All S. fidelis 3313 strains are submitted under the BioProject PRJNA 90327 on NCBI, Accession: SAMN31793880 ID:31793880

Primer sequences on S. flidelis 3313 used for generating Δ SfPat deletion suicide vector and for deletion verification

General prophage deletion scheme. (a) location of upstream, downstream and flanking primers used in the deletion of SfPat, (b) Deletion of SfPat from S. fidelis 3313 identified after assembling Illumina sequenced genomes and mapping on to the improved WT genome.

Effects of prophages on biofilm and swimming in S. fidelis 3313. (a) Effect of prophages on in vitro biofilm formation over 24 hours quantified with crystal violet assay (n=3), (b) role of prophages in swimming (or chemotaxis) quantified as the diameter of spread on soft agar after 24-hours (n=6), and (c) fold-change of pdeB (with Rho as internal control) from 24-hour biofilm (in vitro) (n=4) and 24-hour in vivo (n=4). (*= p-value<0.05, **= p-value<0.01).

Figure 2—figure supplement 1. Supplemental Figure: (a) Cyclic di GMP regulators expression in WT and ΔSfPat in vitro after 24 hours of exposure (n=4), (b) Cyclic di GMP regulators expression in WT and ΔSfPat in vivo of Ciona MS4 after 24 hours of exposure (n=4).

The influence of SfPat prophage on gut colonization in Ciona. (a) colonization of MS4 juvenile gut by WT and ΔSfPat after one hour of exposure to strains (n=3). (b) after 24 hours of exposure (n=6). MS4 juveniles reveal differential colonization of WT and ΔSfPat after one hour of exposure (c-e), where WT strain stained with BacLight Green (c) is seen localized in the lower esophagus to anterior stomach, while the ΔSfPat deletion strain, stained with BacLight Red, localized to the hindgut, while (d) the WT stained with BacLight Red is seen localized mostly as a fecal pellet in the center of the stomach while ΔSfPat stained in the same way prefers to adhere to the stomach folds. The WT strain, stained in BacLight Red (e), also prefers to localize within the pyloric cecum (En = Endostyle, E= Esophagus, S= Stomach, MG= Mid Gut, HG = Hind Gut, PC = Pyloric Cecum)

Figure 3—figure supplement 1. Supplemental Figure 3: WT stained in BacLight Red exposed to Ciona MS4 for one hour c) ΔSfPat stained in BacLight Red exposed to Ciona MS4 for one hour. d) ΔfPat stained in BacLight Green exposed to Ciona MS4 for one hour

The influence of prophages on host gene expression. (a) VCBP-C gene expression in MS4 juveniles after one hour of exposure to S. fidelis 3313 strains (n=4), (b) Survey of additional innate immune gene expression in MS4 juveniles after 24-hour exposure to WT or ΔSfPat mutant strains (n=3). Actin is the internal control. (*= p-value<0.05, **= p-value<0.01.).

Lysogen gene expression in response to host immune effector binding. Gene expression of SfPat structural protein p5, recA and lexA of WT strain grown as a 24-hour biofilm while exposed to 50 μg/ml VCBP-C. Rho is the internal control (n=4). (*= p-value<0.05).

Figure 5—figure supplement 1. Supplemental Figure 2: Rho is the most stable gene across strains when tested in vitro (n=3)

Plasmids used in the study

Ciona Genes targeted and the necessary reverse transcription-qPCR primers

S. fidelis 3313 Genes targeted and the necessary reverse transcription-qPCR primers

Supplemental Figure: (a) Cyclic di GMP regulators expression in WT and ΔSfPat in vitro after 24 hours of exposure (n=4), (b) Cyclic di GMP regulators expression in WT and ΔSfPat in vivo of Ciona MS4 after 24 hours of exposure (n=4).

Supplemental Figure 3: WT stained in BacLight Red exposed to Ciona MS4 for one hour c) ΔSfPat stained in BacLight Red exposed to Ciona MS4 for one hour. d) ΔfPat stained in BacLight Green exposed to Ciona MS4 for one hour

Supplemental Figure 2: Rho is the most stable gene across strains when tested in vitro (n=3)