Dissipation of Proton Motive Force is not Sufficient to Induce the Phage Shock Protein Response in Escherichia coli

Phage shock proteins (Psp) and their homologues are found in species from the three domains of life: Bacteria, Archaea and Eukarya (e.g. higher plants). In enterobacteria, the Psp response helps to maintain the proton motive force (PMF) of the cell when the inner membrane integrity is impaired. The presumed ability of ArcB to sense redox changes in the cellular quinone pool and the strong decrease of psp induction in ΔubiG or ΔarcAB backgrounds suggest a link between the Psp response and the quinone pool. The authors now provide evidence indicating that the physiological signal for inducing psp by secretin-induced stress is neither the quinone redox state nor a drop in PMF. Neither the loss of the H+-gradient nor the dissipation of the electrical potential alone is sufficient to induce the Psp response. A set of electron transport mutants differing in their redox states due to the lack of a NADH dehydrogenase and a quinol oxidase, but retaining a normal PMF displayed low levels of psp induction inversely related to oxidised ubiquinone levels under microaerobic growth and independent of PMF. In contrast, cells displaying higher secretin induced psp expression showed increased levels of ubiquinone. Taken together, this study suggests that not a single but likely multiple signals are needed to be integrated to induce the Psp response

Analysis of Temporal Gene Expression during Bacillus subtilis Spore Germination and Outgrowth

Bacillus subtilis forms dormant spores upon nutrient depletion. Under favorable environmental conditions, the spore breaks its dormancy and resumes growth in a process called spore germination and outgrowth. To elucidate the physiological processes that occur during the transition of the dormant spore to an actively growing vegetative cell, we studied this process in a time-dependent manner by a combination of microscopy, analysis of extracellular metabolites, and a genome-wide analysis of transcription. The results indicate the presence of abundant levels of late sporulation transcripts in dormant spores. In addition, the results suggest the existence of a complex and well-regulated spore outgrowth program, involving the temporal expression of at least 30% of the B. subtilis genome

Challenges and advances in systems biology analysis of Bacillus spore physiology; molecular differences between an extreme heat resistant spore forming Bacillus subtilis food isolate and a laboratory strain

Bacterial spore formers are prime organisms of concern in the food industry. Spores from the genus Bacillus are extremely stress resistant, most notably exemplified by high thermotolerance. This sometimes allows surviving spores to germinate and grow out to vegetative cells causing food spoilage and possible intoxication. Similar issues though more pending toward spore toxigenicity are observed for the anaerobic Clostridia. The paper indicates the nature of stress resistance and highlights contemporary molecular approaches to analyze the mechanistic basis of it in Bacilli. A molecular comparison between a laboratory strain and a food borne isolate, very similar at the genomic level to the laboratory strain but generating extremely heat resistant spores, is discussed. The approaches cover genome-wide genotyping, proteomics and genome-wide expression analyses studies. The analyses aim at gathering sufficient molecular information to be able to put together an initial framework for dynamic modelling of spore germination and outgrowth behaviour. Such emerging models should be developed both at the population and at the single spore level. Tools and challenges in achieving the latter are succinctly discussed

Live Cell Imaging of Germination and Outgrowth of Individual <em>Bacillus subtilis</em> Spores; the Effect of Heat Stress Quantitatively Analyzed with SporeTracker

<div><p>Spore-forming bacteria are a special problem for the food industry as some of them are able to survive preservation processes. <i>Bacillus</i> spp. spores can remain in a dormant, stress resistant state for a long period of time. Vegetative cells are formed by germination of spores followed by a more extended outgrowth phase. Spore germination and outgrowth progression are often very heterogeneous and therefore, predictions of microbial stability of food products are exceedingly difficult. Mechanistic details of the cause of this heterogeneity are necessary. In order to examine spore heterogeneity we made a novel closed air-containing chamber for live imaging. This chamber was used to analyze <i>Bacillus subtilis</i> spore germination, outgrowth, as well as subsequent vegetative growth. Typically, we examined around 90 starting spores/cells for ≥4 hours per experiment. Image analysis with the purposely built program “SporeTracker” allows for automated data processing from germination to outgrowth and vegetative doubling. In order to check the efficiency of the chamber, growth and division of <i>B. subtilis</i> vegetative cells were monitored. The observed generation times of vegetative cells were comparable to those obtained in well-aerated shake flask cultures. The influence of a heat stress of 85°C for 10 min on germination, outgrowth, and subsequent vegetative growth was investigated in detail. Compared to control samples fewer spores germinated (41.1% less) and fewer grew out (48.4% less) after the treatment. The heat treatment had a significant influence on the average time to the start of germination (increased) and the distribution and average of the duration of germination itself (increased). However, the distribution and the mean outgrowth time and the generation time of vegetative cells, emerging from untreated and thermally injured spores, were similar.</p> </div

Schematic picture of the top and side view of the designed closed air-containing chamber for live cell imaging.

<p>A chamber was prepared by attaching a Gene Frame® to a standard microscope slide and cover slip. A thin, semisolid matrix pad (160 µm) of 1% agarose – medium was made. The pad was loaded with exponentially growing vegetative cells or heat-activated spores. The cover slip was placed in upside down position onto the Gene Frame® (See Materials and Methods for details).</p

Time-resolved images showing heterogeneous germination and outgrowth of <i>B. subtilis</i> 1A700 spores on minimal medium.

<p>Heat-activated spores (70°C for 30 min) were spotted on 100% defined minimal (MOPS-buffered) medium including AGFK and followed in time using phase-contrast microscopy. The spore marked in the square (Panel <b>A</b>) becomes phase-dark (germinates) within 60 min (Panel <b>B</b>), grows out, and forms a microcolony (Panel <b>F</b>). The spore marked in the circle (Panel <b>A</b>) becomes phase-dark (Panel <b>B</b>) but does not grow out within 5 hours (Panel <b>F</b>). The spore marked in the triangle (Panel <b>A</b>) remains phase-bright throughout the experiment (Panel <b>F</b>).</p