Aims
A protein termed 2Duf greatly increases wet heat resistance of Bacillus subtilis spores. The current work examines the effects of 2Duf on spore resistance to other sporicides, including ...chemicals that act on or must cross spores’ inner membrane (IM), where 2Duf is likely present. The overall aim was to gain a deeper understanding of how 2Duf affects spore resistance, and of spore resistance itself.
Methods and Results
2Duf's presence increased spore resistance to chemicals that damage or must cross the IM to kill spores. Spore coat removal decreased 2Duf‐spore resistance to chemicals and wet heat, and 2Duf‐spores made at higher temperatures were more resistant to wet heat and chemicals. 2Duf‐less spores lacking coats and Ca‐dipicolinic acid were also extremely sensitive to wet heat and chemicals that transit the IM to kill spores.
Conclusions
The new work plus previous results lead to a number of important conclusions as follows. (1) 2Duf may influence spore resistance by decreasing the permeability of and lipid mobility in spores’ IM. (2) Since wet heat‐killed spores that germinate do not accumulate ATP, wet heat may inactivate some spore IM protein essential in ATP production which is stabilized in a more rigid IM. (3) Both Ca‐dipicolinic acid and the spore coat play an important role in the permeability of the spore IM, and thus in many spore resistance properties.
Significance and Impact of the Study
The work in this manuscript gives a new insight into mechanisms of spore resistance to chemicals and wet heat, to the understanding of spore wet heat killing, and the role of Ca‐dipicolinic acid and the coat in spore resistance.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Spores are the major infectious particle of the Gram-positive nosocomial pathogen
(formerly
), but the molecular details of how this organism forms these metabolically dormant cells remain poorly ...characterized. The composition of the spore coat in
differs markedly from that defined in the well-studied organism
, with only 25% of the ∼70 spore coat proteins being conserved between the two organisms and with only 2 of 9 coat assembly (morphogenetic) proteins defined in
having homologs in
We previously identified SipL as a clostridium-specific coat protein essential for functional spore formation. Heterologous expression analyses in
revealed that SipL directly interacts with
SpoIVA, a coat-morphogenetic protein conserved in all spore-forming organisms, through SipL's C-terminal LysM domain. In this study, we show that SpoIVA-SipL binding is essential for
spore formation and identify specific residues within the LysM domain that stabilize this interaction. Fluorescence microscopy analyses indicate that binding of SipL's LysM domain to SpoIVA is required for SipL to localize to the forespore while SpoIVA requires SipL to promote encasement of SpoIVA around the forespore. Since we also show that clostridial LysM domains are functionally interchangeable at least in
, the basic mechanism for SipL-dependent assembly of clostridial spore coats may be conserved.
The metabolically dormant spore form of the major nosocomial pathogen
is its major infectious particle. However, the mechanisms controlling the formation of this resistant cell type are not well understood, particularly with respect to its outermost layer, the spore coat. We previously identified two spore-morphogenetic proteins in
: SpoIVA, which is conserved in all spore-forming organisms, and SipL, which is conserved only in the clostridia. Both SpoIVA and SipL are essential for heat-resistant spore formation and directly interact through SipL's C-terminal LysM domain. In this study, we demonstrate that the LysM domain is critical for SipL and SpoIVA function, likely by helping recruit SipL to the forespore during spore morphogenesis. We further identified residues within the LysM domain that are important for binding SpoIVA and, thus, functional spore formation. These findings provide important insight into the molecular mechanisms controlling the assembly of infectious
spores.
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•Quantification of heterogeneity in spore size, shape and size distribution.•Spore size can be equally measured with microscopy and coulter counter.•Coulter counter and flow cytometry ...showed a bimodal spore size distribution.•Spores size, shape and size distribution correlate with conidial heat resistance.
