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3.
Food Chem ; 453: 139601, 2024 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-38754350

RESUMO

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.


Assuntos
Antibacterianos , Bacillus cereus , Leite , Esporos Bacterianos , Bacillus cereus/crescimento & desenvolvimento , Bacillus cereus/efeitos dos fármacos , Bacillus cereus/metabolismo , Esporos Bacterianos/efeitos dos fármacos , Esporos Bacterianos/crescimento & desenvolvimento , Esporos Bacterianos/metabolismo , Leite/química , Leite/microbiologia , Antibacterianos/farmacologia , Antibacterianos/química , Animais , Bebidas/análise , Bebidas/microbiologia , Testes de Sensibilidade Microbiana , Lactatos/farmacologia , Lactatos/química , Lactatos/metabolismo
4.
Int J Food Microbiol ; 418: 110730, 2024 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-38714095

RESUMO

Aerobic spore-forming (ASF) bacteria have been reported to cause ropiness in bread. Sticky and stringy degradation, discoloration, and an odor reminiscent of rotting fruit are typical characteristics of ropy bread spoilage. In addition to economic losses, ropy bread spoilage may lead to health risks, as virulent strains of ASF bacteria are not uncommon. However, the lack of systematic approaches to quantify physicochemical spoilage characteristics makes it extremely difficult to assess rope formation in bread. To address this problem, the aim of this study was to identify, characterize and objectively assess the spoilage potential of ASF bacteria associated with ropy bread. Hence, a set of 82 ASF bacteria, including isolates from raw materials and bakery environments as well as strains from international culture collections, were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and their species identity confirmed by 16S rRNA and gyrA or panC gene sequencing. A standardized approach supported by objective colorimetric measurements was developed to assess the rope-inducing potential (RIP) of a strain by inoculating autoclaved bread slices with bacterial spores. In addition, the presence of potential virulence factors such as swarming motility or hemolysis was investigated. This study adds B. velezensis, B. inaquosorum and B. spizizenii to the species potentially implicated of causing ropy bread spoilage. Most importantly, this study introduces a standardized classification protocol for assessing the RIP of a bacterial strain. Colorimetric measurements are used to objectively quantify the degree of breadcrumb discoloration. Furthermore, our results indicate that strains capable of inducing rope spoilage in bread often exhibit swarming motility and virulence factors such as hemolysis, raising important food quality considerations.


Assuntos
Pão , Microbiologia de Alimentos , Pão/microbiologia , Esporos Bacterianos/crescimento & desenvolvimento , Bactérias Aeróbias/isolamento & purificação , Bactérias Aeróbias/classificação , Bactérias Aeróbias/genética , Bactérias Aeróbias/crescimento & desenvolvimento , RNA Ribossômico 16S/genética , Fatores de Virulência/genética , Contaminação de Alimentos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
5.
Int J Food Microbiol ; 418: 110731, 2024 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-38733637

RESUMO

Alicyclobacillus spp. is the cause of great concern for the food industry due to their spores' resistance (thermal and chemical) and the spoilage potential of some species. Despite this, not all Alicyclobacillus strains can spoil fruit juices. Thus, this study aimed to identify Alicyclobacillus spp. strains isolated from fruit-based products produced in Argentina, Brazil, and Italy by DNA sequencing. All Alicyclobacillus isolates were tested for guaiacol production by the peroxidase method. Positive strains for guaiacol production were individually inoculated at concentration of 103 CFU/mL in 10 mL of orange (pH 3.90) and apple (pH 3.50) juices adjusted to 11°Brix, following incubation at 45 °C for at least 5 days to induce the production of the following spoilage compounds: Guaiacol, 2,6-dichlorophenol (2,6-DCP) and 2,6-dibromophenol (2,6-DBP). The techniques of micro-solid phase extraction by headspace (HS-SPME) and gas-chromatography with mass spectrometry (GC-MS) were used to identify and quantify the spoilage compounds. All GC-MS data was analyzed by principal component analysis (PCA). The effects of different thermal shock conditions on the recovery of Alicyclobacillus spores inoculated in orange and apple juice (11°Brix) were also tested. A total of 484 strains were isolated from 48 brands, and the species A. acidocaldarius and A. acidoterrestris were the most found among all samples analyzed. In some samples from Argentina, the species A. vulcanalis and A. mali were also identified. The incidence of these two main species of Alicyclobacillus in this study was mainly in products from pear (n = 108; 22.3 %), peach (n = 99; 20.5 %), apple (n = 86; 17.8 %), and tomato (n = 63; 13 %). The results indicated that from the total isolates from Argentina (n = 414), Brazil (n = 54) and Italy (n = 16) were able to produce guaiacol: 107 (25.8 %), 33 (61.1 %) and 13 (81.2 %) isolates from each country, respectively. The PCA score plot indicated that the Argentina and Brazil isolates correlate with higher production of guaiacol and 2,6-DCP/2,6-DBP, respectively. Heatmaps of cell survival after heat shock demonstrated that strains with different levels of guaiacol production present different resistances according to spoilage ability. None of the Alicyclobacillus isolates survived heat shocks at 120 °C for 3 min. This work provides insights into the incidence, spoilage potential, and thermal shock resistance of Alicyclobacillus strains isolated from fruit-based products.


