Abstract: Tubastraea is a representative hexacoral in mesophotic coral reefs. Although it lacks photosynthetic symbiotic algae, studies have shown that its symbiotic microbial community plays an important role in helping corals adapt to environmental changes. Coral symbiotic microorganisms mostly colonize the interior of coral tissues or mucus in the form of biofilms. This attached growth mode facilitates the generation of new microbial mutations and the formation of phenotypic diversity. However, the specific link between the occurrence of mutations within biofilms and the adaptation of coral microorganisms to specific environments remains largely unexplored. This study conducted functional research on adaptive mutations screened from the biofilm of Alteromonas macleodii isolated from Tubastraea corals in the mesophotic habitat of the Xisha Islands. Three of the mutations occurred in genes with known functions, related to c-di-GMP synthesis, polysaccharide synthesis, and cell wall synthesis, respectively, all of which affected biofilm formation. Another mutation was located in a gene of unknown function containing a DUF3450 domain, which is proposed to name as bfmS; this deletion mutation resulted in the truncation of the second α-helix of the BfmS protein. This mutation significantly enhanced the biofilm-forming capacity of the strain while reducing its motility, thereby promoting bacterial attachment and colonization within coral tissues. Additionally, BfmS exhibits a clear co-distribution pattern with the TonB system across multiple microbial genera, suggesting its potential involvement in the transmembrane transport of substances such as iron ions. Notably, mutations in this gene also conferred resistance to lytic phage infection in the strain. These results indicate that a single mutation generated within the biofilm can provide the strain with multiple survival advantages, thereby collectively enhancing the environmental tolerance and stability of the coral symbiotic system.

Key words: Tubastraea, Alteromonas, Biofilm, DUF3450 domain, TonB system, phage receptor