Transcriptome analysis and prediction of the metabolic state of stress-induced viable but non-culturable Bacillus subtilis cells

Abstract

Many bacteria adapt their physiology and enter the viable but non-culturable state to survive prolonged exposure to adverse environmental conditions. The VBNC cells maintain active metabolism, membrane integrity and gene transcription. However, they lose the ability to form colonies on a conventional culture media. Thus, standard colony counting methods cannot detect these alive but dormant cells. The Gram-positive bacterium Bacillus subtilis was found to enter the VBNC state when pre-exposed to osmotic stress and treated with a lethal dose of kanamycin. These cells reduced their metabolic activity, ceased growth and division and became kanamycin-tolerant. Interestingly, despite active metabolism, the majority of the kanamycin tolerant cells could not be revived on LB agar. In this study, we use a robust RNA-Seq technique to elucidate the differences in transcriptional profiles of B. subtilis VBNC cells. A comparative analysis of differently expressed genes and operons performed in this study indicates high similarities in transcriptional responses of VBNC and kanamycin-sensitive cells to antibiotic treatment. Moreover, this work reveals that VBNC cells strongly upregulate genes involved in proline uptake and catabolism, suggesting a putative role of proline as nutrient in VBNC cells.

Phenotypic and transcriptional characterization of F. tularensis LVS during transition into a viable but non-culturable state

“…The physical changes seen in the outer membrane of VBNC F. tularensis LVS may also affect permeability for various antibiotics. It will also be interesting to test if antibiotic challenge can promote entry into the VBNC state, as has been observed for B. subtilis ( Morawska and Kuipers, 2022 ). Also, whilst we show that there is no change in optical density, fluorescence, or shape in the VBNC cells when challenged with gentamicin, we cannot exclude the possibility that these VBNC bacteria are, in fact being killed, but in contrast to culturable cells do not manifest any changes in morphology or optical density.…”

“…Transcriptomics was first applied as a tool to analyze VBNC populations of Vibrio cholerae ( Asakura et al, 2007 ), and is increasingly being used to investigate the molecular mechanisms involved in the transition into and maintenance of the VBNC state in bacteria ( Liu et al, 2016 ; Su et al, 2016 ; Zhong and Zhao, 2019 ; Morawska and Kuipers, 2022 ). We used two sequencing platforms, Oxford Nanopore and Illumina to analyze the transcriptional changes in F. tularensis LVS as it enters into the VBNC state ( Figure 6 ).…”

Unveiling the mechanisms of black phosphorus nanosheets-induced viable but non-culturable state inBacillus tropicus

“…26,27 When bacteria encounter harsh environmental conditions, they can enter a VBNC state, which allows them to sustain basal metabolic rate by down-regulating the expression of genes involved in central metabolism. 17 Under BP nanosheet stress, a comprehensive analysis identified 72 pathways, with 69 showing up-regulation and 178 showing down-regulation of DEGs compared to the control group. Among these pathways, 51 were found to be related to metabolism, with 60 (which was not certified by peer review) is the author/funder.…”

“…20 Furthermore, the induction mechanisms of the VBNC state have been elucidated in various bacterial strains, including E. coli, Lactobacillus acetotolerans, and Rhodococcus biphenylivorans, with studies demonstrating that changes in gene expression in VBNC cells depend on the specific inducer. 17 Several studies have shown that nanomaterials can induce bacteria to enter the VBNC state. For example, silver nanoparticles have been identified as inducers of the VBNC state in Pseudomonas aeruginosa (P. aeruginosa).…”

Comparable antibacterial effects and action mechanisms of ethyl cyanoacrylate nanoparticles on Bacillus subtilis and Escherichia coli evaluated by transcriptome and morphological changes

“…33 Meanwhile, it has been proved that oxidative damage observed in B. subtilis leads to increased expression of katA (a catalase encoding gene) and namA (an antioxidant enzyme gene), which protect the cell from oxidative stress. Osmotic stress 42 and antibiotic treatment 24 have been previously reported to lead to transcriptional upregulation of these genes.…”