Assuntos
Alicyclobacillus , Sucos de Frutas e Vegetais , Frutas , Cromatografia Gasosa-Espectrometria de Massas , Guaiacol , Esporos Bacterianos , Alicyclobacillus/isolamento & purificação , Alicyclobacillus/genética , Alicyclobacillus/classificação , Alicyclobacillus/crescimento & desenvolvimento , Sucos de Frutas e Vegetais/microbiologia , Guaiacol/análogos & derivados , Guaiacol/metabolismo , Guaiacol/farmacologia , Frutas/microbiologia , Esporos Bacterianos/crescimento & desenvolvimento , Esporos Bacterianos/isolamento & purificação , Microbiologia de Alimentos , Contaminação de Alimentos/análise , Brasil , Microextração em Fase Sólida , Argentina , Malus/microbiologia , Itália , Temperatura Alta , Citrus sinensis/microbiologia
6.
Biotechnol Lett ; 46(3): 355-371, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38607603

RESUMO

OBJECTIVES: Bacillus subtilis is a plant growth promoting bacterium (PGPB) that acts as a microbial fertilizer and biocontrol agent, providing benefits such as boosting crop productivity and improving nutrient content. It is able to produce secondary metabolites and endospores simultaneously, enhancing its ability to survive in unfavorable conditions and eliminate competing microorganisms. Optimizing cultivation methods to produce B. subtilis MSCL 897 spores on an industrial scale, requires a suitable medium, typically made from food industry by-products, and optimal temperature and pH levels to achieve high vegetative cell and spore densities with maximum productivity. RESULTS: This research demonstrates successful pilot-scale (100 L bioreactor) production of a biocontrol agent B. subtilis with good spore yields (1.5 × 109 spores mL-1) and a high degree of sporulation (>80%) using a low-cost cultivation medium. Culture samples showed excellent antifungal activity (1.6-2.3 cm) against several phytopathogenic fungi. An improved methodology for inoculum preparation was investigated to ensure an optimal seed culture state prior to inoculation, promoting process batch-to-batch repeatability. Increasing the molasses concentration in the medium and operating the process in fed-batch mode with additional molasses feed, did not improve the overall spore yield, hence, process operation in batch mode with 10 g molasses L-1 is preferred. Results also showed that the product quality was not significantly impacted for up to 12 months of storage at room temperature. CONCLUSION: An economically-feasible process for B. subtilis-based biocontrol agent production was successfully developed at the pilot scale.


Assuntos
Bacillus subtilis , Biomassa , Reatores Biológicos , Meios de Cultura , Esporos Bacterianos , Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/metabolismo , Esporos Bacterianos/crescimento & desenvolvimento , Esporos Bacterianos/metabolismo , Meios de Cultura/química , Reatores Biológicos/microbiologia , Antifúngicos/metabolismo , Antifúngicos/farmacologia , Projetos Piloto
7.
mBio ; 15(5): e0056224, 2024 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-38564667

RESUMO

Spores of Bacillus subtilis germinate in response to specific germinant molecules that are recognized by receptors in the spore envelope. Germinants signal to the dormant spore that the environment can support vegetative growth, so many germinants, such as alanine and valine, are also essential metabolites. As such, they are also required to build the spore. Here we show that these germinants cause premature germination if they are still present at the latter stages of spore formation and beyond, but that B. subtilis metabolism is configured to prevent this: alanine and valine are catabolized and cleared from wild-type cultures even when alternative carbon and nitrogen sources are present. Alanine and valine accumulate in the spent media of mutants that are unable to catabolize these amino acids, and premature germination is pervasive. Premature germination does not occur if the germinant receptor that responds to alanine and valine is eliminated, or if wild-type strains that are able to catabolize and clear alanine and valine are also present in coculture. Our findings demonstrate that spore-forming bacteria must fine-tune the concentration of any metabolite that can also function as a germinant to a level that is high enough to allow for spore development to proceed, but not so high as to promote premature germination. These results indicate that germinant selection and metabolism are tightly linked, and suggest that germinant receptors evolve in tandem with the catabolic priorities of the spore-forming bacterium. IMPORTANCE: Many bacterial species produce dormant cells called endospores, which are not killed by antibiotics or common disinfection practices. Endospores pose critical challenges in the food industry, where endospore contaminations cause food spoilage, and in hospitals, where infections by pathogenic endospore formers threaten the life of millions every year. Endospores lose their resistance properties and can be killed easily when they germinate and exit dormancy. We have discovered that the enzymes that break down the amino acids alanine and valine are critical for the production of stable endospores. If these enzymes are absent, endospores germinate as they are formed or shortly thereafter in response to alanine, which can initiate the germination of many different species' endospores, or to valine. By blocking the activity of alanine dehydrogenase, the enzyme that breaks down alanine and is not present in mammals, it may be possible to inactivate endospores by triggering premature and unproductive germination.


