobacter
. By constructing
asaA
mutant, we delineated its relative contributions to bacterial competition and secretion of T6SS effector Hcp. Subsequently, we studied the localization of AsaA and potential proteins that may have interactions with AsaA. Our results showed that AsaA in
Acinetobacter baumannii
(
A
.
baumannii
) localized in the bacterial periplasmic space. Results based on bacterial two-hybrid system and protein pull-down assays indicated that it was most likely to affect the assembly or stability of T6SS by interacting with the T6SS core protein TssM. Collectively, our findings of AsaA is most likely a key step in understanding of the T6SS functions in
A
.
baumannii
.
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