Pumping toxic substances through a cytoplasmic membrane by protein transporters known as efflux pumps represents one bacterial mechanism involved in the stress response to the presence of toxic compounds. The active efflux might also take part in exporting low-molecular-weight alcohols produced by intrinsic cell metabolism; in the case of solventogenic clostridia, predominantly acetone, butanol and ethanol (ABE). However, little is known about this active efflux, even though some evidence exists that membrane pumps might be involved in solvent tolerance. In this study, we investigated changes in overall active efflux during ABE fermentation, employing a flow cytometric protocol adjusted for Clostridia and using ethidium bromide (EB) as a fluorescence marker for quantification of direct efflux. A fluctuation in efflux during the course of standard ABE fermentation was observed, with a maximum reached during late acidogenesis, a high efflux rate during early and mid-solventogenesis and an apparent decrease in EB efflux rate in late solventogenesis. The fluctuation in efflux activity was in accordance with transcriptomic data obtained for various membrane exporters in a former study. Surprisingly, under altered cultivation conditions, when solvent production was attenuated, and extended acidogenesis was promoted, stable low efflux activity was reached after an initial peak that appeared in the stage comparable to standard ABE fermentation. This study confirmed that efflux pump activity is not constant during ABE fermentation and suggests that undisturbed solvent production might be a trigger for activation of pumps involved in solvent efflux. KEY POINTS: • Flow cytometric assay for efflux quantification in Clostridia was established. • Efflux rate peaked in late acidogenesis and in early solventogenesis. • Impaired solventogenesis led to an overall decrease in efflux.

• Klíčová slova
• ABE fermentation, Clostridium, Efflux pump, Ethidium bromide, Flow cytometry,
• MeSH
• aceton MeSH
• butanoly MeSH
• Clostridium beijerinckii * MeSH
• Clostridium MeSH
• ethanol MeSH
• fermentace MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• aceton MeSH
• butanoly MeSH
• ethanol MeSH

The main bottleneck in the return of industrial butanol production from renewable feedstock through acetone-butanol-ethanol (ABE) fermentation by clostridia, such as Clostridium beijerinckii, is the low final butanol concentration. The problem is caused by the high toxicity of butanol to the production cells, and therefore, understanding the mechanisms by which clostridia react to butanol shock is of key importance. Detailed analyses of transcriptome data that were obtained after butanol shock and their comparison with data from standard ABE fermentation have resulted in new findings, while confirmed expected population responses. Although butanol shock resulted in upregulation of heat shock protein genes, their regulation is different than was assumed based on standard ABE fermentation transcriptome data. While glucose uptake, glycolysis, and acidogenesis genes were downregulated after butanol shock, solventogenesis genes were upregulated. Cyclopropanation of fatty acids and formation of plasmalogens seem to be significant processes involved in cell membrane stabilization in the presence of butanol. Surprisingly, one of the three identified Agr quorum-sensing system genes was upregulated. Upregulation of several putative butanol efflux pumps was described after butanol addition and a large putative polyketide gene cluster was found, the transcription of which seemed to depend on the concentration of butanol.

• Klíčová slova
• Clostridium beijerinckii, ABE fermentation, butanol shock, transcriptome analysis,
• MeSH
• biologický transport genetika   MeSH
• bioreaktory mikrobiologie   MeSH
• buněčná membrána metabolismus   MeSH
• butanoly toxicita   MeSH
• Clostridium beijerinckii účinky léků   genetika   metabolismus   MeSH
• fyziologický stres genetika   MeSH
• glukosa metabolismus   MeSH
• glykolýza genetika   fyziologie   MeSH
• mastné kyseliny metabolismus   MeSH
• plasmalogeny biosyntéza   MeSH
• proteiny teplotního šoku metabolismus   MeSH
• quorum sensing genetika   MeSH
• stanovení celkové genové exprese MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• butanoly MeSH
• glukosa MeSH
• mastné kyseliny MeSH
• plasmalogeny MeSH
• proteiny teplotního šoku MeSH

BACKGROUND: One of the main obstacles preventing solventogenic clostridia from achieving higher yields in biofuel production is the toxicity of produced solvents. Unfortunately, regulatory mechanisms responsible for the shock response are poorly described on the transcriptomic level. Although the strain Clostridium beijerinckii NRRL B-598, a promising butanol producer, has been studied under different conditions in the past, its transcriptional response to a shock caused by butanol in the cultivation medium remains unknown. RESULTS: In this paper, we present a transcriptional response of the strain during a butanol challenge, caused by the addition of butanol to the cultivation medium at the very end of the acidogenic phase, using RNA-Seq. We resequenced and reassembled the genome sequence of the strain and prepared novel genome and gene ontology annotation to provide the most accurate results. When compared to samples under standard cultivation conditions, samples gathered during butanol shock represented a well-distinguished group. Using reference samples gathered directly before the addition of butanol, we identified genes that were differentially expressed in butanol challenge samples. We determined clusters of 293 down-regulated and 301 up-regulated genes whose expression was affected by the cultivation conditions. Enriched term "RNA binding" among down-regulated genes corresponded to the downturn of translation and the cluster contained a group of small acid-soluble spore proteins. This explained phenotype of the culture that had not sporulated. On the other hand, up-regulated genes were characterized by the term "protein binding" which corresponded to activation of heat-shock proteins that were identified within this cluster. CONCLUSIONS: We provided an overall transcriptional response of the strain C. beijerinckii NRRL B-598 to butanol shock, supplemented by auxiliary technologies, including high-pressure liquid chromatography and flow cytometry, to capture the corresponding phenotypic response. We identified genes whose regulation was affected by the addition of butanol to the cultivation medium and inferred related molecular functions that were significantly influenced. Additionally, using high-quality genome assembly and custom-made gene ontology annotation, we demonstrated that this settled terminology, widely used for the analysis of model organisms, could also be applied to non-model organisms and for research in the field of biofuels.

