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It is reported that a wide range of bacterial infections are polymicrobial, and the members in a local microcommunity can influence the growth of neighbors through physical and chemical interactions.  Pseudomonas aeruginosa  is an important opportunistic pathogen that normally causes a variety of acute and chronic infections, and clinical evidences suggest that  P. aeruginosa  can be frequently coisolated with other pathogens from the patients with chronic infections. However, the interspecific interaction and the coexisting mechanism of  P. aeruginosa  with coinfecting bacterial species during evolution still remain largely unclear. In this study, the relationships of  P. aeruginosa  with other Gram-positive ( Staphylococcus aureus ) and Gram-negative ( Klebsiella pneumoniae ) are investigated by using a series of on-plate proximity assay,  in vitro  coevolution assay, and RNA-sequencing. We find that although the development of a quorum-sensing system contributes  P. aeruginosa  a significant growth advantage to compete with  S. aureus  and  K. pneumoniae , the quorum-sensing regulation of  P. aeruginosa  will be decreased during evolution and thus provides a basis for the formation of interspecific coexistence. The results of comparative transcriptomic analyses suggest that the persistent survival of  S. aureus  in the microcommunity has no significant effect on the intracellular transcriptional pattern of  P. aeruginosa , while a more detailed competition happens between  P. aeruginosa  and  K. pneumoniae . Specifically, the population of  P. aeruginosa  with decreased quorum-sensing regulation can still restrict the proportion increase of  K. pneumoniae  by enhancing the type VI secretion system-elicited cell aggressivity during further coevolution. These findings provide a general explanation for the formation of a dynamic stable microcommunity consisting of more than two bacterial species, and may contribute to the development of population biology and clinical therapy.

Pseudomonas aeruginosa Quorum-Sensing and Type VI Secretion System Can Direct Interspecific Coexistence During Evolution