The spore coat is essential for Bacillus subtilis spore resistance to pulsed light, and pulsed light treatment eliminates some spore coat proteins.

Microbial surface contamination of equipment or of food contact material is a recurring problem in the food industry. Spore-forming bacteria are far more resistant to a wide variety of treatments than their vegetative forms. Understanding the mechanisms underlying decontamination processes is needed to improve surface decontamination strategies against endospores potentially at the source of foodborne diseases or food-spoilage. Pulsed light (PL) with xenon lamps delivers high-energy short-time pulses of light with wavelengths in the range 200 nm-1100 nm and a high UV-C fraction. Bacillus subtilis spores were exposed to either PL or to continuous UV-C. Gel electrophoresis and western blotting revealed elimination of various proteins of the spore coat, an essential outer structure that protects spores from a wide variety of environmental conditions and inactivation treatments. Proteomic analysis confirmed the elimination of some spore coat proteins after PL treatment. Transmission electron microscopy of PL treated spores revealed a gap between the lamellar inner spore coat and the outer spore coat. Overall, spores of mutant strains with defects in genes coding for spore coat proteins were more sensitive to PL than to continuous UV-C. This study demonstrates that radiations delivered by PL contribute to specific damage to the spore coat, and overall to spore inactivation.

Listeria monocytogenes Biofilm Adaptation to Different Temperatures Seen Through Shotgun Proteomics.

Listeria monocytogenes is a foodborne pathogen that can cause invasive severe human illness (listeriosis) in susceptible patients. Most human listeriosis cases appear to be caused by consumption of refrigerated ready-to-eat foods. Although initial contamination levels in foods are usually low, the ability of these bacteria to survive and multiply at low temperatures allows it to reach levels high enough to cause disease. This study explores the set of proteins that might have an association with L. monocytogenes adaptation to different temperatures. Cultures were grown in biofilm, the most widespread mode of growth in natural and industrial realms. Protein extractions were performed from three different growth temperatures (10, 25, and 37°C) and two growth phases (early stage and mature biofilm). L. monocytogenes subproteomes were targeted using three extraction methods: trypsin-enzymatic shaving, biotin-labeling and cell fractionation. The different subproteomes obtained were separated and analyzed by shotgun proteomics using high-performance liquid chromatography combined with tandem mass spectrometry (LC-OrbiTrap LTQVelos, ThermoFisher Scientific). A total of 141 (biotinylation), 98 (shaving) and 910 (fractionation) proteins were identified. Throughout the 920 unique proteins identified, many are connected to basic cell functions, but some are linked with thermoregulation. We observed some noteworthy protein abundance shifts associated with the major adaptation to cold mechanisms present in L. monocytogenes, namely: the role of ribosomes and the stressosome with a higher abundance of the general stress protein Ctc (Rl25) and the general stress transcription factor sigma B (σB), changes in cell fluidity and motility seen by higher levels of foldase protein PrsA2 and flagellin (FlaA), the uptake of osmolytes with a higher abundance of glycine betaine (GbuB) and carnitine transporters (OpucA), and the relevance of the overexpression of chaperone proteins such as cold shock proteins (CspLA and Dps). As for 37°C, we observed a significantly higher percentage of proteins associated with transcriptional or translational activity present in higher abundance upon comparison with the colder settings. These contrasts of protein expression throughout several conditions will enrich databases and help to model the regulatory circuitry that drives adaptation of L. monocytogenes to environments.

Comparison of three methods for cell surface proteome extraction of Listeria monocytogenes biofilms.