Assuntos
Alanina , Aminoácidos , Bacillus subtilis , Esporos Bacterianos , Bacillus subtilis/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/crescimento & desenvolvimento , Esporos Bacterianos/metabolismo , Esporos Bacterianos/crescimento & desenvolvimento , Esporos Bacterianos/genética , Alanina/metabolismo , Aminoácidos/metabolismo , Valina/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Meios de Cultura/química
8.
Toxins (Basel) ; 16(4)2024 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-38668620

RESUMO

The CPR1953 and CPR1954 orphan histidine kinases profoundly affect sporulation initiation and Clostridium perfringens enterotoxin (CPE) production by C. perfringens type F strain SM101, whether cultured in vitro (modified Duncan-Strong sporulation medium (MDS)) or ex vivo (mouse small intestinal contents (MIC)). To help distinguish whether CPR1953 and CPR1954 act independently or in a stepwise manner to initiate sporulation and CPE production, cpr1953 and cpr1954 null mutants of SM101 were transformed with plasmids carrying the cpr1954 or cpr1953 genes, respectively, causing overexpression of cpr1954 in the absence of cpr1953 expression and vice versa. RT-PCR confirmed that, compared to SM101, the cpr1953 mutant transformed with a plasmid encoding cpr1954 expressed cpr1954 at higher levels while the cpr1954 mutant transformed with a plasmid encoding cpr1953 expressed higher levels of cpr1953. Both overexpressing strains showed near wild-type levels of sporulation, CPE toxin production, and Spo0A production in MDS or MIC. These findings suggest that CPR1953 and CPR1954 do not function together in a step-wise manner, e.g., as a novel phosphorelay. Instead, it appears that, at natural expression levels, the independent kinase activities of both CPR1953 and CPR1954 are necessary for obtaining sufficient Spo0A production and phosphorylation to initiate sporulation and CPE production.


Assuntos
Proteínas de Bactérias , Clostridium perfringens , Enterotoxinas , Histidina Quinase , Esporos Bacterianos , Clostridium perfringens/genética , Clostridium perfringens/enzimologia , Esporos Bacterianos/genética , Esporos Bacterianos/crescimento & desenvolvimento , Enterotoxinas/genética , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Histidina Quinase/genética , Histidina Quinase/metabolismo , Regulação Bacteriana da Expressão Gênica , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Camundongos
9.
Int J Food Microbiol ; 418: 110709, 2024 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-38663147

RESUMO

Wet heat treatment is a commonly applied method in the food and medical industries for the inactivation of microorganisms, and bacterial spores in particular. While many studies have delved into the mechanisms underlying wet heat killing and spore resistance, little attention has so far been dedicated to the capacity of spore-forming bacteria to tune their resistance through adaptive evolution. Nevertheless, a recent study from our group revealed that a psychrotrophic strain of the Bacillus cereus sensu lato group (i.e. Bacillus weihenstephanensis LMG 18989) could readily and reproducibly evolve to acquire enhanced spore wet heat resistance without compromising its vegetative cell growth ability at low temperatures. In the current study, we demonstrate that another B. cereus strain (i.e. the mesophilic B. cereus sensu stricto ATCC 14579) can acquire significantly increased spore wet heat resistance as well, and we subjected both the previously and currently obtained mutants to whole genome sequencing. This revealed that five out of six mutants were affected in genes encoding regulators of the spore coat and exosporium pathway (i.e. spoIVFB, sigK and gerE), with three of them being affected in gerE. A synthetically constructed ATCC 14579 ΔgerE mutant likewise yielded spores with increased wet heat resistance, and incurred a compromised spore coat and exosporium. Further investigation revealed significantly increased spore DPA levels and core dehydration as the likely causes for the observed enhanced spore wet heat resistance. Interestingly, deletion of gerE in Bacillus subtilis 168 did not impose increased spore wet heat resistance, underscoring potentially different adaptive evolutionary paths in B. cereus and B. subtilis.


