The Streptococcus mutans IrvR repressor is a CI-like regulator that functions through autocleavage and Clp-dependent proteolysis

Abstract

Previous work has shown that irvR is required for the proper regulation of genetic competence and dextran-dependent aggregation due to its ability to repress the transcription regulator irvA. In this study, we determined the mechanism used to relieve the repression of irvA. We demonstrate that IrvR is a "LexA-like" protein with four conserved amino acid residues likely required for IrvR autocleavage activity. Furthermore, recombinant IrvR protein purified from Escherichia coli was competent to undergo autocleavage in vitro. Using several truncated IrvR constructs, we show that the amino acids adjacent to the autocleavage site are essential for relieving irvA repression and engaging the irvA-dependent regulatory pathway primarily through the ClpXP and ClpCP proteases. By extending the IrvR C terminus with an epitope derived from the autocleavage site, we were also able to create a constitutive Clp-dependent degradation of the full-length IrvR protein. This suggests that the derepression of irvA occurs through a two-step mechanism involving the initial autocleavage of IrvR and exposure of a proteolytic degradation sequence followed by Clp-dependent degradation of the IrvR DNA binding domain. Thus, irvA derepression is highly analogous to the genetic switch mechanism used to regulate lysogeny in bacteriophages.

FIG. 1.

Determination of the IrvR translation start site. (A) The region surrounding the annotated irvR translation start site is shown. Dashed arrows represent potential alternative start sites, while the solid arrow is the annotated start site. Potential start codons are shown in bold, and the corresponding ribosome binding sites (RBS) are underlined. (B) Using the amended sequence of IrvR, an N-terminal ClustalW alignment with several phage repressors is shown. Arrows indicate the position of potential IrvR N termini. The middle arrow indicates the annotated translation start site. The first three phage repressors are from streptococcal phages, while BK5-T infects species of Lactococcus.

FIG. 2.

Alignment of IrvR and LexA-like proteins. Shown are the partial alignment results from a ClustalW analysis of IrvR, the CI repressor from the streptococcal phage EJ-1, HdiR from Lactococcus lactis, and LexA from E. coli. Residues required for the autocleavage ability of LexA-like proteins are shaded. Residues required for LexA proteolytic degradation are underlined, while similar residues in other LexA-like proteins are boxed.

FIG. 3.

IrvR in vitro autocleavage assay. As described in Materials and Methods, IrvR and its derivatives were expressed in E. coli. ClearedE. coli lysate (120 μg) from each sample was incubated for 2 h before the reaction was stopped. Lysate (12 μg) was separated by SDS-PAGE and probed with α-IrvR polyclonal antibodies. The top arrow indicates the full-length IrvR protein, followed by the N-terminal autocleavage fragment (N), and the C-terminal autocleavage fragment (C). Lane 1, wild-type IrvR; lane 2, IrvR^A191D mutant; lane 3, IrvR^K260A mutant; lane 4, purified His₆-tagged IrvR truncated at Ala191. This experiment was performed three times with similar results. A representative result is shown.

FIG. 4.

Analysis of irvA expression in truncated irvR backgrounds. (A) The domain architecture and the location of the autocleavage site VSA epitope are illustrated in the wild-type UA159. Both the full-length IrvR and its truncated derivatives supplied in trans are presented with their relative sizes shown. The truncated IrvR strains also have the three C-terminal amino acids marked with an asterisk to indicate the location of an engineered stop codon. (B) The expression of irvA in the various irvR backgrounds is presented relative to that of the wild type (UA159), which was arbitrarily assigned a value of 1. These data are the average of results of three independent experiments.

FIG. 5.

Transformation efficiency assays of IrvR truncation constructs. The transformation efficiency values are presented relative to the wild-type UA159 value (1.0 × 10⁻⁶), which was arbitrarily assigned as 100%. The actual transformation efficiency of the IrvR^1-191(VSA) strain could not be calculated because the value is below our detection limit (<1.33 × 10⁻⁹). These data are the average of results of three independent experiments.

FIG. 6.

Dextran-dependent aggregation of IrvR truncation constructs. (A) The effects of different truncations of IrvR are compared. Sample 1, the wild-type UA159; sample 2, full-length IrvR; sample 3, IrvR^1-122; sample 4, IrvR^1-191(VSA); sample 5, IrvR^1-191(VSD); sample 6, IrvR^1-191(VDD). (B) The effects of the clpP mutation upon dextran-dependent aggregation are compared. Sample 1, full-length IrvR; sample 2, IrvR^1-191(VSA); sample 3, IrvR^1-191(VSA) in the clpP background; sample 4, clpP mutant. These experiments were repeated three times with similar results. A representative set is shown.

FIG. 7.

In vivo stability of truncated IrvR strains. S. mutans strains expressing irvR truncated at the autocleavage site were detected with α-IrvR polyclonal antibodies. Lane 1, IrvR^1-191(VSA); lane 2, IrvR^1-191(VSD); lane 3, IrvR^1-191(VDD); lane 4, IrvR^1-191(VSA) in the clpP background; lane 5, IrvR^1-191(VSA) in the clpX/clpC background. This experiment was performed three times with similar results. A representative result is shown.

FIG. 8.

In vivo stability of full-length IrvR containing additional C-terminal epitopes. Various epitopes from the autocleavage site of IrvR were added to the C terminus of the full-length protein. These strains were assayed for in vivo stability with α-IrvR polyclonal antibodies. Lane 1, wild-type UA159; lane 2, irvR deletion mutant; lane 3, full-length IrvR; lane 4, IrvR^+VSA; lane 5, IrvR^+3VSA; lane 6, IrvR^+3VSA in the clpP background; lane 7, IrvR^+8VSA; lane 8, IrvR^+8VSA in the clpP background. This experiment was performed three times with similar results. A representative result is shown.