A Bacillus subtilis Mutant Requiring Dipicolinic Acid for the Development of Heat-resistant Spores

Balassa, G; Milhaud, Pierre G.; Raulet, E.; Silva, M. T.; Sousa, Joana

doi:10.1099/00221287-110-2-365

Cited by 66 publications

(35 citation statements)

References 11 publications

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“…tion or lead to fragile, but functional, spores) (3,27,37). This wave of expression seems to represent the final stages of sporulation, as many of these loci would not be necessary until just before release from the mother cell and exposure to a potentially harsh environment.…”

Section: Resultsmentioning

confidence: 99%

Formation and Composition of the Bacillus anthracis Endospore

Liu

Bergman

Thomason³

et al. 2004

J Bacteriol

192

206

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The endospores of Bacillus anthracis are the infectious particles of anthrax. Spores are dormant bacterial morphotypes able to withstand harsh environments for decades, which contributes to their ability to be formulated and dispersed as a biological weapon. We monitored gene expression in B. anthracis during growth and sporulation using full genome DNA microarrays and matched the results against a comprehensive analysis of the mature anthrax spore proteome. A large portion (ϳ36%) of the B. anthracis genome is regulated in a growth phase-dependent manner, and this regulation is marked by five distinct waves of gene expression as cells proceed from exponential growth through sporulation. The identities of more than 750 proteins present in the spore were determined by multidimensional chromatography and tandem mass spectrometry. Comparison of data sets revealed that while the genes responsible for assembly and maturation of the spore are tightly regulated in discrete stages, many of the components ultimately found in the spore are expressed throughout and even before sporulation, suggesting that gene expression during sporulation may be mainly related to the physical construction of the spore, rather than synthesis of eventual spore content. The spore also contains an assortment of specialized, but not obviously related, metabolic and protective proteins. These findings contribute to our understanding of spore formation and function and will be useful in the detection, prevention, and early treatment of anthrax. This study also highlights the complementary nature of genomic and proteomic analyses and the benefits of combining these approaches in a single study.Entry of Bacillus anthracis spores into the host from the environment, or by events brought about by human design, is the initial event of anthrax infections (8). Vegetative bacilli are not believed to be the disease contagion for any form of anthrax. The route of spore entry into the host dictates the specific pathology and severity of the disease; e.g., cutaneous anthrax is generally far less severe than either the gastrointestinal or inhalational form (8). Endospores are produced in response to nutrient deprivation via an alternative developmental cascade by two known genera of gram-positive bacteria, Clostridium and Bacillus (34). During sporulation, vegetative metabolism is minimized, and a series of alternative sigma factors are sequentially expressed and activated to coordinate the expression of mRNAs responsible for spore development (22).Mature spores are metabolically inactive and have a highly ordered structure. This structure provides the protection required for survival over long periods, even in the face of harsh environmental conditions (48). Spore germination, outgrowth, and initiation of a vegetative cycle occur when small molecules, often nutrients and/or ions, are sensed in the context of aqueous environments. B. anthracis spores recognize specific signals provided by the local environment of a mammalian host and rapidly germinate when ass...

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Section: Resultsmentioning

confidence: 99%

Formation and Composition of the Bacillus anthracis Endospore

Liu

Bergman

Thomason³

et al. 2004

J Bacteriol

192

206

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“…Spores are also very resistant to ethanol, again a common disinfectant (Russell 1990). There have been a few studies examining the acquisition of resistance to some of these agents during sporulation (Milhaud and Balassa 1973;Balassa et al 1979), as well as their effects on spores (Russell 1990;Bloomfield and Arthur 1994;McDonnell and Russell 1999). However, the mechanisms of spore resistance to acid, alkali and ethanol are not known and neither are the precise mechanisms of spore killing by these agents.…”