Contamination by spores is often the cause of fungal food spoilage. Some distinct strains of the food spoilage fungus Paecilomyces variotii are able to produce airborne conidia that are more heat-resistant than similar species. These ellipsoid asexual spores can vary in size between strains, but also within strains. Here, we compared four measurement techniques to measure conidia size and distribution of five heat-sensitive and five heat-resistant P. variotii strains. Light microscopy (LM), Scanning Electron Microscopy (SEM) and Coulter Counter (CC) were used to measure and compare the spherical equivalent diameter, while CC and flow cytometry were used to study spore size distributions. The flow cytometry data was useful to study spore size distributions, but only relative spore sizes were obtained. There was no statistic difference between the method used of spore size measurement between LM, SEM and CC, but spore size was significantly different between strains with a 2.4-fold volume difference between the extremes. Various size distribution and shape parameters were correlated with conidial heat resistance. We found significant correlations in mean spore size, aspect ratio, roundness and skewness in relation to heat resistance, which suggests that these parameters are indicative for the conidial heat resistance of a P. variotii strain.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Sporeforming bacteria are ubiquitous in the environment and exhibit a wide range of diversity leading to their natural prevalence in foodstuff. The state of the art of sporeformer prevalence in ...ingredients and food was investigated using a multiparametric PCR-based tool that enables simultaneous detection and identification of various genera and species mostly encountered in food, i.e. Alicyclobacillus, Anoxybacillus flavithermus, Bacillus, B. cereus group, B. licheniformis, B. pumilus, B. sporothermodurans, B. subtilis, Brevibacillus laterosporus, Clostridium, Geobacillus stearothermophilus, Moorella and Paenibacillus species. In addition, 16S rDNA sequencing was used to extend identification to other possibly present contaminants. A total of 90 food products, with or without visible trace of spoilage were analysed, i.e. 30 egg-based products, 30 milk and dairy products and 30 canned food and ingredients. Results indicated that most samples contained one or several of the targeted genera and species. For all three tested food categories, 30 to 40% of products were contaminated with both Bacillus and Clostridium. The percentage of contaminations associated with Clostridium or Bacillus represented 100% in raw materials, 72% in dehydrated ingredients and 80% in processed foods. In the last two product types, additional thermophilic contaminants were identified (A. flavithermus, Geobacillus spp., Thermoanaerobacterium spp. and Moorella spp.). These results suggest that selection, and therefore the observed (re)-emergence of unexpected sporeforming contaminants in food might be favoured by the use of given food ingredients and food processing technologies.
► A validated PCR-based tool was used to track sporeformers in 90 foodstuffs. ► Occurrence was presented for 5 genera and 9 species of Bacillus species. ► Multiple contaminations were detected for most food samples with or without spoilage. ► Emergence of thermophilic species was reported in ingredient and processed food.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
can survive in the form of spores for prolonged periods posing a serious problem for the manufacture of safe shelf-stable foods of optimal quality. Our study aims at increasing knowledge of
spores ...focusing primarily on germination mechanisms to develop novel milder food preservation strategies. Major features of
spores are a core with the genetic material encased by multiple protective layers, an important one being the spores' inner membrane (IM), the location of many important germination proteins. To study mechanisms involved in germination of
spores, we have examined the organization of germinant receptors (GRs) in spores' IM. Previous studies have indicated that in spores of
ATCC 14579 the L-alanine responsive GR, GerR, plays a major role in the germination process. In our study, the location of the GerR GR subunit, GerRB, in spores was examined as a C-terminal SGFP2 fusion protein expressed under the control of the
