The cis-encoded antisense RNA IsrA from Salmonella Typhimurium represses the expression of STM0294.1n (iasE), an SOS-induced gene coding for an endoribonuclease activity

https://doi.org/10.1016/j.bbrc.2020.03.131Get rights and content

Highlights

  • IasE belongs to the SymE toxin superfamily.

  • The expression of iasE is induced in response to mitomycin C, an SOS-inducing agent.

  • The iasE overexpression affects the bacterial growth.

  • The bacterial lysate from an iasE overexpressing strain exhibits RNase activity.

  • The iasE/isrA module constitutes a functional type I toxin-antitoxin system.

Abstract

Toxin-antitoxin systems are known to be involved in many bacterial functions that can lead to growth arrest and cell death in response to stress. Typically, toxin and antitoxin genes of type I systems are located in opposite strands, where the antitoxin is a small antisense RNA (sRNA). In the present work we show that the sRNA IsrA from Salmonella Typhimurium down-regulates the expression of its overlapping gene STM0294.1n. Multiple sequence alignment and comparative structure analysis indicated that STM0294.1n belongs to the SymE toxin superfamily, and the gene was renamed iasE (IsrA-overlapping gene with similarity to SymE). The iasE expression was induced in response to mitomycin C, an SOS-inducing agent; conversely, IsrA overexpression repressed the iasE expression even in the presence of mitomycin C. Accordingly, the inactivation of IsrA with an anti-IsrA RNA expressed in trans abrogated the repressive effect of IsrA on the iasE expression. On the other hand, iasE overexpression, as well as the blockage of the antisense IsrA function, negatively affected bacterial growth, arguing for a toxic effect of the iasE gene product. Besides, a bacterial lysate obtained from the iasE-overexpressing strain exhibited endoribonuclease activity, as determined by a fluorometric assay based on fluorescent reporter RNAs. Together, these results indicate that the IasE/IsrA pair of S. Typhimurium constitutes a functional type I toxin-antitoxin system.

Introduction

Bacteria are regularly exposed to environmental stress conditions and have accordingly evolved global regulatory systems that contributes to bacterial survival by adjusting the rates of intracellular metabolic processes. Among these systems, the toxin-antitoxin (TA) modules play essential roles in bacterial physiology in response to stress [1]. The TA systems encode a toxin that either kills cells or confers growth stasis by interfering with vital processes, and an antitoxin that protects the cell from the toxin. At present, the TA systems are classified on six different types based on the nature of the antitoxin (RNA or protein) and the mechanism by which it controls the activity of its cognate toxin (types I through VI) [2]. In type I TA systems the antitoxin is an antisense RNA, typically of 50–200 nucleotides, that impedes the translation of the toxin mRNA via base pairing, avoiding toxin production under favorable growth conditions. Toxin genes are usually inducible under stress conditions and higher mRNA levels are only observed upon unfavorable conditions. If mRNA levels of the toxin rise enough to overcome the antisense activity of the RNA antitoxin, the toxin production will take place [3].

Most toxins of type I TA systems are small hydrophobic proteins that are proposed to induce pores into bacterial cell membranes, disrupting the ATP synthesis and consequently reducing protein synthesis. Well-characterized type I TA system families in E. coli further include LdrD/RdlD, Hok/Sok, and SymE/SymR which are regulated by cis-encoded [[4], [5], [6]]. SymE system includes an atypical toxin because SymE is not a hydrophobic protein and does not show functional homology to other type I toxin proteins [7]. The SymE/SymR pair is an SOS-responsive system activated upon DNA damage where SymE promotes RNA degradation of mRNAs. These data have led to hypothesize about the participation of SymE in the recycling of RNAs damaged under SOS-inducing conditions [6,7].

The number of type I TA loci significantly varies in different bacteria, and even between closely related microorganisms. For example, E. coli K-12 encodes at least 19 type I TA loci [2]. Conversely, only 6 functional type I TA loci were identified in the closely related genome of S. Typhimurium. Even more striking, some type I TA loci identified in S. Typhimurium SL1344 display a narrow distribution in other Salmonella species [8]. Even though a homolog SymE/SymR TA module was identified in S. Typhimurium in that study, this behaved as a non-functional system.

