Characterization of sporulation dynamics of Pseudoclostridium thermosuccinogenes using flow cytometry

Single-cell analysis of microbial population heterogeneity is a fast growing research area in microbiology due to its potential to identify and quantify the impact of subpopulations on microbial performance in, for example, industrial biotechnology, environmental biology, and pathogenesis. Although several tools have been developed, determination of population heterogenity in anaerobic bacteria, especially spore-forming clostridia species has been amply studied. In this study we applied single cell analysis techniques such as flow cytometry (FCM) and fluorescence-assisted cell sorting (FACS) on the spore-forming succinate producer Pseudoclostridium thermosuccinogenes. By combining FCM and FACS with fluorescent staining, we differentiated and enriched all sporulation-related morphologies of P. thermosuccinogenes. To evaluate the presence of metabolically active vegetative cells, a blend of the dyes propidium iodide (PI) and carboxy fluorescein diacetate (cFDA) tested best. Side scatter (SSC-H) in combination with metabolic indicator cFDA dye provided the best separation of sporulation populations. Based on this protocol, we successfully determined culture heterogeneity of P. thermosuccinogenes by discriminating between mature spores, forespores, dark and bright phase endospores, and vegetative cells populations. Henceforth, this methodology can be applied to further study sporulation dynamics and its impact on fermentation performance and product formation by P. thermosuccinogenes.</p

Comparative transcriptome analysis of biofilm and planktonic cells of Bacillus cereus ATCC 10987

Planktonic and biofilm cells of Bacillus cereus ATCC 14579 and ATCC 10987 were studied using microscopy and transcriptome analysis. By microscopy, clear differences could be observed between biofilm and planktonic cells as well as between the two strains. By using hierarchical clustering of the transcriptome data, little difference was observed between the biofilm cells of B. cereus ATCC 14579 and ATCC 10987. Different responses between biofilm and planktonic cells could be identified using transcriptome analysis. Biofilm formation seemed to cause a shift in metabolism with up- or down-regulation of genes involved in different metabolic pathways. Genes involved in motility were down-regulated. No clear up-regulation related to capsular or extracellular polysaccharides was observed. Sporulation was observed in biofilm cells using microscopy, which was corroborated with up-regulation of genes involved in sporulation in biofilm cells. The results obtained in this study provide insight in general and strain specific behavior of B. cereus cells in multicellular communities

Comparative transcriptome analysis of biofilm and planktonic cells of Bacillus cereus ATCC 14579

Planktonic and biofilm cells of Bacillus cereus ATCC 14579 and ATCC 10987 were studied using microscopy and transcriptome analysis. By microscopy, clear differences could be observed between biofilm and planktonic cells as well as between the two strains. By using hierarchical clustering of the transcriptome data, little difference was observed between the biofilm cells of B. cereus ATCC 14579 and ATCC 10987. Different responses between biofilm and planktonic cells could be identified using transcriptome analysis. Biofilm formation seemed to cause a shift in metabolism with up- or down-regulation of genes involved in different metabolic pathways. Genes involved in motility were down-regulated. No clear up-regulation related to capsular or extracellular polysaccharides was observed. Sporulation was observed in biofilm cells using microscopy, which was corroborated with up-regulation of genes involved in sporulation in biofilm cells. The results obtained in this study provide insight in general and strain specific behavior of B. cereus cells in multicellular communities

Heterogeneity in single-cell outgrowth of Listeria monocytogenes in half Fraser enrichment broth is affected by strain variability and physiological state

The behaviour of pathogens at the single-cell level can be highly variable and can thus affect the detection efficacy of enrichment-based detection methods. The outgrowth of single cells of three Listeria monocytogenes strains was monitored after fluorescence-activated single-cell sorting in non-selective brain heart infusion (BHI) broth and selective half Fraser enrichment broth (HFB) to quantify outgrowth heterogeneity and its effect on the detection probability. Single-cell heterogeneity was higher in HFB compared to non-selective BHI and heterogeneity increased further when cells were heat-stressed. The increase in heterogeneity was also strain-dependent because the fast-recovering strain Scott A showed less outgrowth heterogeneity than the slower-recovering strains EGDe and H7962. Modelling of the outgrowth kinetics during the primary enrichment demonstrated that starting at low cell concentrations could fail detection of L. monocytogenes at least partly due to cell heterogeneity. This highlights that it is important to take single-cell heterogeneity into account when optimizing enrichment formulations and procedures when L. monocytogenes contamination levels are low

Analysis of the Role of RsbV, RsbW, and RsbY in Regulating σ(B) Activity in Bacillus cereus

The alternative sigma factor σ(B) is an important regulator of the stress response of Bacillus cereus. Here, the role of the regulatory proteins RsbV, RsbW, and RsbY in regulating σ(B) activity in B. cereus is analyzed. Functional characterization of RsbV and RsbW showed that they act as an anti-sigma factor antagonist and an anti-sigma factor, respectively. RsbW can also act as a kinase on RsbV. These data are in line with earlier functional characterizations of RsbV and RsbW homologs in B. subtilis. The rsbY gene is unique to B. cereus and its closest relatives and is predicted to encode a protein with an N-terminal CheY domain and a C-terminal PP2C domain. In an rsbY deletion mutant, the σ(B) response upon stress exposure was almost completely abolished, but the response could be restored by complementation with full-length rsbY. Expression analysis showed that rsbY is transcribed from both a σ(A)-dependent promoter and a σ(B)-dependent promoter. The central role of RsbY in regulating the activity of σ(B) indicates that in B. cereus, the σ(B) activation pathway is markedly different from that in other gram-positive bacteria

Analysis of germination and outgrowth of sorbic acid-stressed Bacillus cereus ATCC 14579 spores.

