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.

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.

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.

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. This article is categorized under: Infectious Diseases > Molecular and Cellular Physiology.

Antibiotic tolerance in environmentally stressed Bacillus subtilis: physical barriers and induction of a viable but nonculturable state.

Bacterial communities exposed to rapid changes in their habitat encounter different forms of stress. Fluctuating conditions of the microenvironment drive microorganisms to develop several stress responses to sustain growth and division, like altering gene expression and changing the cell's physiology. It is commonly known that these protection systems may give rise to differently adapted subpopulations and indirectly impact bacterial susceptibility to antimicrobials. This study focuses on the adaptation of a soil-dwelling bacterium, Bacillus subtilis, to sudden osmotic changes, including transient and sustained osmotic upshift. Here, we demonstrate that physiological changes caused by pre-exposure to osmotic stress facilitate B. subtilis' entry into a quiescent state, helping them survive when exposed to a lethal antibiotic concentration. We show that the adaptation to transient osmotic upshift with 0.6 M NaCl causes decreased metabolic rates and lowered antibiotic-mediated ROS production when cells were exposed to the aminoglycoside antibiotic kanamycin. Using a microfluidic platform combined with time-lapse microscopy, we followed the uptake of fluorescently labelled kanamycin and examined the metabolic activity of differently preadapted populations at a single-cell level. The microfluidics data revealed that under the conditions tested, B. subtilis escapes from the bactericidal activity of kanamycin by entering into a nongrowing dormant state. Combining single-cell studies and population-wide analysis of differently preadapted cultures, we demonstrate that kanamycin-tolerant B. subtilis cells are entrapped in a viable but nonculturable (VBNC) state.

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.