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.

The conserved transcriptional regulator CdnL is required for metabolic homeostasis and morphogenesis in Caulobacter

“…M. tuberculosis CarD is essential for growth in culture, persistence in mice, and survival during genotoxic stress and starvation [2,4]. The CdnL homolog in Bacillus cereus is upregulated during heat treatment and B. cereus spores lacking CdnL do not recover as well as wild-type (WT) [8]. Additionally, the Borrelia burgdorferi CdnL homolog LtpA is important for infection and is upregulated during cold shock [9,10].…”

Recovery of Heat Treated Bacillus cereus Spores Is Affected by Matrix Composition and Factors with Putative Functions in Damage Repair

“…A double deletion mutant (Δ cdnL1 /Δ cdnL2 ) was obtained as described above with the exception that the cdnL2 knock out plasmid was transformed into a B. cereus Δ cdnL1 (BC4714) mutant strain constructed previously ( Warda et al, 2016 ) and 5 μg/ml chloramphenicol was included as selective pressure preventing excision of the chloramphenicol resistance cassette that disrupted the cdnL1 .…”

“…To evaluate the degree of spore damage, the method previously reported by Warda et al (2016) was used. Briefly, 50 μl of decimally diluted heat treated samples were enumerated in duplicate on BHI plates and BHI plates supplemented with 1.5 and 5.5% salt following incubation at 30°C.…”

“…Using a transcriptome approach, we previously identified 21 genes putatively involved in heat damage repair in B. cereus. For one of these candidate genes, cdnL (now referred as cdnL1 ), a role in spore damage repair was further confirmed using a targeted deletion mutant ( Warda et al, 2016 ). Here we report on behavior of eight newly and one previously ( Warda et al, 2016 ) constructed mutant to assess respective putative roles in recovery efficiency of heat treated B. cereus spores.…”

“…Heterogeneity in germination and outgrowth of surviving C. botulinum and Bacillus cereus spore populations is more pronounced in the presence of a secondary mild stress factor such as low pH without and with sorbic acid, and increasing levels of salt ( Stringer et al, 2011 ; van Melis et al, 2014 ; Warda et al, 2015 ) or the natural components of food media ( Warda et al, 2015 ). In general, damaged spores were shown to be more sensitive to secondary stresses including sodium chloride, pH, sorbic acid compared to undamaged spores ( Feeherry et al, 1987 ; Faille et al, 1997 ; Cazemier et al, 2001 ; Cortezzo et al, 2004 ; Samapundo et al, 2014 ; van Melis et al, 2014 ; Warda et al, 2015 , 2016 ). Some studies suggest a pre-plating recovery step in optimal (perhaps strain and treatment specific) conditions to allow recovery of injured cells ( Wu, 2008 ) or spores ( Rodriguez-Palacios and Lejeune, 2011 ).…”