Project description:Biofilms are structured communities of tightly associated cells that constitute thepredominant state of bacterial growth in naturaland human-madeenvironments. Although the core genetic circuitry that controls biofilm formation in model bacteria such as Bacillus subtilishas been well characterized, little is known about the role that metabolism plays in this complex developmental process. Here, weperformed a time-resolved analysisof the metabolic changes associated with pellicle biofilm formation and development inB. subtilisby combining metabolomic, transcriptomic, and proteomic analyses. We report a surprisingly widespread and dynamic remodeling of metabolism affecting central carbon metabolism, primary biosynthetic pathways, fermentation pathways, and secondary metabolism. Most of these metabolic alterations were hithertounrecognized as biofilm-associated.For example, we observed increased activity of the tricarboxylic acid (TCA) cycle during early biofilm growth, a shift from fatty acid biosynthesis to fatty acid degradation, reorganization of iron metabolism and transport, and a switch from acetate to acetoin fermentation. Close agreement between metabolomic, transcriptomic, and proteomic measurements indicated that remodeling of metabolism during biofilm development was generally controlled at the transcriptional level. Our resultsalsoprovide insights into the transcription factors and regulatory networks involved in thiscomplexmetabolic remodeling. Following upon these results, we demonstrate that acetoin production via acetolactate synthase is essential for robust biofilm growthand has the dual role of conservingredox balance and maintaining extracellularpH.This study represents a comprehensive systems-level investigation of the metabolic remodeling occurring during B. subtilisbiofilm development that will serve as a useful roadmap for future studies on biofilm physiology.

Project description: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. Per acid down-shock three exposure times (i.e., 10, 30 and 60 min) were each compared with non-exposed cells (i.e., t0). In total 4 different pH 5.5 acid down-shocks were applied. pH 5.5 was reached by adding different acidulants i.e., hydrochloric acid (HCl), lactic acid (HL) resulting in 2 mM undissociated HL, acetic acid (HAc) resulting in 15 mM undissociated HAc, and a combination of acetic acid and hydrochloric acid (HAc/HCl) resulting in 2 mM undissociated HAc. The experiments were performed in duplicate and the duplicate samples were hybridised with a dye-swap.

Project description:Pathogens that grow during infections must cope with the reactive oxygen species (ROS) released by host immune cells. Among the different strategies to prevent oxidative damage during infections, pathogenic bacteria have evolved mechanisms to reduce respiration and other cellular processes that are particularly sensitive to free radicals, obtaining energy preferentially by undergoing to fermentative metabolism. As fermentation produces fewer ATP per glucose than respiration, bacteria ensure an appropriate supply of energy by increasing the glycolytic flux. The opportunistic human pathogen Staphylococcus aureus is one such microbe but the underlying mechanism that enables S. aureus to induce fermentation during infections is still unclear. Here we show that the ComK-like regulator of natural competence that is present in many gram-positive bacteria is crucial to redirect S. aureus metabolism to fermentation during infection. ComK is cryptic in laboratory conditions but highly induced during infections or in response to infection-related cues, such as ROS. ComK induces the glycolytic flux and glucose consumption rate, a key step to redirect ATP production to fermentation. This licenses fermenting S. aureus to reduce oxidative damage while increasing DNA uptake by natural transformation. As a consequence, a comK-defective mutant shows an accumulation of ROS as well as DNA mutations that lower bacterial survival. This mutant shows no distinctive phenotype in laboratory conditions but is unable to cause infection in vertebrates or invertebrate infection models. ComK-mediated synchronization of natural transformation to fermentative metabolism may allow S. aureus, and probably other gram-positive bacteria, to use the fermentation acid end products to eliminate bacterial competitors while assimilating their DNA. Assimilated DNA may serve as a source of nucleotides for DNA repair or to promote genetic variability, thereby enabling a successful host colonization of this bacterium.

Project description: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.

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CHCC2862_Respiratory_TimeCourse_EntryIntoPlusStationary

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