Development and Application of Flow-Cytometric Techniques for Analyzing and Sorting Endospore-Forming Clostridia

Tracy, Bryan P.; Gaida, Stefan M.; Papoutsakis, Eleftherios T.

doi:10.1128/aem.01626-08

Cited by 73 publications

(61 citation statements)

References 44 publications

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“…TEM image analysis of the sigF inactivation mutant revealed that the cells failed to initiate asymmetric division (Fig. 5) (88), with no identifiable clostridial forms or endospores (23). This is in contrast to the B. subtilis sigF mutant strain, which develops asymmetric septa (91).…”

Section: Sporulation-specific Sigma Factors That Are Activated Downstmentioning

confidence: 73%

“…Clostridial-form cells (from which the name of the genus Clostridium is derived) are recognizable by their cigar-shaped and swollen phenotype, with accumulating granulose vesicles (storage vesicles made of amylopectin) (22). These clostridial-form cells were originally thought to be the ones that are responsible for solvent formation (22), but more recent studies have shown that solventogenesis is initiated before these cell types are formed (23). The clostridial form is a morphological phenotype unique to Clostridium and is not encountered among the morphological structures of B. subtilis.…”

Section: Figmentioning

confidence: 99%

“…A second phenotype is the low, variable, and stochastic-like frequency of sporulation of solventogenic Clostridium organisms (23). According to the proposed model (Fig.…”

Section: Phenotypic Variations Of Note In Clostridium Organismsmentioning

confidence: 99%

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The Clostridium Sporulation Programs: Diversity and Preservation of Endospore Differentiation

Al-Hinai

Jones

Papoutsakis

2015

Microbiol Mol Biol Rev

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SUMMARY Bacillus and Clostridium organisms initiate the sporulation process when unfavorable conditions are detected. The sporulation process is a carefully orchestrated cascade of events at both the transcriptional and posttranslational levels involving a multitude of sigma factors, transcription factors, proteases, and phosphatases. Like Bacillus genomes, sequenced Clostridium genomes contain genes for all major sporulation-specific transcription and sigma factors ( spo0A , sigH , sigF , sigE , sigG , and sigK ) that orchestrate the sporulation program. However, recent studies have shown that there are substantial differences in the sporulation programs between the two genera as well as among different Clostridium species. First, in the absence of a Bacillus -like phosphorelay system, activation of Spo0A in Clostridium organisms is carried out by a number of orphan histidine kinases. Second, downstream of Spo0A, the transcriptional and posttranslational regulation of the canonical set of four sporulation-specific sigma factors (σ F , σ E , σ G , and σ K ) display different patterns, not only compared to Bacillus but also among Clostridium organisms. Finally, recent studies demonstrated that σ K , the last sigma factor to be activated according to the Bacillus subtilis model, is involved in the very early stages of sporulation in Clostridium acetobutylicum , C. perfringens , and C. botulinum as well as in the very late stages of spore maturation in C. acetobutylicum . Despite profound differences in initiation, propagation, and orchestration of expression of spore morphogenetic components, these findings demonstrate not only the robustness of the endospore sporulation program but also the plasticity of the program to generate different complex phenotypes, some apparently regulated at the epigenetic level.

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Section: Sporulation-specific Sigma Factors That Are Activated Downstmentioning

confidence: 73%

Section: Figmentioning

confidence: 99%

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The Clostridium Sporulation Programs: Diversity and Preservation of Endospore Differentiation

Al-Hinai

Jones

Papoutsakis

2015

Microbiol Mol Biol Rev

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158

190

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“…We chose spo0A as the perturbation target because of its central role in controlling cellular phase transition from acidogenesis to solventogenesis. The knockout was implemented by setting the concentration of Spo0A to zero; the spo0A overexpression was implemented by assigning a higher production rate (31). Simultaneously, we increased the specific maintenance rate (SI Appendix, Eq.…”

Section: Resultsmentioning

confidence: 99%

Integrated, systems metabolic picture of acetone-butanol-ethanol fermentation by Clostridium acetobutylicum

Liao

Seo

Celik

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Microbial metabolism involves complex, system-level processes implemented via the orchestration of metabolic reactions, gene regulation, and environmental cues. One canonical example of such processes is acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum, during which cells convert carbon sources to organic acids that are later reassimilated to produce solvents as a strategy for cellular survival. The complexity and systems nature of the process have been largely underappreciated, rendering challenges in understanding and optimizing solvent production. Here, we present a system-level computational framework for ABE fermentation that combines metabolic reactions, gene regulation, and environmental cues. We developed the framework by decomposing the entire system into three modules, building each module separately, and then assembling them back into an integrated system. During the model construction, a bottom-up approach was used to link molecular events at the singlecell level into the events at the population level. The integrated model was able to successfully reproduce ABE fermentations of the WT C. acetobutylicum (ATCC 824), as well as its mutants, using data obtained from our own experiments and from literature. Furthermore, the model confers successful predictions of the fermentations with various network perturbations across metabolic, genetic, and environmental aspects. From foundation to applications, the framework advances our understanding of complex clostridial metabolism and physiology and also facilitates the development of systems engineering strategies for the production of advanced biofuels.integrated modeling | ABE fermentation | clostridial physiology | systems biology | metabolic engineering M icrobial metabolism is a means by which a microbe uses nutrients and generates energy to live and reproduce. As one of the most fundamental cellular characteristics, it typically involves complex biochemical processes implemented through the orchestration of metabolic reactions and gene regulation, as well as their interactions with environmental cues (1-3). One representative example of such complex processes is solvent production by Clostridium acetobutylicum, a Gram-positive, anaerobic bacterium that is considered to be one of the most prominent species for industrial biofuel production (4).Solvent [acetone-butanol-ethanol (ABE)] fermentation of the species involves two physiological phases (5-8): During the first phase, the bacterium grows exponentially, and organic acids (acetic acid and butyric acid) are produced with the release of energy-the acidogenic phase. This process causes a dramatic drop in extracellular pH. In response to the substantial decrease of the pH, cells enter the stationary phase, and the organic acids formed are reassimilated to produce solvents including acetone, butanol, and ethanol-the solventogenic phase-thereby helping the bacterium to relieve the stress as a strategy for survival. Solventogenesis is subsequently accompanied by the onset of sporulati...

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“…Tracy et al (2008) described the development and application of flow-cytometric and fluorescence assisted cell-sorting (FACS) techniques for study endospore-forming bacteria. In particular, they showed that by combining flow-cytometry light scattering with nucleic acid staining it's possible discriminate, quantify, and enrich all sporulation associated morphologies exhibited by the endospore-forming anaerobe Clostridium acetobutylicum.…”

Section: Wwwintechopencommentioning

confidence: 99%

Applications of Flow Cytometry to Clinical Microbiology

Pieretti¹,

Masucci²,

Moretti³

2012

Clinical Flow Cytometry - Emerging Applications

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Development and Application of Flow-Cytometric Techniques for Analyzing and Sorting Endospore-Forming Clostridia

Cited by 73 publications

References 44 publications

The Clostridium Sporulation Programs: Diversity and Preservation of Endospore Differentiation

The Clostridium Sporulation Programs: Diversity and Preservation of Endospore Differentiation

Integrated, systems metabolic picture of acetone-butanol-ethanol fermentation by Clostridium acetobutylicum

Applications of Flow Cytometry to Clinical Microbiology

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