Chance and Necessity in
            <i>Bacillus subtilis</i>
            Development

Mirouze, Nicolas; Dubnau, David

doi:10.1128/microbiolspectrum.tbs-0004-2012

Cited by 18 publications

(11 citation statements)

References 110 publications

(184 reference statements)

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“…Soil-dwelling B. subtilis is a model organism for studying the various facets of bacterial life (17,18) and an important producer strain for industrial enzymes and valuable low-molecular-weight substances (19). Various tools, like markerless gene deletion and insertion systems, are already available for genome editing.…”

mentioning

confidence: 99%

Editing of the Bacillus subtilis Genome by the CRISPR-Cas9 System

Altenbuchner

2016

Appl Environ Microbiol

278

246

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The clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) systems are adaptive immune systems of bacteria. A type II CRISPR-Cas9 system from Streptococcus pyogenes has recently been developed into a genome engineering tool for prokaryotes and eukaryotes. Here, we present a single-plasmid system which allows efficient genome editing of Bacillus subtilis. The plasmid pJOE8999 is a shuttle vector that has a pUC minimal origin of replication for Escherichia coli, the temperature-sensitive replication origin of plasmid pE194 ts for B. subtilis, and a kanamycin resistance gene working in both organisms. For genome editing, it carries the cas9 gene under the control of the B. subtilis mannose-inducible promoter P manP and a single guide RNA (sgRNA)-encoding sequence transcribed via a strong promoter. This sgRNA guides the Cas9 nuclease to its target. The 20-nucleotide spacer sequence at the 5= end of the sgRNA sequence, responsible for target specificity, is located between BsaI sites. Thus, the target specificity is altered by changing the spacer sequences via oligonucleotides fitted between the BsaI sites. Cas9 in complex with the sgRNA induces double-strand breaks (DSBs) at its target site. Repair of the DSBs and the required modification of the genome are achieved by adding homology templates, usually two PCR fragments obtained from both sides of the target sequence. Two adjacent SfiI sites enable the ordered integration of these homology templates into the vector. The function of the CRISPR-Cas9 vector was demonstrated by introducing two large deletions in the B. subtilis chromosome and by repair of the trpC2 mutation of B. subtilis 168. IMPORTANCEIn prokaryotes, most methods used for scarless genome engineering are based on selection-counterselection systems. The disadvantages are often the lack of a suitable counterselection marker, the toxicity of the compounds needed for counterselection, and the requirement of certain mutations in the target strain. CRISPR-Cas systems were recently developed as important tools for genome editing. The single-plasmid system constructed for the genome editing of B. subtilis overcomes the problems of counterselection methods. It allows deletions and introduction of point mutations. It is easy to handle and very efficient, and it may be adapted for use in other firmicutes.

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mentioning

confidence: 99%

Editing of the Bacillus subtilis Genome by the CRISPR-Cas9 System

Altenbuchner

2016

Appl Environ Microbiol

278

246

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“…Regarding the transformation procedure, B. subtilis was grown overnight on Luria-Bertani plates (Difco, Becton, Dickinson and Company) at 37°C. After incubation, a colony was resuspended in MG1 medium composed of MG medium containing 2 g/liter of (NH 4 ) 2 SO 4 (Merck, Germany), 1 g/liter of Na 3 (47). A total of 200 l of the suspension in MG2 was added to 0.1 l of genomic DNA extracted from strain AC699 with a High Pure PCR template extraction kit (Roche Dignostics, Meylan, France) and incubated for 30 min at 37°C.…”

Section: Methodsmentioning

confidence: 99%

“…Preventive means are also used in order to limit the bacterial growth by applying the cold chain or to limit the formation of biofilms with cleaning and disinfection steps. However, little attention has been paid to the formation of spores during food processes, on food lines, whereas sporulation has been observed in milk powder processes (3,4). The sporulation leads to an increase of the spore yield in foods.…”

Section: Importancementioning

confidence: 99%

Differentiation of Vegetative Cells into Spores: a Kinetic Model Applied to Bacillus subtilis

