Activation of TnSmu1, an integrative and conjugative element, is lethal to Streptococcus mutans

Abstract

Integrative and conjugative elements (ICEs) are chromosomally encoded mobile genetic elements that can transfer DNA between bacterial strains. They are important for generating microbial diversity and can encode for antibiotic, virulence, and metabolism determinants. Recently, as part of efforts to determine hypothetical essential 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 transcriptional regulator we are naming essential regulatory factor (erfR) and a metalloprotease (erfP). The efrR gene cannot be deleted and repression by CRISPR interference (CRISPRi) causes a severe growth defect. We used a bypass of essentiality (BoE) screen, with transposon mutagenesis allied to genome sequencing, to discover genes that allow deletion of the regulatory module. This revealed that conjugation genes, located within TnSmu1, can partially restore the viability of an erfR mutant. Loss of erfR also leads to production of a circular intermediate form of TnSmu1, that is also inducible by the genotoxic agent mitomycin C. To gain further insights into potential regulation of TnSmu1 by ErfR and broader effects on S. mutans UA159 physiology we used a combination of CRISPRi and RNA-seq. The RNA-seq analysis identified 134 genes whose log₂ fold change was ≥2 after repression of erfR. 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 a newly characterized ICE is strongly deleterious to S. mutans, highlighting the complex interplay between mobile elements and their host. We are continuing to investigate TnSmu1 as a model to understand ICE distribution, and effects on host fitness and cell biology in oral streptococci.