Section: Introductionmentioning

confidence: 99%

Mechanisms of killing spores of Bacillus subtilis by acid, alkali and ethanol

et al. 2002

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Aims: To determine the mechanisms of killing of Bacillus subtilis spores by ethanol or strong acid or alkali. Methods and Results: Killing of B. subtilis spores by ethanol or strong acid or alkali was not through DNA damage and the spore coats did not protect spores against these agents. Spores treated with ethanol or acid released their dipicolinic acid (DPA) in parallel with spore killing and the core wet density of ethanol-or acid-killed spores fell to a value close to that for untreated spores lacking DPA. The core regions of spores killed by these two agents were stained by nucleic acid stains that do not penetrate into the core of untreated spores and acid-killed spores appeared to have ruptured. Spores killed by these two agents also did not germinate in nutrient and nonnutrient germinants and were not recovered by lysozyme treatment. Spores killed by alkali did not lose their DPA, did not exhibit a decrease in their core wet density and their cores were not stained by nucleic acid stains. Alkali-killed spores released their DPA upon initiation of spore germination, but did not initiate metabolism and degraded their cortex very poorly. However, spores apparently killed by alkali were recovered by lysozyme treatment. Conclusions: The data suggest that spore killing by ethanol and strong acid involves the disruption of a spore permeability barrier, while spore killing by strong alkali is due to the inactivation of spore cortex lytic enzymes. Significance and Impact of the Study: The results provide further information on the mechanisms of spore killing by various chemicals.

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“…As this phenotype is reverted by supplementation of the sporulation medium with dipicolinate, it seems likely that dpa-2 affects dipicolinate synthase (Balassa et al, 1979). Since, in addition, AKI and dipicolinate synthase belong to the same metabolic pathway, we assessed the linkage of the above markers by transformation.…”

Section: Genetic Linkage Between Dapg and Spovfmentioning

confidence: 99%

Genes involved in meso-diaminopimelate synthesis in Bacillus subtilis: identification of the gene encoding aspartokinase I

Roten¹,

Brandt²,

Karamata³

1991

Journal of General Microbiology

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Thermosensitive mutants of Bacillus subtilis deficient in peptidoglycan synthesis were screened for mutations in the rneso-diaminopimelate ( L D -A~~~) metabolic pathway. Mutations in two out of five relevant linkage groups, lssB and IssD, were shown to induce, at the restrictive temperature, a deficiency in LD-A,pm synthesis and accumulation of UDP-MurNAc-dipeptide. Group IssB is heterogeneous; it encompasses mutations that confer deficiency in the deacylation of N-acetyI-~~-A,pm and accumulation of this precursor. Accordingly, these mutations are assigned to the previously identified locus dapE. Mutations in linkage group lssD entail a thermosensitive aspartokinase I. Therefore, they are most likely to affect the structural gene of this enzyme, which we propose to designate dapC. Mutation pyc-1476, previously reported to affect the pyruvate carboxylase, was shown to confer a deficiency in aspartokinase I, not in the carboxylase, and to belong to the dapC locus. dapG is closely linked to spoVF, the putative gene of dipicolinate synthase. In conclusion, mutations affecting only two out of eight steps known to be involved in LD-A,pm synthesis were uncovered in a large collection of thermosensitive mutants obtained by indirect selection. We propose that this surprisingly restricted distribution of the thermosensitive dap mutations isolated so far is due to the existence, in each step of the pathway, of isoenzymes encoded by separate genes. The biological role of different aspartokinases was investigated with mutants deficient in dapE and dapC genes. Growth characteristics of these mutants in the presence of various combinations of aspartate family amino acids allow a reassessment of a metabolic channel hypothesis, i.e. the proposed existence of multienzyme complexes, each specific for a given end product.

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A Bacillus subtilis Mutant Requiring Dipicolinic Acid for the Development of Heat-resistant Spores

Cited by 66 publications

References 11 publications

Formation and Composition of the Bacillus anthracis Endospore

Formation and Composition of the Bacillus anthracis Endospore

Mechanisms of killing spores of Bacillus subtilis by acid, alkali and ethanol

Genes involved in meso-diaminopimelate synthesis in Bacillus subtilis: identification of the gene encoding aspartokinase I

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