The cell surface proteome of the foodborne pathogen Listeria monocytogenes, the etiological agent of listeriosis, is critical for understanding the physiological processes associated with stress resistance and persistence in the environment. In this context, the most widespread mode of growth for bacterial cells in natural and industrial environments is in biofilms. Cell surface proteins are, however, challenging to characterize because of their low abundance and poor solubility. Moreover, cell surface protein extracts are usually contaminated with cytoplasmic proteins that constitute the main signal in proteomic analysis. This study aimed to compare the efficiency of three methods to extract and explore surface proteins of L. monocytogenes growing in a biofilm: trypsin shaving, biotinylation, and cell fractionation. Peptide separation and identification were performed by shotgun proteomics using high-performance liquid chromatography combined with tandem mass spectrometry (LC-MS/MS). The biotinylation method was the most effective in extracting surface proteins, with the lowest rate of contamination by cytoplasmic proteins. Although presenting a higher contamination rate in cytoplasmic proteins, the other two techniques allowed the identification of additional surface proteins. Seven proteins were commonly retrieved by the three methods. The extracted proteins belong to several functional classes, involved in virulence, transport, or metabolic pathways. Finally, the three extraction methods seemed complementary and their combined use improved the exploration of the bacterial surface proteome. These new findings collectively inform future discovery and translational proteomics for clinical, environmental health, and industrial applications.

MALDI mass spectrometry imaging and in situ microproteomics of Listeria monocytogenes biofilms.

MALDI-TOF Mass spectrometry Imaging (MSI) is a surface-sampling technology that can determine spatial information and relative abundance of analytes directly from biological samples. Human listeriosis cases are due to the ingestion of contaminated foods with the pathogenic bacteria Listeria monocytogenes. The reduction of water availability in food workshops by decreasing the air relative humidity (RH) is one strategy to improve the control of bacterial contamination. This study aims to develop and implement an MSI approach on L. monocytogenes biofilms and proof of concept using a dehumidified stress condition. MSI allowed examining the distribution of low molecular weight proteins within the biofilms subjected to a dehumidification environment, mimicking the one present in a food workshop (10 °C, 75% RH). Furthermore, a LC-MS/MS approach was made to link the dots between MSI and protein identification. Five identified proteins were assigned to registered MSI m/z, including two cold-shock proteins and a ligase involved in cell wall biogenesis. These data demonstrate how imaging can be used to dissect the proteome of an intact bacterial biofilm giving new insights into protein expression relating to a dehumidification stress adaptation. Data are available via ProteomeXchange with identifier PXD010444. BIOLOGICAL SIGNIFICANCE: The ready-to-eat food processing industry has the daily challenge of controlling the contamination of surfaces and machines with spoilage and pathogenic microorganisms. In some cases, it is a lost cause due to these microorganisms' capacity to withstand the cleaning treatments, like desiccation procedures. Such a case is the ubiquitous Gram-positive Bacterium Listeria monocytogenes. Its surface proteins have particular importance for the interaction with its environment, being important factors contributing to adaptation to stress conditions. There are few reproducibly techniques to obtain the surface proteins of Gram-positive cells. Here, we developed a workflow that enables the use of MALDI imaging on Gram-positive bacterium biofilms to study the impact of dehumidification on sessile cells. It will be of the most interest to test this workflow with different environmental conditions and potentially apply it to other biofilm-forming bacteria.

Desiccation: An environmental and food industry stress that bacteria commonly face.

Water is essential for all living organisms, for animals as well as for plants and micro-organisms. For these latter, the presence of water or a humid environment with a high air relative humidity (RH) is necessary for their survival and growth. Thus, variations in the availability of water or in the air relative humidity constitute widespread environmental stresses which challenge microorganisms, and especially bacteria. Indeed, in their direct environment, bacteria are often faced with conditions that remove cell-bound water through air-drying of the atmosphere. Bacterial cells are subject to daily or seasonal environmental variations, sometimes going through periods of severe desiccation. This is also the case in the food industry, where air dehumidification treatments are applied after the daily cleaning-disinfection procedures. In plants producing low-water activity products, it is also usual to significantly reduce or eliminate water usage. Periodic desiccation exposure affects bacteria viability and so they require strategies to persist. Negative effects of desiccation are wide ranging and include direct cellular damage but also changes in the biochemical and biophysical properties of cells for which planktonic cells are more exposed than cells in biofilm. Understanding the mechanisms of desiccation adaptation and tolerance has a biological and biotechnological interest. This review gives an overview of the factors influencing desiccation tolerance and the biological mechanisms involved in this stress response.