Gauvry

Mathot

Couvert

et al. 2019

Appl Environ Microbiol

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Spore-forming bacteria are natural contaminants of food raw materials, and sporulation can occur in many environments from farm to fork. In order to characterize and to predict spore formation over time, we developed a model that describes both the kinetics of growth and the differentiation of vegetative cells into spores. The model is based on a classical growth model and enables description of the kinetics of sporulation with the addition of three parameters specific to sporulation. Two parameters are related to the probability of each vegetative cell to commit to sporulation and to form a spore, and the last one is related to the time needed to form a spore once the cell is committed to sporulation. The goodness of fit of this growth-sporulation model was assessed using growth-sporulation kinetics at various temperatures in laboratory medium or in whey for Bacillus subtilis, Bacillus cereus, and Bacillus licheniformis. The model accurately describes the kinetics in these different conditions, with a mean error lower than 0.78 log 10 CFU/ml for the growth and 1.08 log 10 CFU/ml for the sporulation. The biological meaning of the parameters was validated with a derivative strain of Bacillus subtilis 168 which produces green fluorescent protein at the initiation of sporulation. This model provides physiological information on the spore formation and on the temporal abilities of vegetative cells to differentiate into spores and reveals the heterogeneity of spore formation during and after growth. Citation Gauvry E, Mathot A-G, Couvert O, Leguérinel I, Jules M, Coroller L. 2019. Differentiation of vegetative cells into spores: a kinetic model applied to Bacillus subtilis. Appl Environ Microbiol 85:e00322-19. https://doi .on July 10, 2020 by guest http://aem.asm.org/ Downloaded from RESULTSModel development and experimental strategy. The growth of vegetative cells was described by a modified logistic model (equation 1) and their differentiation into spores over time with specific sporulation parameters: the probability (t max , , and P max ; see below) of vegetative cells to give a mature spore (heat resistant) during the incubation and the time needed for the spore formation (t f

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“…However, it is well known that isogenic bacteria growing under identical conditions do not necessarily display the same pattern of gene expression during competence. This phenomenon is termed "bistability", and is not associated with genome rearrangements or mutations, but alternatively involves reversible switching between varying states of competency in the absence of any genetic modification [12,23,28]. Consequently, it is possible that bistability gives rise to a mixed population of competent "predators" and non-competent target cells.…”

Section: Fratricide In Streptococcusmentioning

confidence: 99%

“…When exposed to environmentally stressful conditions, for example starvation, this microorganism initiates a complex survival strategy which is called sporulation leading to the formation of endospores that are very resistant to adverse environmental conditions. It allows B. subtilis to survive and wait for favorable conditions for growth [9,28,54]. The entrance into the sporulation pathway is a multistep process associated with a diversification into distinct sub-populations of specialized cell types that try to extract different types of nutrients from the environment [55,56].…”

Section: Cannibalism In Bacillus Subtilismentioning

confidence: 99%

Harnessing the Potential of Killers and Altruists within the Microbial Community: A Possible Alternative to Antibiotic Therapy?

et al. 2019

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In the context of a post-antibiotic era, the phenomenon of microbial allolysis, which is defined as the partial killing of bacterial population induced by other cells of the same species, may take on greater significance. This phenomenon was revealed in some bacterial species such as Streptococcus pneumoniae and Bacillus subtilis, and has been suspected to occur in some other species or genera, such as enterococci. The mechanisms of this phenomenon, as well as its role in the life of microbial populations still form part of ongoing research. Herein, we describe recent developments in allolysis in the context of its practical benefits as a form of cell death that may give rise to developing new strategies for manipulating the life and death of bacterial communities. We highlight how such findings may be viewed with importance and potential within the fields of medicine, biotechnology, and pharmacology.

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Chance and Necessity in Bacillus subtilis Development

Cited by 18 publications

References 110 publications

Editing of the Bacillus subtilis Genome by the CRISPR-Cas9 System

Editing of the Bacillus subtilis Genome by the CRISPR-Cas9 System

Differentiation of Vegetative Cells into Spores: a Kinetic Model Applied to Bacillus subtilis

Harnessing the Potential of Killers and Altruists within the Microbial Community: A Possible Alternative to Antibiotic Therapy?

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