9 research outputs found
Cell-to-cell molecular transfer and environmental stress adaptation in Bacillus subtilis
In this thesis, we approached two different subjects, both focusing on a popular Gram-positive model organism - Bacillus subtilis. In the first experimental part of this thesis (Chapters 2 and 3), we addressed the intra- and interspecies transfer of DNA and protein transfer between B. subtilis and members of lactic acid bacteria (LAB). Here, we showed the ubiquity of horizontal gene transfer (HGT) in mixed co-cultures and proposed novel applications for natural strain improvement which are highly desired in producing novel starter cultures for food fermentation. The second part of this dissertation explores the physiological and transcriptional responses of B. subtilis to osmotic and antibiotic stress on a population-wide and single-cell level (Chapters 4, 5 and 6). This work sheds new light on how preadaptation to adverse conditions can modulate metabolic activity, affect cell membrane polarization, and finally increase tolerance to certain antibiotics in bacteria. We showed that B. subtilis, after being pretreated with osmotic stress, enters the viable but non-culturable state (VBNC) and becomes resistant to aminoglycoside antibiotics. Besides, the observations made in this work draw attention to the typical methods of cell enumeration and their error-prone outcomes and highlight the importance of VBNC bacteria when testing new antibiotic compounds for clinical applications
Transcriptome analysis and prediction of the metabolic state of stress-induced viable but non-culturable Bacillus subtilis cells
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
Diversity of bet-hedging strategies in microbial communities-Recent cases and insights
Microbial communities are continuously exposed to unpredictable changes in their environment. To thrive in such dynamic habitats, microorganisms have developed the ability to readily switch phenotypes, resulting in a number of differently adapted subpopulations expressing various traits. In evolutionary biology, a particular case of phenotypic heterogeneity that evolved in an unpredictably changing environment has been defined as betâhedging. Betâhedging is a riskâspreading strategy where isogenic populations stochastically (randomly) diversify their phenotypes, often resulting in maladapted individuals that suffer lower reproductive success. This fitness tradeâoff in a specific environment may have a selective advantage upon the sudden environmental shift. Thus, a betâhedging strategy allows populations to persist in very dynamic habitats, but with a particular fitness cost. In recent years, numerous examples of phenotypic heterogeneity in different microorganisms have been observed, some suggesting betâhedging. Here, we highlight the latest reports concerning betâhedging phenomena in various microorganisms to show how versatile this strategy is within the microbial realms. This article is categorized under: Infectious Diseases > Molecular and Cellular Physiology
Stress-induced activation of the proline biosynthetic pathway in Bacillus subtilis:A population-wide and single-cell study of the osmotically controlled proHJ promoter
Bacillus subtilis, in its natural habitat, is regularly exposed to rapid changes in the osmolarity of its surrounding. As its primary survival strategy, it accumulates large amounts of the compatible solute proline by activating the de novo proline biosynthesis pathway and exploiting the glutamate pools. This osmotically-induced biosynthesis requires activation of a SigA-type promoter that drives the expression of the proHJ operon. Population-wide studies have shown that the activity of the proHJ promoter correlates with the increased osmotic pressure of the environment. Therefore, the activation of the proHJ transcription should be an adequate measure of the adaptation to osmotic stress through proline synthesis in the absence of other osmoprotectants. In this study, we investigate the kinetics of the proHJ promoter activation and the early adaptation to mild osmotic upshift at the single-cell level. Under these conditions, we observed a switching point and heterogeneous proline biosynthesis gene expression, where the subpopulation of cells showing active proHJ transcription is able to continuously divide, and those unresponsive to osmotic stress remain dormant. Additionally, we demonstrate that bactericidal antibiotics significantly upregulate proHJ transcription in the absence of externally imposed osmotic pressure, suggesting that the osmotically-controlled proline biosynthesis pathway is also involved in the antibiotic-mediated stress response
Analyses of competent and non-competent subpopulations of Bacillus subtilis reveal yhfW, yhxC and ncRNAs as novel players in competence