Assuntos
Bacillus cereus , Temperatura Alta , Esporos Bacterianos , Esporos Bacterianos/genética , Esporos Bacterianos/crescimento & desenvolvimento , Bacillus cereus/genética , Bacillus cereus/crescimento & desenvolvimento , Bacillus cereus/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Mutação , Termotolerância , Adaptação Fisiológica , Sequenciamento Completo do Genoma , Microbiologia de Alimentos , Genoma Bacteriano , Evolução Biológica
10.
Int J Food Microbiol ; 418: 110716, 2024 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-38669747

RESUMO

Anoxybacillus flavithermus, Geobacillus stearothermophilus and Bacillus licheniformis are the main contaminants found in dairy powders. These spore-forming thermophilic bacteria, rarely detected in raw milk, persist, and grow during the milk powder manufacturing process. Moreover, in the form of spores, these species resist and concentrate in the powders during the processes. The aim of this study was to determine the stages of the dairy powder manufacturing processes that are favorable to the growth of such contaminants. A total of 5 strains were selected for each species as a natural contaminant of dairy pipelines in order to determine the minimum and maximum growth enabling values for temperature, pH, and aw and their optimum growth rates in milk. These growth limits were combined with the environmental conditions of temperature, pH and aw encountered at each step of the manufacture of whole milk, skim milk and milk protein concentrate powders to estimate growth capacities using cardinal models and the Gamma concept. These simulations were used to theoretically calculate the population sizes reached for the different strains studied at each stage in between two successive cleaning in place procedures. This approach highlights the stages at which risk occurs for the development of spore-forming thermophilic bacterial species. During the first stages of production, i.e. pre-treatment, pasteurization, standardization and pre-heating before concentration, physico-chemical conditions encountered are suitable for the development and growth of A. flavithermus, G. stearothermophilus and B. licheniformis. During the pre-heating stage and during the first effects in the evaporators, the temperature conditions appear to be the most favorable for the growth of G. stearothermophilus. The temperatures in the evaporator during the last evaporator effects are favorable for the growth of B. licheniformis. In the evaporation stage, low water activity severely limits the development of A. flavithermus.


Assuntos
Leite , Pós , Esporos Bacterianos , Esporos Bacterianos/crescimento & desenvolvimento , Leite/microbiologia , Animais , Geobacillus stearothermophilus/crescimento & desenvolvimento , Microbiologia de Alimentos , Bacillus licheniformis/crescimento & desenvolvimento , Bacillus licheniformis/metabolismo , Concentração de Íons de Hidrogênio , Anoxybacillus/crescimento & desenvolvimento , Manipulação de Alimentos/métodos , Temperatura , Contaminação de Alimentos/análise , Indústria de Laticínios/métodos , Laticínios/microbiologia
11.
Microbiol Spectr ; 12(5): e0401023, 2024 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-38501822

RESUMO

The actinomycete Actinoplanes missouriensis forms branched substrate mycelia during vegetative growth and produces terminal sporangia, each of which contains a few hundred spherical flagellated spores, from the substrate mycelia through short sporangiophores. Based on the observation that remodeling of membrane lipid composition is involved in the morphological development of Streptomyces coelicolor A3(2), we hypothesized that remodeling of membrane lipid composition is also involved in sporangium formation in A. missouriensis. Because some acyltransferases are presumably involved in the remodeling of membrane lipid composition, we disrupted each of the 22 genes annotated as encoding putative acyltransferases in the A. missouriensis genome and evaluated their effects on sporangium formation. The atsA (AMIS_52390) null mutant (ΔatsA) strain formed irregular sporangia of various sizes. Transmission electron microscopy revealed that some ΔatsA sporangiospores did not mature properly. Phase-contrast microscopy revealed that sporangium dehiscence did not proceed properly in the abnormally small sporangia of the ΔatsA strain, whereas apparently normal sporangia opened to release the spores. Consistently, the number of spores released from ΔatsA sporangia was lower than that released from wild-type sporangia. These phenotypic changes were recovered by introducing atsA with its own promoter into the ΔatsA strain. These results demonstrate that AtsA is required for normal sporangium formation in A. missouriensis, although the involvement of AtsA in the remodeling of membrane lipid composition is unlikely because AtsA is an acyltransferase_3 (AT3) protein, which is an integral membrane protein that usually catalyzes the acetylation of cell surface structures.IMPORTANCEActinoplanes missouriensis goes through a life cycle involving complex morphological development, including mycelial growth, sporangium formation and dehiscence, swimming as zoospores, and germination to mycelial growth. In this study, we carried out a comprehensive gene disruption experiment of putative acyltransferase genes to search for acyltransferases involved in the morphological differentiation of A. missouriensis. We revealed that a stand-alone acyltransferase_3 domain-containing protein, named AtsA, is required for normal sporangium formation. Although the molecular mechanism of AtsA in sporangium formation, as well as the enzymatic activity of AtsA, remains to be elucidated, the identification of a putative acyltransferase involved in sporangium formation is significant in the study of morphological development of A. missouriensis. This finding will contribute to our understanding of a complex system for producing sporangia, a rare multicellular organism in bacteria.


