In Vitro Evolution of Listeria monocytogenes Reveals Selective Pressure for Loss of SigB and AgrA Function at Different Incubation Temperatures

Abstract

The alternative sigma factor B (σ^B) contributes to the stress tolerance of the foodborne pathogen Listeria monocytogenes by upregulating the general stress response. We previously showed that σ^B loss-of-function mutations arise frequently in strains of L. monocytogenes and suggested that mild stresses might favor the selection of such mutations. In this study, we performed in vitro evolution experiments (IVEE) where L. monocytogenes was allowed to evolve over 30 days at elevated (42°C) or lower (30°C) incubation temperatures. Isolates purified throughout the IVEE revealed the emergence of sigB operon mutations at 42°C. However, at 30°C, independent alleles in the agr locus arose, resulting in the inactivation of Agr quorum sensing. Colonies of both sigB mutants and agr mutants exhibited a greyer coloration on 7-days-old agar plates than those of the parental strain. Scanning electron microscopy revealed a more complex colony architecture in the wild type than in the mutant strains. sigB mutant strains outcompeted the parental strain at 42°C but not at 30°C, while agr mutant strains showed a small increase in competitive fitness at 30°C. Analysis of 40,080 L. monocytogenes publicly available genome sequences revealed a high occurrence rate of premature stop codons in both the sigB and agrCA loci. An analysis of a local L. monocytogenes strain collection revealed 5 out of 168 strains carrying agrCA alleles. Our results suggest that the loss of σ^B or Agr confer an increased competitive fitness in some specific conditions and this likely contributes to the emergence of these alleles in strains of L. monocytogenes. IMPORTANCE To withstand environmental aggressions, L. monocytogenes upregulates a large regulon through the action of the alternative sigma factor B (σ^B). However, σ^B becomes detrimental for L. monocytogenes growth under mild stresses, which confer a competitive advantage to σ^B loss-of-function alleles. Temperatures of 42°C, a mild stress, are often employed in mutagenesis protocols of L. monocytogenes and promote the emergence of σ^B loss-of-function alleles in the sigB operon. In contrast, lower temperatures of 30°C promote the emergence of Agr loss-of-function alleles, a cell-cell communication mechanism in L. monocytogenes. Our findings demonstrate that loss-of-function alleles emerge spontaneously in laboratory-grown strains. These alleles rise in the population as a consequence of the trade-off between growth and survival imposed by the activation of σ^B in L. monocytogenes. Additionally, our results demonstrate the importance of identifying unwanted hitchhiker mutations in newly constructed mutant strains.

Keywords: Listeria monocytogenes; agr system; bacterial competition; general stress response; in vitro evolution; quorum sensing; σB.

FIG 1

Differential emergence of grey colony phenotypic variants during in vitro evolution experiments at 42°C and 30°C. In vitro evolution experiments where cultures of L. monocytogenes EGD-e wild-type strain were grown in BHI at either 42°C (A) or 30°C (B) overnight. Passages were made every 24 h for a total of 30 days by diluting the cultures in a 1:100 ratio into fresh BHI. Every 5 days, the number of white and grey colonies was used to calculate the percentage of relative abundance. Panels C and D show that the colony coloration between the whiter WT and the greyish ΔsigB (C) and ΔagrA (D) strains in BHI agar plates were incubated at 37°C for 24 h and a further 6 days at 30°C. At least two independent biological replicates were performed for each incubation temperature. Statistical analysis was performed using a paired Student's t test relative to passage 0 (**, P value of <0.01; ***, P value of <0.001).