• Klíčová slova
• ABE fermentation, Butanol shock, Clostridium beijerinckii NRRL B-598, RNA-Seq transcriptome,
• Publikační typ
• časopisecké články MeSH

Clostridium beijerinckii NRRL B-598 is a sporulating, butanol and hydrogen producing strain that utilizes carbohydrates by the acetone-butanol-ethanol (ABE) fermentative pathway. The pathway consists of two metabolic phases, acidogenesis and solventogenesis, from which the latter one can be coupled with sporulation. Thorough transcriptomic profiling during a complete life cycle and both metabolic phases completed with flow cytometry, microscopy and a metabolites analysis helped to find out key genes involved in particular cellular events. The description of genes/operons that are closely involved in metabolism or the cell cycle is a necessary condition for metabolic engineering of the strain and will be valuable for all C. beijerinckii strains and other Clostridial species. The study focused on glucose transport and catabolism, hydrogen formation, metabolic stress response, binary fission, motility/chemotaxis and sporulation, which resulted in the composition of the unique image reflecting clostridial population changes. Surprisingly, the main change in expression of individual genes was coupled with the sporulation start and not with the transition from acidogenic to solventogenic metabolism. As expected, solvents formation started at pH decrease and the accumulation of butyric and acetic acids in the cultivation medium.

• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• Clostridium beijerinckii cytologie   genetika   MeSH
• fermentace genetika   MeSH
• fyziologický stres * genetika   MeSH
• glukosa metabolismus   MeSH
• kyseliny metabolismus   MeSH
• mastné kyseliny metabolismus   MeSH
• proteiny teplotního šoku genetika   metabolismus   MeSH
• regulace genové exprese u bakterií * MeSH
• rozpouštědla metabolismus   MeSH
• spory bakteriální metabolismus   MeSH
• transkriptom genetika   MeSH
• vodík metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• glukosa MeSH
• kyseliny MeSH
• mastné kyseliny MeSH
• proteiny teplotního šoku MeSH
• rozpouštědla MeSH
• vodík MeSH

BACKGROUND: Butanol is currently one of the most discussed biofuels. Its use provides many benefits in comparison to bio-ethanol, but the price of its fermentative production is still high. Genetic improvements could help solve many problems associated with butanol production during ABE fermentation, such as its toxicity, low concentration achievable in the cultivation medium, the need for a relatively expensive substrate, and many more. Clostridium pasteurianum NRRL B-598 is non-type strain producing butanol, acetone, and a negligible amount of ethanol. Its main benefits are high oxygen tolerance, utilization of a wide range of carbon and nitrogen sources, and the availability of its whole genome sequence. However, there is no established method for the transfer of foreign DNA into this strain; this is the next step necessary for progress in its use for butanol production. RESULTS: We have described functional protocols for conjugation and transformation of the bio-butanol producer C. pasteurianum NRRL B-598 by foreign plasmid DNA. We show that the use of unmethylated plasmid DNA is necessary for efficient transformation or successful conjugation. Genes encoding DNA methylation and those for restriction-modification systems and antibiotic resistance were searched for in the whole genome sequence and their homologies with other clostridial bacteria were determined. Furthermore, activity of described novel type I restriction system was proved experimentally. The described electrotransformation protocol achieved an efficiency 1.2 × 10(2) cfu/μg DNA after step-by-step optimization and an efficiency of 1.6 × 10(2) cfu/μg DNA was achieved by the sonoporation technique using a standard laboratory ultrasound bath. The highest transformation efficiency was achieved using a combination of these approaches; sono/electroporation led to an increase in transformation efficiency, to 5.3 × 10(2) cfu/μg DNA. CONCLUSIONS: Both Dam and Dcm methylations are detrimental for transformation of C. pasteurianum NRRL B-598. Methods for conjugation, electroporation, sonoporation, and a combined method for sono/electroporation were established for this strain. The methods described could be used for genetic improvement of this strain, which is suitable for bio-butanol production.

• Klíčová slova
• Butanol, Clostridium, Conjugation, Dam, Dcm, Electroporation, Methylation, Sonoporation, Transformation,
• Publikační typ
• časopisecké články MeSH