Assuntos
Actinoplanes , Aciltransferases , Esporângios , Actinoplanes/genética , Actinoplanes/metabolismo , Actinoplanes/crescimento & desenvolvimento , Actinoplanes/enzimologia , Aciltransferases/genética , Aciltransferases/metabolismo , Esporângios/crescimento & desenvolvimento , Esporângios/genética , Esporângios/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Esporos Bacterianos/genética , Esporos Bacterianos/crescimento & desenvolvimento , Esporos Bacterianos/enzimologia , Esporos Bacterianos/metabolismo , Lipídeos de Membrana/metabolismo
12.
Mol Microbiol ; 121(5): 1002-1020, 2024 05.
Artigo em Inglês | MEDLINE | ID: mdl-38525557

RESUMO

Upon starvation, rod-shaped Myxococcus xanthus bacteria form mounds and then differentiate into round, stress-resistant spores. Little is known about the regulation of late-acting operons important for spore formation. C-signaling has been proposed to activate FruA, which binds DNA cooperatively with MrpC to stimulate transcription of developmental genes. We report that this model can explain regulation of the fadIJ operon involved in spore metabolism, but not that of the spore coat biogenesis operons exoA-I, exoL-P, and nfsA-H. Rather, a mutation in fruA increased the transcript levels from these operons early in development, suggesting negative regulation by FruA, and a mutation in mrpC affected transcript levels from each operon differently. FruA bound to all four promoter regions in vitro, but strikingly each promoter region was unique in terms of whether or not MrpC and/or the DNA-binding domain of Nla6 bound, and in terms of cooperative binding. Furthermore, the DevI component of a CRISPR-Cas system is a negative regulator of all four operons, based on transcript measurements. Our results demonstrate complex regulation of sporulation genes by three transcription factors and a CRISPR-Cas component, which we propose produces spores suited to withstand starvation and environmental insults.


Assuntos
Proteínas de Bactérias , Sistemas CRISPR-Cas , Regulação Bacteriana da Expressão Gênica , Myxococcus xanthus , Óperon , Regiões Promotoras Genéticas , Esporos Bacterianos , Fatores de Transcrição , Myxococcus xanthus/genética , Myxococcus xanthus/metabolismo , Myxococcus xanthus/crescimento & desenvolvimento , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Óperon/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Esporos Bacterianos/genética , Esporos Bacterianos/metabolismo , Esporos Bacterianos/crescimento & desenvolvimento , Regiões Promotoras Genéticas/genética , Mutação , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética
13.
Microb Genom ; 8(2)2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35166653

RESUMO

Members of the genus Clostridium are frequently associated with meat spoilage. The ability for low numbers of spores of certain Clostridium species to germinate in cold-stored vacuum-packed meat can result in blown pack spoilage. However, little is known about the germination process of these clostridia, despite this characteristic being important for their ability to cause spoilage. This study sought to determine the genomic conditions for germination of 37 representative Clostridium strains from seven species (C. estertheticum, C. tagluense, C. frigoris, C. gasigenes, C. putrefaciens, C. aligidicarnis and C. frigdicarnis) by comparison with previously characterized germination genes from C. perfringens, C. sporogenes and C. botulinum. All the genomes analysed contained at least one gerX operon. Seven different gerX operon configuration types were identified across genomes from C. estertheticum, C. tagluense and C. gasigenes. Differences arose between the C. gasigenes genomes and those belonging to C. tagluense/C. estertheticum in the number and type of genes coding for cortex lytic enzymes, suggesting the germination pathway of C. gasigenes is different. However, the core components of the germination pathway were conserved in all the Clostridium genomes analysed, suggesting that these species undergo the same major steps as Bacillus subtilis for germination to occur.