operon's promoter. Our results showed that: i) the fluorescence maxima and integrated intensity in spores with plasmid-borne expression of GerRB-SGFP2 were significantly higher than in wild-type spores; ii) western blot analysis confirmed the expression of the GerRB-SGFP2 fusion protein in spores; and iii) fluorescence microscopy visualized GerRB-SGFP2 specific bright foci in ~30% of individual dormant spores if only GerRB-SGFP2 was expressed, but, noticeably, in ~85% of spores upon co-expression with GerRA and GerRC. Our data corroborates the notion that co-expression of GR subunits improves their stability. Finally, all spores displayed bright fluorescent foci upon expression of GerD-mScarlet-I under the control of the
promoter. We termed all fluorescent foci observed germinosomes, the term used for the IM foci of GRs in
spores. Our data are the first evidence for the existence of germinosomes in
spores.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Conventional thermal and chemical treatments used in food preservation have come under scrutiny by consumers who demand minimally processed foods free from chemical agents but microbiologically safe. ...As a result, antimicrobial peptides (AMPs) such as bacteriocins and nisin that are ribosomally synthesised by bacteria, more prominently by the lactic acid bacteria (LAB) have appeared as a potent alternative due to their multiple biological activities. They represent a powerful strategy to prevent the development of spore-forming microorganisms in foods. Unlike thermal methods, they are natural without an adverse impact on food organoleptic and nutritional attributes. AMPs such as nisin and bacteriocins are generally effective in eliminating the vegetative forms of spore-forming bacteria compared to the more resilient spore forms. However, in combination with other non-thermal treatments, such as high pressure, supercritical carbon dioxide, electric pulses, a synergistic effect with AMPs such as nisin exists and has been proven to be effective in the inactivation of microbial spores through the disruption of the spore structure and prevention of spore outgrowth. The control of microbial spores in foods is essential in maintaining food safety and extension of shelf-life. Thus, exploration of the mechanisms of action of AMPs such as nisin is critical for their design and effective application in the food industry. This review harmonises information on the mechanisms of bacteria inactivation from published literature and the utilisation of AMPs in the control of microbial spores in food. It highlights future perspectives in research and application in food processing.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
cells produce lipid bodies containing triacylglycerides during fruiting body development. Fatty acid β-oxidation is the most energy-efficient pathway for lipid body catabolism. In this study, we used ...mutants in
(MXAN_5371 and MXAN_6987) and
(MXAN_5372) homologs to examine whether β-oxidation serves an essential developmental function. These mutants contained more lipid bodies than the wild-type strain DK1622 and 2-fold more flavin adenine dinucleotide (FAD), consistent with the reduced consumption of fatty acids by β-oxidation. The β-oxidation pathway mutants exhibited differences in fruiting body morphogenesis and produced spores with thinner coats and a greater susceptibility to thermal stress and UV radiation. The MXAN_5372/5371 operon is upregulated in sporulating cells, and its expression could not be detected in
,
, or
mutants. Lipid bodies were found to persist in mature spores of DK1622 and wild strain DK851, suggesting that the roles of lipid bodies and β-oxidation may extend to spore germination.
Lipid bodies act as a reserve of triacylglycerides for use when other sources of carbon and energy become scarce. β-Oxidation is essential for the efficient metabolism of fatty acids associated with triacylglycerides. Indeed, the disruption of genes in this pathway has been associated with severe disorders in animals and plants.
, a model organism for the study of development, is ideal for investigating the complex effects of altered lipid metabolism on cell physiology. Here, we show that β-oxidation is used to consume fatty acids associated with lipid bodies and that the disruption of the β-oxidation pathway is detrimental to multicellular morphogenesis and spore formation.
•Inhibitory effect of PLA against the spores of Bacillus cereus were first reported.•Inactivation mechanism of PLA against germinated spores was proposed.•PLA was evidenced to exhibit preservative ...effect against spores in milk beverage.•This investigation could provide scientific basis for the application of PLA in food.
