General prophage deletion scheme.

(a) Location of upstream, downstream, and flanking primers used in the deletion of SfPat, primer orientation shown with respect to the prophage. (b) Deletion of SfPat from S. fidelis 3313 identified after assembling Illumina (short-read) sequenced genomes and mapping onto the improved (short and long-read, PacBio, sequencing) WT genome. The figure illustrates SfPat deletion as revealed by subsequent Illumina sequencing. The solid gray areas on the SfPat genome indicate regions that share identity with the WT.

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.

Effects of prophages on biofilm and swimming in S. fidelis 3313.

(a) Influence of prophages on in vitro biofilm formation over 24 hours quantified with crystal violet assay (n=3), (b) role of prophages in swimming 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; ns= not significant).

The influence of SfPat prophage on gut colonization in Ciona.

(a) Results of six biological replicates (n=6, each replicate being a pool of ten juvenile tunicates) of the experimental exposure of Ciona MS4 juveniles to either WT or ΔSfPat strains for 1 hr and 24 hrs; retention quantification displayed as a Beeswarm plot of colony-forming units (CFUs). There is significant retention observed in WT after 24 hours. The MS4 juveniles reveal differential colonization of WT and ΔSfPat after one hour of exposure (b-e), where WT strain is stained with BacLight Red and ΔSfpat is stained with BacLight Green reveal (b) WT is seen localized in the lower esophagus to anterior stomach, while the ΔSfPat deletion strain localized to the hindgut, while (C) the WT is seen localized mostly as a fecal pellet in the center of the stomach while ΔSfPat prefers to localize to the stomach wall. (d) The WT strain is retained in the pyloric cecum. (e) Summary schematic of asymmetric bilaterial views of MS4 animals; top of image is anterior and stomach is posterior. The ventral side is the ‘En’ side, and the dorsal side is the opposite side. The findings can be summarized as such: WT is retained in E and S, in PC, and also in the HG, while the ΔSfPat is retained in the stomach folds, MG, and portions of the HG. Some overlap in signal is noted with yellow coloring. (En = Endostyle, E= Esophagus, S= Stomach, MG= Mid Gut, HG = Hind Gut, PC = Pyloric Cecum)

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, ns = not significant).

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, ns = not significant).

Plasmids and strains 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.

qPCR showing relative fold change gene expression with Rho as an endogenous control.

(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). (ns= not significant)

WT stained with BacLight Red exposed to Ciona MS4 for one hour.

(a) WT found in esophagus (E), stomach (S), and fecal pellet (FP). (b) WT also found to occupy the hind gut (HG). (c) WT is retained in the center of the stomach but not the stomach walls.

ΔSfPat localization in Ciona MS4 after one hour exposure.

(a) Stained with BacLight Red, ΔSfPat is found adhered to the stomach (S) and mid gut (MG). (b) Stained with BacLight Green, ΔSfPat is found in the mid gut and hind gut (HG). (c) ΔSfPat is more localized in the stomach fold than the center of the stomach, with presence in the mid gut.

Rho is the most stable gene across strains when tested.