Sorbic acid (SA) is widely used as a preservative, but the effect of SA on spore germination and outgrowth has gained limited attention up to now. Therefore, the effect of sorbic acid on germination of spores of B. cereus strain ATCC 14579 was analyzed both at phenotype and transcriptome level. Spore germination and outgrowth was assessed at pH 5.5 without and with 0.75, 1.5 and 3.0mM (final concentrations) undissociated sorbic acid (HSA). This resulted in distinct HSA concentration-dependent phenotypes, varying from delays in germination and outgrowth to complete blockage of germination at 3.0mM HSA. The phenotypes reflecting different stages in the germination process could be confirmed using flow cytometry and could be recognized at transcriptome level by distinct expression profiles. In the absence and presence of 0.75 and 1.5mM HSA, similar cellular ATP levels were found up to the initial stage of outgrowth, suggesting that HSA-induced inhibition of outgrowth is not caused by depletion of ATP. Transcriptome analysis revealed the presence of a limited number of transcripts in dormant spores, outgrowth related expression, and genes specifically associated with sorbic acid stress, including alterations in cell envelope and multi-drug resistance. The potential role of these HSA-stress associated genes in spore outgrowth is discussed

Analysis of germination and outgrowth of sorbic acid-stressed Bacillus cereus ATCC 14579 spores.

Sorbic acid (SA) is widely used as a preservative, but the effect of SA on spore germination and outgrowth has gained limited attention up to now. Therefore, the effect of sorbic acid on germination of spores of B. cereus strain ATCC 14579 was analyzed both at phenotype and transcriptome level. Spore germination and outgrowth was assessed at pH 5.5 without and with 0.75, 1.5 and 3.0mM (final concentrations) undissociated sorbic acid (HSA). This resulted in distinct HSA concentration-dependent phenotypes, varying from delays in germination and outgrowth to complete blockage of germination at 3.0mM HSA. The phenotypes reflecting different stages in the germination process could be confirmed using flow cytometry and could be recognized at transcriptome level by distinct expression profiles. In the absence and presence of 0.75 and 1.5mM HSA, similar cellular ATP levels were found up to the initial stage of outgrowth, suggesting that HSA-induced inhibition of outgrowth is not caused by depletion of ATP. Transcriptome analysis revealed the presence of a limited number of transcripts in dormant spores, outgrowth related expression, and genes specifically associated with sorbic acid stress, including alterations in cell envelope and multi-drug resistance. The potential role of these HSA-stress associated genes in spore outgrowth is discussed

Spore formation.

<p>Number of spores and total viable counts in BHI formed by <i>B</i>. <i>cereus</i> ATCC 14579 WT, Δ<i>rpoN</i> and Δ<i>rpoN</i>-comp in BHI following aerated growth at 30°C for 48 h. The asterisk indicates significant difference (student’s t test, p<0.05) of the Δ<i>rpoN</i> compared to both the WT and Δ<i>rpoN</i>-comp.</p

Transcriptomic and physiological responses of Bacillus cereus to organic and inorganic acid down-shocks

Comparative phenotype and transcriptome analyses were performed with Bacillus cereus ATCC 14579 exposed to acid down-shock to pH 5.5 set with different acidulants. When acidified with hydrochloric acid (HCl), growth was diminished, whereas 2 mM undissociated lactic acid (HL) or acetic acid (HAc) stopped growth without inactivation (bacteriostatic condition), and 15 mM undissociated HAc caused growth arrest and, finally, cell death, as reflected by a 3 to 4 log inactivation (bactericidal condition). Within the first 60 min after pH down-shock, the intracellular ATP levels of cultures shocked with HCl were increased. The bacteriostatic pH shocks did not result in increased nor decreased intracellular ATP levels, indicating that the high energy status within the stressed aerobically grown B. cereus cells could be maintained. In contrast, exposure to 15 mM undissociated HAc resulted in significant lower ATP levels, which was in accordance with the observed inactivation. The transcriptomic responses pH down-shocked cultures were studied in the same time frame. The analyses revealed general and specific responses coupled to the different phenotypes and the acidulant used. The general acid stress response, shown in all different pH shocks, involves modulation of pyruvate metabolism and an oxidative stress response. The shifts in pyruvate metabolism include induction dehydrogenases of a butanediol fermentation pathway under non-lethal acid stress conditions and of lactate, formate, and ethanol fermentation pathways under 15 mM HAc stress. Other 15 mM HAc-specific responses were induction of the alternative electron-transport systems, including cydAB, and fatty acid biosynthesis genes. Differences in gene expression for the bacteriostatic organic acid stress conditions compared to the growth-retarded inorganic stress condition indicated a more stringent oxidative stress response, including induction of an additional catalase gene and a gene encoding a Dps-like protein. Moreover, modulations in amino acid and oligopeptide transport were also found for the 2 mM HAc and HL shocks. HL-specific and HAc-specific responses both involve amino acid metabolism. Our study on the genome-wide responses of aerobically grown B. cereus pH 5.5 shocks provides a unique overview of the different responses induced by three acidulants relevant for food preservation