The human gastrointestinal (GI) tract is colonized by a dense consortium of microorganisms, including bacteria, viruses, archaea, and fungi, which together make up the gut microbiome. While the bacterial component of the microbiome has been studied for decades, providing a wealth of information on how this community impacts human health and disease, other community members including viruses are only beginning to gain attention. The viral component of the gut microbiome (or the virome) is primarily composed of bacteriophages (phages) and is highly individualized, but stable over time. Preliminary studies have found shifts in virome composition during various diseases such as inflammatory bowel disease, but whether these virome shifts play a causal role in disease development or are consequences of disease remains unclear. Whether by bacterial lysis during the phage lytic cycle, or by altering bacterial phenotypes during the lysogenic cycle, it is clear that phages have the potential to alter both the composition and function of the gut microbiome community in diverse ways; distinguishing the effect of these two very different lifestyles and how they are determined is important to our understanding of what roles phages might play in the gut microbiome.

Most bacteria found in the mouse gut contain at least one prophage, underscoring the importance of studying temperate phages in the context of the GI tract. Interestingly, it has also been found that rates of lysogeny vary by bacterial taxa, and that all sequenced Pseudomonadota were lysogens, which differed from Bacteroidota where only about 20% of this phylum were identified as lysogens. This suggests that lysogeny may be more inherent to the lifestyle and niche of Pseudomonadota in the gut, as compared to other taxa. It is not well understood, however, whether these temperate phages are undergoing the productive lytic cycle or the dormant lysogenic cycle in the GI tract. Studies using germ-free mice have found high rates of prophage induction in the GI tract, so much so that the ability of the lysogen to colonize the gut was impaired. On the other hand, a recent study following the fecal virome of one individual for over two years found that prophage induction was steadily maintained at a low rate, perhaps explaining the previously observed stability of the human gut virome. The molecular genetics of the lytic/lysogenic switch have been extensively characterized in model phages such as Lambda, revealing how phages integrate their regulatory circuitry with the status of their host. Notably however, these studies have primarily been done in laboratory-adapted bacterial strains and under laboratory conditions, calling into question whether these mechanisms are relevant in vivo, or if additional regulatory processes might control prophage induction in the GI tract.

To begin to address these questions, we previously isolated and characterized a novel temperate phage Kapi1, which infects a strain of commensal Escherichia coli MP1 isolated from a healthy mouse. Using luminescent reporter assays to monitor the activity of the Kapi1 CI promoter (the master repressor of the phage lytic cycle), we found that Kapi1 favors lysogeny in simulated intestinal fluid (SIF), compared to standard LB growth media. These results were further supported by in vivo studies of lysogen stability during colonization of specific pathogen-free (SPF) mice; approximately 85% of fecal MP1 shed from SPF mice retained the Kapi1 prophage and were stable at these levels for 28 days, suggesting that lysogeny with Kapi1 is favored in the GI environment.

It was unexpected that Kapi1 favored lysogeny in SIF, as this media contains bile salts which are known to cause DNA damage, and digestive enzymes which would presumably be stressful for the bacterial cell. Accordingly, we noted a slight induction of recA-lux reporters and a minor growth defect of ∆recA mutants in SIF, indicating certain levels of DNA damage. The fact that Kapi1 favors lysogeny despite evidence of DNA damage in SIF is at-odds with the classical phage induction paradigm, where DNA damage leads to an activation of RecA to RecA*, which mediates proteolysis of CI, resulting in de-repression of lytic genes and entrance into the lytic cycle. Western blots comparing the stability of Kapi1 and Lambda CI-6xHis in cells treated with the genotoxic agent mitomycin C showed that in contrast to Lambda CI, there was no detectable degradation of Kapi1 CI. This suggests that Kapi1 may instead use phage-encoded antirepressors for its induction, as has been shown for other phages. Antirepressors bind to CI and inhibit its DNA-binding activity, rather than degrading CI altogether; still, this induction mechanism would not explain lysogeny being promoted in DNA-damaging conditions, as antirepressor synthesis is typically directly regulated by LexA, which itself is regulated by RecA*. Interestingly, we found that LexA-6xHis strongly accumulated during bacterial growth in SIF, and that the levels of LexA-6xHis were identical between wild-type and ∆recA mutants in SIF, indicating that it is not getting cleaved by RecA* as it normally does during DNA damage. We therefore hypothesized that an unidentified protein is induced during growth in SIF which alters the normal SOS cascade, resulting in increased repression of Kapi1 beyond basal levels observed in LB.

Moving forward, we will first confirm whether LexA accumulation in SIF occurs in isogenic non-lysogens as it does in Kapi1 lysogens, to determine whether this putative SOS-modulating factor is host- or phage-encoded. RNAseq will be subsequently performed to identify genes upregulated during bacterial growth in SIF and interrogate the general status of the SOS regulon, to determine what step in the pathway is being modulated. Overall, this project will significantly contribute to our fundamental understanding of phage molecular biology, as reports of lysogeny being actively promoted in SOS-inducing conditions are extremely rare. Further, understanding these mechanisms may provide insight into the temporal stability of the human gut virome. Increasing our fundamental knowledge of the organisms that inhabit humans will have important future implications in understanding how the microbiome as a whole (including phages) contributes to health and disease.