Inactivation by Pulsed Light of Bacillus subtilis Spores with Impaired Protection Factors.

The resistance to pulsed light (PL) of spores of Bacillus subtilis strain 168 and of strains with mutations increasing sensitivity to UV-C or affecting spore structure was evaluated and compared to resistance to continuous UV-C and moist heat, in order to reveal original mechanisms of inactivation by PL. Spores of B. subtilis strain 168 (1A1) and eight mutant strains (sspA, sspB, sspAB, cotA, gerE, cotE, uvrA and recA) were exposed to PL (up to 1.77 J cm-2 ), continuous UV-C (up to 147 mJ cm-2 ) and moist heat at 90°C. Spores of the strains lacking proteins linked to coat formation or structure (cotA, gerE and cotE) were markedly more sensitive to PL than 1A1, while their sensitivity to continuous UV-C or to moist heat was similar to the one of strain 1A1. Coat proteins had a major contribution to the resistance of B. subtilis spores to PL irradiation characterized by short-time and high-energy pulses of white light in the wavelengths 200-1100 nm. In contrast the role of coat proteins to UV-C or to moist heat resistance was marginal or null.

Role of pigmentation in protecting Aspergillus niger conidiospores against pulsed light radiation.

The photoprotective potential of fungus pigments was investigated by irradiating conidiospores of three Aspergillus niger strains possessing the same genetic background, but differing in their degree of pigmentation with pulsed light (PL) and monochromatic (254 nm) UV-C radiation. Spores of A. niger MA93.1 and JHP1.1 presenting, respectively, a fawn and a white pigmentation were more sensitive to PL and continuous UV-C radiation than the wild-type A. niger strain N402 possessing a dark pigment. Both spores of the dark A. niger N402 and the fawn-color mutant were equally resistant to moist heat at 56°C while spores of the white-color mutant were highly sensitive. These results indicate that melanin protects pigmented spores of A. niger from PL.

Bacillus cereus Fnr binds a [4Fe-4S] cluster and forms a ternary complex with ResD and PlcR.

BACKGROUND: Bacillus cereus is a facultative anaerobe that causes diarrheal disease in humans. Diarrheal syndrome may result from the secretion of various virulence factors including hemolysin BL and nonhemolytic enterotoxin Nhe. Expression of genes encoding Hbl and Nhe is regulated by the two redox systems, ResDE and Fnr, and the virulence regulator PlcR. B. cereus Fnr is a member of the Crp/Fnr family of iron-sulfur (Fe-S) proteins. Only its apo-form has so far been studied. A major goal in deciphering the Fnr-dependent regulation of enterotoxin genes is thus to obtain and characterize holoFnr. RESULTS: Fnr has been subjected to in vitro Fe-S cluster reconstitution under anoxic conditions. UV-visible and EPR spectroscopic analyses together with the chemical estimation of the iron content indicated that Fnr binds one [4Fe-4S]2+ cluster per monomer. Atmospheric O2 causes disassembly of the Fe-S cluster, which exhibited a half-life of 15 min in air. Holo- and apoFnr have similar affinities for the nhe and hbl promoter regions, while holoFnr has a higher affinity for fnr promoter region than apoFnr. Both the apo- and holo-form of Fnr interact with ResD and PlcR to form a ternary complex. CONCLUSIONS: Overall, this work shows that incorporation of the [4Fe-4S]2+ cluster is not required for DNA binding of Fnr to promoter regions of hbl and nhe enterotoxin genes or for the formation of a ternary complex with ResD and PlcR. This points to some new unusual properties of Fnr that may have physiological relevance in the redox regulation of enterotoxin gene regulation.

Differential translocation of green fluorescent protein fused to signal sequences of Ruminococcus albus cellulases by the Tat and Sec pathways of Escherichia coli.

