Activation of TnSmu1, an integrative and conjugative element, by an ImmR-like transcriptional regulator in Streptococcus mutans

Abstract

Integrative and conjugative elements (ICEs) are chromosomally encoded mobile genetic elements that can transfer DNA between bacterial strains. Recently, as part of efforts to determine hypothetical gene functions, we have discovered an important regulatory module encoded on an ICE known as TnSmu1 on the Streptococcus mutans chromosome. The regulatory module consists of a cI-like repressor with a helix-turn-helix DNA binding domain immR Smu (immunity repressor) and a metalloprotease immA Smu (anti-repressor). It is not possible to create an in-frame deletion mutant of immR Smu and repression of immR Smu with CRISPRi (CRISPR interference) causes substantial cell defects. We used a bypass of essentiality (BoE) screen to discover genes that allow deletion of the regulatory module. This revealed that conjugation genes, located within TnSmu1, can restore the viability of an immR Smu mutant. Deletion of immR Smu also leads to production of a circular intermediate form of TnSmu1, which is also inducible by the genotoxic agent mitomycin C. To gain further insights into potential regulation of TnSmu1 by ImmRSmu and broader effects on S. mutans UA159 physiology, we used CRISPRi and RNA-seq. Strongly induced genes included all the TnSmu1 mobile element, genes involved in amino acid metabolism, transport systems and a type I-C CRISPR-Cas system. Lastly, bioinformatic analysis shows that the TnSmu1 mobile element and its associated genes are well distributed across S. mutans isolates. Taken together, our results show that activation of TnSmu1 is controlled by the immRA Smu module, and that activation is deleterious to S. mutans , highlighting the complex interplay between mobile elements and their host.

TnSmu1 is a functional integrative and conjugative element in Streptococcus mutans that when expressed causes growth arrest of host bacteria

“…The sequences of the various sites also allowed us to identify a 17 bp sequence present in both attB and att Tn Smu1 that is likely the recombination site used by the tyrosine recombinase encoded by int ( smu_191c ) in Tn Smu1 (Figure 3b ). This is consistent with excision noted by King et al ( 2022 ) when examining read coverage in an immR Smu mutant. Together, these data demonstrate that Tn Smu1 is capable of excision from the chromosome of host cells and that a small proportion of cells had “spontaneously” activated Tn Smu1 in the absence of any exogenous treatment.…”

“…The sequences of the various sites also allowed us to identify a 17 bp sequence present in both attB and attTnSmu1 that is likely the recombination site used by the tyrosine recombinase encoded by int (smu_191c) in TnSmu1 (Figure 3b). This is consistent with excision noted by King et al (2022) when examining read coverage in an immR Smu mutant.…”

“…However, unlike Tn 916 and ICE clc , our results indicate that Tn Smu1 does not cause host cell death. Additionally, a CRISPRi knockdown of immR Smu caused an arrest in growth of the entire population (King et al, 2022 ). It is possible that the cells with an activated Tn Smu1 die following growth arrest, but we have not observed this, nor have assays measuring death been reported.…”

“…Together, our results illuminate the functionality of Tn Smu1 and demonstrate that Tn Smu1 affects the physiology of its host cells by halting host cell growth. As our work was nearing completion, an independent study identified the putative repressor of Tn Smu1 and reported that the element can be activated to excise from the chromosome by conditional inactivation of the repressor and also by DNA damage (King et al, 2022 ).…”

Aspects of oral streptococcal metabolic diversity: Imagining the landscape beneath the fog

“…An alternative delivery system could take advantage of the recently discovered integrative conjugative elements (ICE) in S. mutans (King et al, 2022;McLellan et al, 2022). In this case, the ICE can be engineered with a cas9 fusion construct, which could then be disseminated to other members of the biofilm.…”