Alternative sigma factor σ 54 (RpoN) of Vibrio fischeri is required during initial squid light organ colonization but its continued induction is not required for maintenance

ABSTRACT Animals depend on bacterial symbionts that provide functions essential for host physiology and fitness. The marine bacterium Vibrio fischeri establishes a mutualistic association within the light organ of the Hawaiian bobtail squid Euprymna scolopes , where bacterial bioluminescence enables a host camouflage behavior. Successful colonization of the light organ requires the alternative sigma factor σ 54 (RpoN), which activates genes involved in biofilm formation and motility that are essential for bacterial entry into the host. However, whether σ 54 remains important after the initial stages of colonization has been unclear. Here, using an IPTG-inducible rpoN expression system, we demonstrate that induction of rpoN is required for initial entry into the light organ, but continued inducer exposure is not required for subsequent maintenance of colonization and bioluminescence production. Transcriptomic analysis of RNA-sequencing of low-cell-density cultures identified σ 54 -dependent expression of genes involved in flagellar motility, chemotaxis, nitrogen assimilation, molybdenum cofactor (Moco) biosynthesis, and formate metabolism. We further show that the bacterial enhancer-binding protein VF_1401 activates genes required for Moco biosynthesis and formate metabolism but is not required for light organ colonization. Together, these findings define the temporal requirement for σ 54 during symbiosis establishment and reveal a previously unrecognized role for σ 54 in regulating molybdenum-dependent metabolism in V. fischeri . IMPORTANCE Many bacteria encode the alternative sigma factor σ 54 (RpoN), which enables selective activation of specific cellular processes in response to environmental signals. Our findings refine the conditions under which σ 54 contributes to host colonization by a bacterial symbiont, providing new insight into the context-dependent roles of this conserved regulator. In addition, our transcriptomic analysis identifies a previously unrecognized link between σ 54 and molybdenum assimilation, revealing a potential mechanism by which σ 54 coordinates trace element homeostasis with bacterial physiology. These findings broaden our understanding of σ 54 regulatory networks and may inform future strategies to manipulate the physiology of beneficial bacterial symbionts.