Identification of CdnL, a Putative Transcriptional Regulator Involved in Repair and Outgrowth of Heat-Damaged Bacillus cereus Spores

Abstract

Spores are widely present in the environment and are common contaminants in the food chain, creating a challenge for food industry. Nowadays, heat treatments conventionally applied in food processing may become milder to comply with consumer desire for products with higher sensory and nutritional values. Consequently subpopulations of spores may emerge that are sublethally damaged rather than inactivated. Such spores may germinate, repair damage, and eventually grow out leading to uncontrolled spoilage and safety issues. To gain insight into both the behaviour of damaged Bacillus cereus spores, and the process of damage repair, we assessed the germination and outgrowth performance using OD595 measurements and microscopy combined with genome-wide transcription analysis of untreated and heat-treated spores. The first two methods showed delayed germination and outgrowth of heat-damaged B. cereus ATCC14579 spores. A subset of genes uniquely expressed in heat-treated spores was identified with putative roles in the outgrowth of damaged spores, including cdnL (BC4714) encoding the putative transcriptional regulator CdnL. Next, a B. cereus ATCC14579 cdnL (BC4714) deletion mutant was constructed and assessment of outgrowth from heat-treated spores under food relevant conditions showed increased damage compared to wild type spores. The approach used in this study allows for identification of candidate genes involved in spore damage repair. Further identification of cellular parameters and characterisation of the molecular processes contributing to spore damage repair may provide leads for better control of spore outgrowth in foods.

Fig 1. Impact of heat-treatment on germination and outgrowth of B. cereus ATCC14579 spores.

(A) Relative change in OD₅₉₅ for untreated (circles) and heat-treated for 1 min at 95°C (squares) dormant spores was monitored in time in BHI broth at 30°C. Closed symbols indicate the sampling points selected for transcriptome analysis. The starting OD₅₉₅ was 0.15–0.2 (B) Microscopy analysis of samples taken before initiation of germination (t0) and at indicated time points (10 up to 150 min) thereafter.

Fig 2. Expression profiles (log2 values) of reported spore specific transcripts during germination and outgrowth of untreated (white diamonds) and heat-treated (black diamonds) B. cereus ATCC14579 spores.

Expression ratio’s for untreated spores are relative to untreated germinating control spores at t10, and for heat-treated spores relative to heat-treated germinating spores at t50.