Assuntos
Clostridium/crescimento & desenvolvimento , Clostridium/genética , Carne/microbiologia , Esporos Bacterianos/crescimento & desenvolvimento , Esporos Bacterianos/genética , Clostridium/classificação , Hibridização Genômica Comparativa , DNA Bacteriano , Microbiologia de Alimentos , Genes Bacterianos , Genoma Bacteriano
14.
Cell ; 185(1): 145-157.e13, 2022 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-34995513

RESUMO

Contrary to multicellular organisms that display segmentation during development, communities of unicellular organisms are believed to be devoid of such sophisticated patterning. Unexpectedly, we find that the gene expression underlying the nitrogen stress response of a developing Bacillus subtilis biofilm becomes organized into a ring-like pattern. Mathematical modeling and genetic probing of the underlying circuit indicate that this patterning is generated by a clock and wavefront mechanism, similar to that driving vertebrate somitogenesis. We experimentally validated this hypothesis by showing that predicted nutrient conditions can even lead to multiple concentric rings, resembling segments. We additionally confirmed that this patterning mechanism is driven by cell-autonomous oscillations. Importantly, we show that the clock and wavefront process also spatially patterns sporulation within the biofilm. Together, these findings reveal a biofilm segmentation clock that organizes cellular differentiation in space and time, thereby challenging the paradigm that such patterning mechanisms are exclusive to plant and animal development.


Assuntos
Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/genética , Biofilmes/crescimento & desenvolvimento , Padronização Corporal/genética , Bacillus subtilis/metabolismo , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Cinética , Modelos Biológicos , Nitrogênio/metabolismo , Transdução de Sinais/genética , Somitos/crescimento & desenvolvimento , Esporos Bacterianos/crescimento & desenvolvimento , Estresse Fisiológico/genética , Fatores de Tempo
15.
J Bacteriol ; 204(2): e0047021, 2022 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-34780301

RESUMO

Bacterial spores can rapidly exit dormancy through the process of germination. This process begins with the activation of nutrient receptors embedded in the spore membrane. The prototypical germinant receptor in Bacillus subtilis responds to l-alanine and is thought to be a complex of proteins encoded by the genes in the gerA operon: gerAA, gerAB, and gerAC. The GerAB subunit has recently been shown to function as the nutrient sensor, but beyond contributing to complex stability, no additional functions have been attributed to the other two subunits. Here, we investigate the role of GerAA. We resurrect a previously characterized allele of gerA (termed gerA*) that carries a mutation in gerAA and show that it constitutively activates germination even in the presence of a wild-type copy of gerA. Using an enrichment strategy to screen for suppressors of gerA*, we identified mutations in all three gerA genes that restore a functional receptor. Characterization of two distinct gerAB suppressors revealed that one (gerAB[E105K]) reduces the GerA complex's ability to respond to l-alanine, while another (gerAB[F259S]) disrupts the germinant signal downstream of l-alanine recognition. These data argue against models in which GerAA is directly or indirectly involved in germinant sensing. Rather, our data suggest that GerAA is responsible for transducing the nutrient signal sensed by GerAB. While the steps downstream of gerAA have yet to be uncovered, these results validate the use of a dominant-negative genetic approach in elucidating the gerA signal transduction pathway. IMPORTANCE Endospore formers are a broad group of bacteria that can enter dormancy upon starvation and exit dormancy upon sensing the return of nutrients. How dormant spores sense and respond to these nutrients is poorly understood. Here, we identify a key step in the signal transduction pathway that is activated after spores detect the amino acid l-alanine. We present a model that provides a more complete picture of this process that is critical for allowing dormant spores to germinate and resume growth.


Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Membrana/genética , Transdução de Sinais/genética , Esporos Bacterianos/genética , Esporos Bacterianos/metabolismo , Alanina/metabolismo , Alelos , Bacillus subtilis/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Mutação , Óperon , Esporos Bacterianos/crescimento & desenvolvimento
16.
Int J Mol Sci ; 22(22)2021 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-34830357

RESUMO

Membrane proteins are fascinating since they play an important role in diverse cellular functions and constitute many drug targets. Membrane proteins are challenging to analyze. The spore, the most resistant form of known life, harbors a compressed inner membrane. This membrane acts not only as a barrier for undesired molecules but also as a scaffold for proteins involved in signal transduction and the transport of metabolites during spore germination and subsequent vegetative growth. In this study, we adapted a membrane enrichment method to study the membrane proteome of spores and cells of the food-borne pathogen Bacillus cereus using quantitative proteomics. Using bioinformatics filtering we identify and quantify 498 vegetative cell membrane proteins and 244 spore inner membrane proteins. Comparison of vegetative and spore membrane proteins showed there were 54 spore membrane-specific and 308 cell membrane-specific proteins. Functional characterization of these proteins showed that the cell membrane proteome has a far larger number of transporters, receptors and proteins related to cell division and motility. This was also reflected in the much higher expression level of many of these proteins in the cellular membrane for those proteins that were in common with the spore inner membrane. The spore inner membrane had specific expression of several germinant receptors and spore-specific proteins, but also seemed to show a preference towards the use of simple carbohydrates like glucose and fructose owing to only expressing transporters for these. These results show the differences in membrane proteome composition and show us the specific proteins necessary in the inner membrane of a dormant spore of this toxigenic spore-forming bacterium to survive adverse conditions.