Upon competence-inducing nutrient-limited conditions, only part of the Bacillus subtilis population becomes competent. Here, we separated the two subpopulations by fluorescence-assisted cell sorting (FACS). Using RNA-seq, we confirmed the previously described ComK regulon. We also found for the first time significantly downregulated genes in the competent subpopulation. The downregulated genes are not under direct control by ComK but have higher levels of corresponding antisense RNAs in the competent subpopulation. During competence, cell division and replication are halted. By investigating the proteome during competence, we found higher levels of the regulators of cell division, MinD and Noc. The exonucleases SbcC and SbcD were also primarily regulated at the post-transcriptional level. In the competent subpopulation, yhfW was newly identified as being highly upregulated. Its absence reduces the expression of comG, and has a modest, but statistically significant effect on the expression of comK. Although expression of yhfW is higher in the competent subpopulation, no ComK-binding site is present in its promoter region. Mutants of yhfW have a small but significant defect in transformation. Metabolomic analyses revealed significant reductions in tricarboxylic acid (TCA) cycle metabolites and several amino acids in a ÎyhfW mutant. RNA-seq analysis of ÎyhfW revealed higher expression of the NAD synthesis genes nadA, nadB and nadC
Cooperação internacional catĂłlica: entre a polĂtica dos movimentos sociais e o ativismo da fĂ© em ação
Cell-to-cell non-conjugative plasmid transfer between Bacillus subtilis and lactic acid bacteria
Bacillus subtilis is a soil-dwelling bacterium that can interact with a plethora of other microorganisms in its natural habitat. Due to the versatile interactions and its ability to form nanotubes, i.e., recently described membrane structures that trade cytoplasmic content between neighbouring cells, we investigated the potential of HGT from B. subtilis to industrially-relevant members of lactic acid bacteria (LAB). To explore the interspecies HGT events, we developed a co-culturing protocol and provided proof of transfer of a small high copy non-conjugative plasmid from B. subtilis to LABs. Interestingly, the plasmid transfer did not involve conjugation nor activation of the competent state by B. subtilis. Moreover, our study shows for the first time non-conjugative cell-to-cell intraspecies plasmid transfer for non-competent Lactococcus lactis sp. cremoris strains. Our study indicates that cell-to-cell transformation is a ubiquitous form of HGT and can be potentially utilized as an alternative tool for natural (non-GMO) strain improvement
Diversity of bet-hedging strategies in microbial communities-Recent cases and insights
Microbial communities are continuously exposed to unpredictable changes in their environment. To thrive in such dynamic habitats, microorganisms have developed the ability to readily switch phenotypes, resulting in a number of differently adapted subpopulations expressing various traits. In evolutionary biology, a particular case of phenotypic heterogeneity that evolved in an unpredictably changing environment has been defined as bet-hedging. Bet-hedging is a risk-spreading strategy where isogenic populations stochastically (randomly) diversify their phenotypes, often resulting in maladapted individuals that suffer lower reproductive success. This fitness trade-off in a specific environment may have a selective advantage upon the sudden environmental shift. Thus, a bet-hedging strategy allows populations to persist in very dynamic habitats, but with a particular fitness cost. In recent years, numerous examples of phenotypic heterogeneity in different microorganisms have been observed, some suggesting bet-hedging. Here, we highlight the latest reports concerning bet-hedging phenomena in various microorganisms to show how versatile this strategy is within the microbial realms
Renaissance of traditional DNA transfer strategies for improvement of industrial lactic acid bacteria
The ever-expanding genomic insight in natural diversity of lactic acid bacteria (LAB) has revived the industrial interest in traditional and natural genetic mobilization methodologies. Here, we review recent advances in horizontal gene transfer processes in LAB, including natural competence, conjugation, and phage transduction. In addition, we envision the possibilities for industrial strain improvement arising from the recent discoveries of molecular exchanges between bacteria through nanotubes and extracellular vesicles, as well as the constantly expanding genome editing possibilities using the CRISPR-Cas technology.</p