Ruminococcus albus is a Gram-positive bacterium that degrades plant cell walls in the rumen of herbivores. It was described to synthesize two major glycoside-hydrolases (Cel9B and Cel48A), which are exported and anchored at the cell surface. In bacteria, proteins destined to cross the cytoplasmic membrane are synthesized as precursors and possess a signal sequence (SS) directing them to the 'Sec' (general secretory) or 'Tat' (twin arginine translocation) pathway. SS composition of Cel9B and Cel48A suggests that these two enzymes translocate using different secretory pathways. In order to confirm this hypothesis, the SSs of Cel9B and Cel48A were fused to the green fluorescent protein (GFP) and expressed in wild-type Escherichia coli and in its Tat and Sec isogenic mutants. The SS cleavage and the formation of the mature protein were then followed by Western blot and fluorescence microscopy. This study shows that the SS of Cel9B directs the preprotein to the 'Tat' translocation pathway while the GFP fused to the SS of Cel48A is exported through the 'Sec' machinery. These observations suggest that R. albus possess a Tat pathway, in addition to the general secretory pathway.

ResDE-dependent regulation of enterotoxin gene expression in Bacillus cereus: evidence for multiple modes of binding for ResD and interaction with Fnr.

In the food-borne pathogen Bacillus cereus F4430/73, the production of major virulence factors hemolysin BL (Hbl) and nonhemolytic enterotoxin (Nhe) is regulated through complex mechanisms. The two-component regulatory system ResDE is involved in the activation of hbl and nhe transcription. Here, the response regulator ResD and the sensor kinase ResE were overexpressed and purified, and autophosphorylation of ResE and transphosphorylation of ResD by ResE were demonstrated in vitro. ResD is mainly monomeric in solution, regardless of its phosphorylation state. ResD was shown to interact directly with promoter regions (p) of the enterotoxin regulator genes resDE, fnr, and plcR and the enterotoxin structural genes nhe and hbl, but with different affinities. Binding of ResD to pplcR, pnhe, and phbl was not dependent on the ResD phosphorylation status. In contrast, ResD phosphorylation significantly increased interactions between ResD and presDE and pfnr. Taken together, these results showed that phosphorylation of ResD results in a different target expression pattern. Furthermore, ResD and the redox activator Fnr were found to physically interact and simultaneously bind their target DNAs. We propose that unphosphorylated ResD acts as an antiactivator of Fnr, while phosphorylated ResD acts as a coactivator of Fnr. Finally, our findings represent the first molecular evidence of the role of ResDE as a sentinel system capable of sensing redox changes and coordinating a response that modulates B. cereus virulence.

ApoFnr binds as a monomer to promoters regulating the expression of enterotoxin genes of Bacillus cereus.

Bacillus cereus Fnr is a member of the Crp/Fnr (cyclic AMP-binding protein/fumarate nitrate reduction regulatory protein) family of helix-turn-helix transcriptional regulators. It is essential for the expression of hbl and nhe enterotoxin genes independently of the oxygen tension in the environment. We studied aerobic Fnr binding to target sites in promoters regulating the expression of enterotoxin genes. B. cereus Fnr was overexpressed and purified as either a C-terminal His-tagged (Fnr(His)) fusion protein or an N-terminal fusion protein tagged with the Strep-tag (IBA BioTAGnology) ((Strep)Fnr). Both recombinant Fnr proteins were produced as apoforms (clusterless) and occurred as mixtures of monomers and oligomers in solution. However, apoFnr(His) was mainly monomeric, while apo(Strep)Fnr was mainly oligomeric, suggesting that the His-tagged C-terminal extremity may interfere with oligomerization. The oligomeric state of apo(Strep)Fnr was dithiothreitol sensitive, underlining the importance of a disulfide bridge for apoFnr oligomerization. Electrophoretic mobility shift assays showed that monomeric apoFnr, but not oligomeric apoFnr, bound to specific sequences located in the promoter regions of the enterotoxin regulators fnr, resDE, and plcR and the structural genes hbl and nhe. The question of whether apoFnr binding is regulated in vivo by redox-dependent oligomerization is discussed.