In this work, we examined the possibility that the IasE/IsrA pair function as a type I toxin-antitoxin module in S. Typhimurium SL1344. IsrA was previously identified as an island-encoded sRNA that overlaps at the 5′-end with the cis-encoded gene STM0294.1n, which encodes a hypothetical protein with no explicit functional annotation [9]. In that study, both genes exhibited inversed expression patterns in bacterial cultures, i.e., IsrA levels were high under conditions in which STM0294.1n levels were low (e.g. log phase) and vice versa (e.g. stationary phase and acid stress). Here we present evidence showing that IsrA fulfills an antitoxin function by repressing the expression of STM0294.1n, which we have renamed IasE (IsrA-overlapping gene with similarity to SymE) based on its homology to SymE toxin. The iasE gene exhibits SOS-responsive expression, and its product has properties of the endonuclease toxins. We propose that the IasE/IsrA pair of S. Typhimurium represents a functional type I TA system. The possible roles under the SOS response are also discussed.

Section snippets

Bacterial strains and growth conditions

The strains and plasmids used in this study are listed in Supplementary Table S1. The module iasE/isrA from S. Typhimurium SL1344 (wild type, WT) was deleted by the technique of phage λ Red recombinase, as previously described [10]. The plasmid for arabinose-inducible expression of the isrA gene into the wild type strain (pIsrA), was constructed using the pBAD plasmid as previously described [11]. The plasmid for arabinose-inducible expression of the antisense-IsrA (pAnti-IsrA) was constructed

The overlapping gene of the IsrA sRNA exhibits homology with the SymE toxin superfamily

In a previous screen for island-encoded sRNAs of S. Typhimurium conducted by Padalon-Brauch et al. [9], it was reported that the sRNA IsrA overlaps at the 5′-end with the cis-encoded gene STM0294.1n exhibiting inversed expression patterns under certain growth conditions. In order to shed light on the function of STM0294.1n, a PSI-BLAST analysis was performed, finding a domain of 54 amino acids (residues interval 21-75) with significant homology (e-value = 10−12) to the SymE toxin superfamily.

Discussion

The most studied prokaryotic sRNA regulators act by base pairing and share either extensive (cis-acting sRNAs) or more limited (trans-acting sRNAs) complementarity with their target transcripts [19]. Most of the chromosomally, cis-encoded bacterial sRNAs have been reported to repress genes that encode small toxic proteins. Such an interaction has been classified in the literature as the type I toxin-antitoxin (TA) pair [20]. In the present work, we present evidence indicating that the cis

Declaration of competing interest

None declared.

Acknowledgments

This work was supported by FONDECYT 1171655.

References (33)

  • M. Kawano

    Divergently overlapping cis-encoded antisense RNA regulating toxin-antitoxin systems from E. coli: hok/sok, ldr/rdl, symE/symR

    RNA Biol.

    (2012)
  • D. Lobato-Márquez et al.

    Distinct type I and type II toxin-antitoxin modules control Salmonella lifestyle inside eukaryotic cells

    Sci. Rep.

    (2015)
  • G. Padalon-Brauch et al.

    Small RNAs encoded within genetic islands of Salmonella typhimurium show host-induced expression and role in virulence

    Nucleic Acids Res.

    (2008)
  • K.A. Datsenko et al.

    One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products

    Proc. Natl. Acad. Sci. U.S.A.

    (2000)
  • D.N. Fuentes et al.

    Motility modulation by the small non-coding RNA SroC in Salmonella Typhimurium

    FEMS Microbiol. Lett.

    (2015)
  • S.F. Altschul et al.

    Gapped BLAST and PSI-BLAST: a new generation of protein database search programs

    Nucleic Acids Res.

    (1997)
  • 1

    These authors contributed equally to the work.

    View full text