Assuntos
Bacillus cereus/genética , Proteínas de Bactérias/genética , Doenças Transmitidas por Alimentos/genética , Proteoma/genética , Bacillus cereus/patogenicidade , Proteínas de Bactérias/classificação , Membrana Celular/genética , Contaminação de Alimentos , Microbiologia de Alimentos , Doenças Transmitidas por Alimentos/microbiologia , Humanos , Proteínas de Membrana/classificação , Proteínas de Membrana/genética , Proteômica , Esporos Bacterianos/genética , Esporos Bacterianos/crescimento & desenvolvimento , Esporos Bacterianos/patogenicidade
17.
Int J Mol Sci ; 22(20)2021 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-34681888

RESUMO

Spores of the bacterium Bacillus cereus can cause disease in humans due to contamination of raw materials for food manufacturing. These dormant, resistant spores can survive for years in the environment, but can germinate and grow when their surroundings become suitable, and spore germination proteins play an important role in the decision to germinate. Since germinated spores have lost dormant spores' extreme resistance, knowledge about the formation and function of germination proteins could be useful in suggesting new preservation strategies to control B. cereus spores. In this study, we confirmed that the GerR germinant receptor's (GR) A, B, and C subunits and GerD co-localize in B. cereus spore inner membrane (IM) foci termed germinosomes. The interaction between these proteins was examined by using fusions to the fluorescent reporter proteins SGFP2 and mScarlet-I and Förster Resonance Energy Transfer (FRET). This work found that the FRET efficiency was 6% between GerR(A-C-B)-SGFP2 and GerD-mScarlet-I, but there was no FRET between GerD-mScarlet-I and either GerRA-SGFP2 or GerRC-SGFP2. These results and that GerD does not interact with a GR C-subunit in vitro suggest that, in the germinosome, GerD interacts primarily with the GR B subunit. The dynamics of formation of germinosomes with GerR(A-C-B)-SGFP2 and GerD-mScarlet-I was also followed during sporulation. Our results showed heterogeneity in the formation of FRET positive foci of GerR(A-C-B)-SGFP2 and GerD-mScarlet-I; and while some foci formed at the same time, the formation of foci in the FRET channel could be significantly delayed. The latter finding suggests that either the GerR GR can at least transiently form IM foci in the absence of GerD, or that, while GerD is essential for GerR foci formation, the time to attain the final germinosome structure with close contacts between GerD and GerR can be heterogeneous.


Assuntos
Bacillus cereus/metabolismo , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Domínios e Motivos de Interação entre Proteínas , Esporos Bacterianos/metabolismo , Bacillus cereus/genética , Bacillus cereus/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Esporos Bacterianos/genética , Esporos Bacterianos/crescimento & desenvolvimento
18.
Microbiol Spectr ; 9(2): e0088121, 2021 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-34612699

RESUMO

Sporulation is an important part of the life cycle of Bacillus thuringiensis and the basis for the production of parasporal crystals. This study identifies and characterizes two homologous spoVS genes (spoVS1 and spoVS2) in B. thuringiensis, both of whose expression is dependent on the σH factor. The disruption of spoVS1 and spoVS2 resulted in defective B. thuringiensis sporulation. Similar to Bacillus subtilis, B. thuringiensis strain HD(ΔspoVS1) mutants showed delayed formation of the polar septa, decreased sporulation efficiency, and blocked spore release. Different from B. subtilis, B. thuringiensis HD(ΔspoVS1) mutants had disporic septa and failed to complete engulfment in some cells. Moreover, HD(ΔspoVS2) mutants had delayed spore release. The effect of spoVS1 deletion on polar septum delay and sporulation efficiency could be compensated by spoVS2. ß-Galactosidase activity analysis showed that the expression of pro-sigE and spoIIE decreased to different degrees in the HD(ΔspoVS1) and HD(ΔspoVS2) mutants. The different effects of the two mutations on the expression of sporulation genes led to decreases in Cry1Ac production of different levels. IMPORTANCE There is only one spoVS gene in B. subtilis, and its effects on sporulation have been reported. In this study, two homologous spoVS genes were found and identified in B. thuringiensis. The different effects on sporulation and parasporal crystal protein production in B. thuringiensis and their relationship were investigated. We found that these two homologous spoVS genes are highly conserved in the Bacillus cereus group, and therefore, the functional characterization of SpoVS is helpful to better understand the sporulation processes of members of the Bacillus cereus group.


