The pneumococcal cell envelope stress-sensing system LiaFSR is activated by murein hydrolases and lipid II-interacting antibiotics

Abstract

In the Firmicutes, two-component regulatory systems of the LiaSR type sense and orchestrate the response to various agents that perturb cell envelope functions, in particular lipid II cycle inhibitors. In the current study, we found that the corresponding system in Streptococcus pneumoniae displays similar properties but, in addition, responds to cell envelope stress elicited by murein hydrolases. During competence for genetic transformation, pneumococci attack and lyse noncompetent siblings present in the same environment. This phenomenon, termed fratricide, increases the efficiency of horizontal gene transfer in vitro and is believed to stimulate gene exchange also under natural conditions. Lysis of noncompetent target cells is mediated by the putative murein hydrolase CbpD, the key effector of the fratricide mechanism, and the autolysins LytA and LytC. To avoid succumbing to their own lysins, competent attacker cells must possess a protective mechanism rendering them immune. The most important component of this mechanism is ComM, an integral membrane protein of unknown function that is expressed only in competent cells. Here, we show that a second layer of self-protection is provided by the pneumococcal LiaFSR system, which senses the damage inflicted to the cell wall by CbpD, LytA, and LytC. Two members of the LiaFSR regulon, spr0810 and PcpC (spr0351), were shown to contribute to the LiaFSR-coordinated protection against fratricide-induced self-lysis.

FIG. 1.

Expression and promoter deletion analysis of the liaFSR operon. (A) Map of the liaFSR operon. The size of the liaR-specific transcript that was detected by Northern analysis is indicated by the arrow. (B) Sequence of the promoter region upstream of liaF (spr0342). (C) Schematic representation of the fragments cloned for the promoter deletion analysis of the liaF promoter. (D) β-Galactosidase activity of the fragments in the absence (light gray bars) and presence (black bars) of 10 μg/ml bacitracin. The numbers correspond to the fragments illustrated in panel C.

FIG. 2.

LiaR binding sites and consensus sequence. (A) LiaR-like binding sites in Bacillus and Listeria species (group 1), as derived from the work of Jordan et al. (21). (B) LiaR/CesR-like binding sites in Lactococcus and Streptococcus species, based on the work of Martinez et al. (29) and this work. LiaR dependently expressed genes, as determined by DNA microarray analysis (Table 2), are highlighted in bold. Core residues (>80% conserved) and highly conserved residues (>60% conserved) are highlighted in black and gray, respectively. WebLogo (5) representations of the position weight matrices derived from these sequences are shown below. The inverted repeat is indicated by the two black arrows.

FIG. 3.

Induction of the LiaSR system by antibiotics. A luciferase (luc) reporter gene was inserted immediately downstream of liaR (strain RH259). Bacterial growth curves are marked by triangles, whereas expression of liaR::luc transcriptional fusions is shown as unmarked curves. Luciferase activity is presented as relative luminescence units (RLU)/optical density (OD₄₉₂). Gray curves, untreated cultures; red curves, cultures grown in 5 μg/ml bacitracin; blue curves, cultures grown in 20 μg/ml nisin; green curves, cultures grown in 20 μg/ml tunicamycin. Antibiotics were added at time zero.

FIG. 4.

Comparison of liaSR activation in competent and noncompetent pneumococci harboring various mutations in genes involved in the fratricide mechanism. The luc reporter was inserted immediately downstream of spr0810, a gene that is part of the LiaSR regulon. Bacterial growth curves are marked by triangles, whereas expression of spr0810::luc transcriptional fusions is shown as unmarked curves. Luciferase activity is presented as relative luminescence units (RLU)/optical density (OD₄₉₂). Gray curves represent noncompetent cultures, while colored curves represent CSP-induced competent cultures. CSP was added at time zero. (A) RH270 (spr0810::luc), (B) RH272 (spr0810::luc comM), (C) RH283 (spr0810::luc comM cbpD), (D) RH284 (spr0810::luc comM cbpD_C75A), (E) RH290 (spr0810::luc comM lytC), (F) RH291 (spr0810::luc comM lytC lytA), (G) RH273 (spr0810::luc comM liaR), (H) RH294 (spr1332::luc comM). Results presented in panels A to H are the means ± standard errors of results of three to five independent experiments.

FIG. 5.

Competence-induced cell lysis in various pneumococcal deletion mutants quantified by means of β-galactosidase release. All strains contain a hirL::lacZ fusion conferring constitutive production of an intracellular β-galactosidase reporter protein. The level of β-galactosidase activity (in Miller units) present in the cell-free supernatants is given as a percentage of the total activity present in supernatants plus intact cells. Strains used were as follows: RH237 (ΔcomM), RH253 (ΔcomM ΔliaR), SPH-5 (ΔcomM Δspr0810), SPH-4 (ΔcomM ΔpcpC), SPH-6 (ΔcomM Δspr0810 ΔpcpC), RH4 (wild type), and RH276 (ΔliaR). Data represent the averages ± standard errors of results of at least three independent experiments. **, P ≤ 0.01; *, P ≤ 0.05.