Phenyllactic acid (PLA) as a natural phenolic acid exhibits antibacterial activity against non-spore-forming bacteria, while the inhibitory effect against bacterial spore remained unknown. Herein, this study investigated the inactivation effect of PLA against Bacillus cereus spores. The results revealed that the minimum inhibitory concentration of PLA was 1.25 mg/mL. PLA inhibited the outgrowth of germinated spores into vegetative cells rather than germination of spores. PLA disrupted the spore coat, and damaged the permeability and integrity of inner membrane. Moreover, PLA disturbed the establishment of membrane potential due to the inhibition of oxidative metabolism. SEM observations further visualized the morphological changes and structural disruption caused by PLA. Besides, PLA caused the degradation of DNA of germinated spores. Finally, PLA was applied in milk beverage, and showed promising inhibitory effect against B. cereus spores. This finding could provide scientific basis for the application of PLA against spore-forming bacteria in food industry.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Changes with time of a population of
KBAB4 and
AD978 dormant spores into germinated spores and vegetative cells were followed by flow cytometry, at pH ranges of 4.7 to 7.4 and temperatures of 10°C to ...37°C for
and 18°C to 59°C for
Incubation conditions lower than optimal temperatures or pH led to lower proportions of dormant spores able to germinate and extended time of germination, a lower proportion of germinated spores able to outgrow, an extension of their times of outgrowth, and an increase of the heterogeneity of spore outgrowth time. A model based on the strain growth limits was proposed to quantify the impact of incubation temperature and pH on the passage through each physiological stage. The heat treatment temperature or time acted independently on spore recovery. Indeed, a treatment at 85°C for 12 min or at 95°C for 2 min did not have the same impact on spore germination and outgrowth kinetics of
despite the fact that they both led to a 10-fold reduction of the population. Moreover, acidic sporulation pH increased the time of outgrowth 1.2-fold and lowered the proportion of spores able to germinate and outgrow 1.4-fold. Interestingly, we showed by proteomic analysis that some proteins involved in germination and outgrowth were detected at a lower abundance in spores produced at pH 5.5 than in those produced at pH 7.0, maybe at the origin of germination and outgrowth behavior of spores produced at suboptimal pH.
Sporulation and incubation conditions have an impact on the numbers of spores able to recover after exposure to sublethal heat treatment. Using flow cytometry, we were able to follow at a single-cell level the changes in the physiological states of heat-stressed spores of
spp. and to discriminate between dormant spores, germinated spores, and outgrowing vegetative cells. We developed original mathematical models that describe (i) the changes with time of the proportion of cells in their different states during germination and outgrowth and (ii) the influence of temperature and pH on the kinetics of spore recovery using the growth limits of the tested strains as model parameters. We think that these models better predict spore recovery after a sublethal heat treatment, a common situation in food processing and a concern for food preservation and safety.
Clostridioides difficile spores are the infective form for this endospore-forming organism. The vegetative cells are intolerant to oxygen and poor competitors with a healthy gut microbiota. ...Therefore, in order for C. difficile to establish infection, the spores have to germinate in an environment that supports vegetative growth. To initiate germination, C. difficile uses Csp-type germinant receptors that consist of the CspC and CspA pseudoproteases as the bile acid and cogerminant receptors, respectively. CspB is a subtilisin-like protease that cleaves the inhibitory propeptide from the pro-SleC cortex lytic enzyme, thereby activating it and initiating cortex degradation. Though several locations have been proposed for where these proteins reside within the spore (i.e., spore coat, outer spore membrane, cortex, and inner spore membrane), these have been based, mostly, on hypotheses or prior data in Clostridium perfringens. In this study, we visualized the germination and outgrowth process using transmission electron microscopy (TEM) and scanning electron microscopy (SEM) and used immunogold labeling to visualize key germination regulators. These analyses localize these key regulators to the spore cortex region for the first time.
Germination by C. difficile spores is the first step in the establishment of potentially life-threatening C. difficile infection (CDI). A deeper understanding of the mechanism by which spores germinate may provide insight for how to either prevent spore germination into a disease-causing vegetative form or trigger germination prematurely when the spore is either in the outside environment or in a host environment that does not support the establishment of colonization/disease.
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