Assuntos
Toxinas de Bacillus thuringiensis/biossíntese , Bacillus thuringiensis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Endotoxinas/biossíntese , Proteínas Hemolisinas/biossíntese , Esporos Bacterianos/crescimento & desenvolvimento , Bacillus thuringiensis/crescimento & desenvolvimento , Deleção de Genes , Regulação Bacteriana da Expressão Gênica/genética , Fator sigma/genética , Fator sigma/metabolismo
19.
PLoS Genet ; 17(9): e1009791, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34570752

RESUMO

Spore-forming pathogens like Clostridioides difficile depend on germination to initiate infection. During gemination, spores must degrade their cortex layer, which is a thick, protective layer of modified peptidoglycan. Cortex degradation depends on the presence of the spore-specific peptidoglycan modification, muramic-∂-lactam (MAL), which is specifically recognized by cortex lytic enzymes. In C. difficile, MAL production depends on the CwlD amidase and its binding partner, the GerS lipoprotein. To gain insight into how GerS regulates CwlD activity, we solved the crystal structure of the CwlD:GerS complex. In this structure, a GerS homodimer is bound to two CwlD monomers such that the CwlD active sites are exposed. Although CwlD structurally resembles amidase_3 family members, we found that CwlD does not bind Zn2+ stably on its own, unlike previously characterized amidase_3 enzymes. Instead, GerS binding to CwlD promotes CwlD binding to Zn2+, which is required for its catalytic mechanism. Thus, in determining the first structure of an amidase bound to its regulator, we reveal stabilization of Zn2+ co-factor binding as a novel mechanism for regulating bacterial amidase activity. Our results further suggest that allosteric regulation by binding partners may be a more widespread mode for regulating bacterial amidase activity than previously thought.


Assuntos
Amidoidrolases/metabolismo , Clostridioides difficile/fisiologia , Lipoproteínas/metabolismo , Esporos Bacterianos/crescimento & desenvolvimento , Regulação Alostérica , Amidoidrolases/química , Catálise , Domínio Catalítico , Cromatografia em Gel , Clostridioides difficile/enzimologia , Cristalografia por Raios X , Lactamas/metabolismo , Estrutura Molecular , Ácidos Murâmicos/metabolismo , Ligação Proteica
20.
Front Immunol ; 12: 688257, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34497601

RESUMO

We present a stochastic mathematical model of the intracellular infection dynamics of Bacillus anthracis in macrophages. Following inhalation of B. anthracis spores, these are ingested by alveolar phagocytes. Ingested spores then begin to germinate and divide intracellularly. This can lead to the eventual death of the host cell and the extracellular release of bacterial progeny. Some macrophages successfully eliminate the intracellular bacteria and will recover. Here, a stochastic birth-and-death process with catastrophe is proposed, which includes the mechanism of spore germination and maturation of B. anthracis. The resulting model is used to explore the potential for heterogeneity in the spore germination rate, with the consideration of two extreme cases for the rate distribution: continuous Gaussian and discrete Bernoulli. We make use of approximate Bayesian computation to calibrate our model using experimental measurements from in vitro infection of murine peritoneal macrophages with spores of the Sterne 34F2 strain of B. anthracis. The calibrated stochastic model allows us to compute the probability of rupture, mean time to rupture, and rupture size distribution, of a macrophage that has been infected with one spore. We also obtain the mean spore and bacterial loads over time for a population of cells, each assumed to be initially infected with a single spore. Our results support the existence of significant heterogeneity in the germination rate, with a subset of spores expected to germinate much later than the majority. Furthermore, in agreement with experimental evidence, our results suggest that most of the spores taken up by macrophages are likely to be eliminated by the host cell, but a few germinated spores may survive phagocytosis and lead to the death of the infected cell. Finally, we discuss how this stochastic modelling approach, together with dose-response data, allows us to quantify and predict individual infection risk following exposure.


Assuntos
Antraz/microbiologia , Bacillus anthracis/patogenicidade , Macrófagos Peritoneais/microbiologia , Modelos Biológicos , Esporos Bacterianos/patogenicidade , Animais , Antraz/imunologia , Antraz/patologia , Bacillus anthracis/crescimento & desenvolvimento , Bacillus anthracis/imunologia , Teorema de Bayes , Morte Celular , Simulação por Computador , Modelos Animais de Doenças , Interações Hospedeiro-Patógeno , Exposição por Inalação , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/patologia , Camundongos , Viabilidade Microbiana , Fagocitose , Densidade Demográfica , Esporos Bacterianos/crescimento & desenvolvimento , Esporos Bacterianos/imunologia , Processos Estocásticos , Fatores de Tempo
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