Single Bacillus Subtilis Research Articles

Classification of antimicrobial mechanism of action using dynamic bacterial morphology imaging

Antimicrobial resistance is a major threat to human health. Basic knowledge of antimicrobial mechanism of action (MoA) is imperative for patient care and for identification of novel antimicrobials. However, the process of antimicrobial MoA identification is relatively laborious. Here, we developed a simple, quantitative time-lapse fluorescence imaging method, Dynamic Bacterial Morphology Imaging (DBMI), to facilitate this process. It uses a membrane dye and a nucleoid dye to track the morphological changes of single Bacillus subtilis cells in response to antimicrobials for up to 60 min. DBMI of bacterial cells facilitated assignment of the MoAs of 14 distinct, known antimicrobial compounds to the five main classes. We conclude that DBMI is a simple method, which facilitates rapid classification of the MoA of antimicrobials in functionally distinct classes.

Biphasic Cell-Size and Growth-Rate Homeostasis by Single Bacillus subtilis Cells

The growth rate of single bacterial cells is continuously disturbed by random fluctuations in biosynthesis rates and by deterministic cell-cycle events, such as division, genome duplication, and septum formation. It is not understood whether, and how, bacteria reject these growth-rate disturbances. Here, we quantified growth and constitutive protein expression dynamics of single Bacillus subtilis cells as a function of cell-cycle progression. We found that, even though growth at the population level is exponential, close inspection of the cell cycle of thousands of single Bacillus subtilis cells reveals systematic deviations from exponential growth. Newborn cells display varying growth rates that depend on their size. When they divide, growth-rate variation has decreased, and growth rates have become birth size independent. Thus, cells indeed compensate for growth-rate disturbances and achieve growth-rate homeostasis. Protein synthesis and growth of single cells displayed correlated, biphasic dynamics from cell birth to division. During a first phase of variable duration, the absolute rates were approximately constant and cells behaved as sizers. In the second phase, rates increased, and growth behavior exhibited characteristics of a timer strategy. These findings demonstrate that, just like size homeostasis, growth-rate homeostasis is an inherent property of single cells that is achieved by cell-cycle-dependent rate adjustments of biosynthesis and growth.

Real-time attack of LL-37 on single Bacillus subtilis cells

Time-lapse fluorescence microscopy of single, growing Bacillus subtilis cells with 2–12s time resolution reveals the mechanisms of antimicrobial peptide (AMP) action on a Gram-positive species with unprecedented detail. For the human cathelicidin LL-37 attacking B. subtilis, the symptoms of antimicrobial stress differ dramatically depending on the bulk AMP concentration. At 2μM LL-37, the mean single-cell growth rate decreases, but membrane permeabilization does not occur. At 4μM LL-37, cells abruptly shrink in size at the same time that Sytox Green enters the cytoplasm and stains the nucleoids. We interpret the shrinkage event as loss of turgor pressure (and presumably the membrane potential) due to permeabilization of the membrane. Movies of Sytox Green staining at 0.5frame/s show that nucleoid staining is initially local, more consistent with pore formation than with global permeabilization models. In a novel “growth recovery” assay, cells are incubated with LL-37 for a variable period and then rinsed with fresh growth medium lacking LL-37. The growth rate attenuation observed at 2μM LL-37 is a recoverable symptom, while the abrupt cell shrinkage observed at 4μM LL-37 is not.

Temporal competition between differentiation programs determines cell fate choice

Multipotent differentiation, where cells adopt one of several possible fates, occurs in diverse systems ranging from bacteria to mammals. This decision-making process is driven by multiple differentiation programs that operate simultaneously in the cell. How these programs interact to govern cell fate choice is poorly understood. To investigate this issue, we simultaneously measured activities of the competing sporulation and competence programs in single Bacillus subtilis cells. This approach revealed that these competing differentiation programs progress independently without cross-regulation before the decision point. Cells seem to arrive at a fate choice through differences in the relative timing between the two programs. To test this proposed dynamic mechanism, we altered the relative timing by engineering artificial cross-regulation between the sporulation and competence circuits. Results suggest a simple model that does not require a checkpoint or intricate cross-regulation before cellular decision-making. Rather, cell fate choice appears to be the outcome of a 'molecular race' between differentiation programs that compete in time, providing a simple dynamic mechanism for decision-making.

Microcalorimetric measurements of the microbial activities of single- and mixed-species with trivalent iron in soil

A microcalorimetric technique was applied to a series of experiments to follow the toxic effect caused by the trivalent iron on the single and mixed microbes in sterilized soil that was inoculated with the single Bacillus subtilis (B. subtilis) (prokaryotic bacterium), single Candida humicola (C. humicola) (eukaryotic fungus) and the mixed-species. The microbial activity was stimulated by the addition of 5.0mg glucose and 5.0mg ammonium sulfate under a 35% controlled humidity in the studied soil samples of 1.2g. The power–time curves from every experiment were analyzed, and from these analyses characteristic parameters, such as growth rate constant (k) and total thermal effect (Q) which can reflect the biochemical reactions were determined. The mixed-species have moderate tolerance to the iron overload, comparing with single species, and exhibit synergistic interaction in exponential growth phase (0–400.0μgmL−1). Meanwhile, there is no much difference in the thermal effect (Q) per gram soil sample for the single and mixed culture. This also validates that the nutrient substances in natural environment determine the organisms’ metabolic activities. Ultraviolet–visible spectrophotometry and dissolved oxygen sensor also were successfully applied to reflect the activities of B. subtilis and C. humicola in the pure culture. The investigation could provide insight into the microbial ecology of bacteria and fungi in ecological niches.

Simultaneous forward- and backward-hemisphere elastic-light-scattering patterns of respirable-size aerosols

Two-dimensional angular optical scattering (TAOS) patterns of aerosols are measured simultaneously from the forward hemisphere 15 degrees < theta < 90 degrees as well as the backward hemisphere 90 degrees < theta < 165 degrees (detecting 63% of the 4pi sr of scattered light) by using an ellipsoidal reflector and an intensified CCD detector. TAOS patterns were obtained from polystyrene-latex spheres (individuals and aggregates) and from single Bacillus subtilis spores. These information-rich patterns, measured with a single laser pulse for individual particles on the fly, suggest that forward-TAOS and backward-TAOS measurements may be used for rapid classification of single aerosol particles.

Angularly-resolved elastic scatter from single particles collected over a large solid angle and with high resolution

Elastic light scattering from a single non-spherical particle of various morphologies has been measured simultaneously with a large angular range (90° < θ < 165° and 0° < ϕ < 360°) and with high angular resolution (1024 pixels in θ and 512 pixels in ϕ). Because the single-shot laser pulse is short (pulse duration of 70 ns), the tumbling and flowing particle can be treated as frozen in space. The large angle two-dimensional angular optical scattering (hereafter referred to as LA TAOS) intensity pattern, I(θ,ϕ), has been measured for a variety of particle morphology, such as the following: (1) single polystyrene latex (PSL) sphere; (2) cluster of PSL spheres; (3) single Bacillus subtilis (BG) spore; (4) cluster of BG spores; (5) dried aggregates of bio-aerosols as well as background clutter aerosols. All these measurements were made using the second harmonic of a Nd:YAG laser (0.532 μm). Islands structures in the LA TAOS patterns seem to be the prominent feature. Efforts are being made to extract metrics from these islands and compare them to theoretical results based on the T-matrix method.