FIG 2

Grey colonies isolated from in vitro evolution experiments at 42°C have acid-sensitive phenotypes. Cultures of L. monocytogenes EGD-e wild-type, ΔsigB, and ΔagrA strains and white and grey colonies isolated from the in vitro evolution experiments at 42°C (A) and 30°C (B) were grown overnight in BHI at 37°C. Cultures were challenged in acidified BHI medium (pH 2.5) at 37°C. Samples were taken at 0 and 30 min for viable counts. Blue bars represent white colony strains, while red bars represent the ΔsigB strain or grey colonies isolated at 42°C and green bars represent the ΔagrA strain or grey colonies isolated at 30°C. Two independent biological replicates were made. Statistical analysis was performed using a paired Student's t test by comparing with the parental strain (*, P value of <0.05; **, P value of <0.01; ***, P value of <0.001).

FIG 3

Strains carrying alleles in sigB or agr operons exhibit reduced activity in each of the respective systems. Stationary-phase cultures of WT, ΔsigB, and ΔagrA strains and colonies isolated from the in vitro evolution experiments at both temperatures were transformed with pKSV7-P_lmo2230::egfp (A) and pGID310-P_agrB::gfp::bgaB (B, C, D). (A) Percentage of eGFP signal normalized relative to the WT. Quantification was obtained from Western blot images. Blue bars represent eGFP levels from the wild-type strain or white-colored colonies, red bars represent levels in the ΔsigB strain and grey-colored colonies isolated at 42°C, and green bars represent levels in the ΔagrA strain and grey-colored colonies isolated at 30°C. Cultures transformed with pGID310-P_agrB::gfp::bgaB were patched in BHI agar plates, supplemented with Chl and X-Gal, and incubated at 30°C (B), 37°C (C), and 42°C (D) for 48 h. For strain details, refer to Table 1. Western blot quantification was generated from two independent biological replicates. Statistical analysis was performed by a paired Student's t test (**, P value of <0.01; ***, P value of <0.001).

FIG 4

Strains carrying sigB or agr alleles exhibit a competitive advantage during mixed culture passaging. Competition experiments were performed with white versus grey colonies (starting ratio 1:1) isolated during the in vitro evolution experiments. Mixed cultures were passaged at 42°C (A, B) or 30°C (C, D, E, F, G). Blue bars represent the parental strain and white-colored colonies, while red and green bars represent the sigB and agr mutants, respectively. Data were generated from two independent biological replicates. For strain details, refer to Table 1. Statistical analysis was performed using a paired Student's t test (*, P value of <0.05; **, P value of <0.01).

FIG 5

Colony architecture is influenced by either ΔsigB or ΔagrA deletions. Images recorded by scanning electron microscopy of 7-day-old colonies of wild type (A, D), ΔsigB (B, E), and ΔagrA (C, F) strains grown in BHI agar plates at 30°C. Under these conditions, the grey colony phenotype was evident in the mutant strains. Images correspond to the colony border (A, B, C) or center (D, E, F). (G) Colonies of L. monocytogenes EGD-e WT, ΔsigB, and ΔagrA strains grown in BHI agar supplemented with Congo red (25 μg · mL⁻¹). Plates of Congo red were incubated for 24 h at 37°C and for three more days at 30°C. Representative data from three biological replicates are shown.

FIG 6

High frequency of PMSCs detected in the agrCA and sigB operons. In silico analysis of 40,080 genomes of L. monocytogenes strains publicly available. (A, B, and C) PMSC rate normalized by 100 bp of the open reading frame length for genes comprising the agr operon (red), two-component systems (pink), sigB operon (blue), mazEF (yellow), MLST genes (orange), sigma factors (black), metabolic genes (purple), rsbR1 paralogues (dark green), and virulence genes (light green). (B and C) Expanded area indicated by the blue and red squares in panel A, respectively.

FIG 7

Field isolates carrying alleles in the agr operon exhibit reduced activity in each of the respective systems. (A) Isolates that integrated pGID310-P_agrB::gfp::bgaB were patched in BHI agar plates supplemented with Chl and X-Gal and incubated at 37°C for 48 h. (B) Expression of agrB and agrA in strain 1389 grown to stationary phase (16 h) at 37°C was compared to strain 1371 by RT-qPCR experiment.