RRNPP-type quorum sensing affects solvent formation and sporulation in Clostridium acetobutylicum

The strictly anaerobic bacterium Clostridium acetobutylicum is well known for its ability to convert sugars into organic acids and solvents, most notably the potential biofuel butanol. However, the regulation of its fermentation metabolism, in particular the shift from acid to solvent production, remains poorly understood. The aim of this study was to investigate whether cell–cell communication plays a role in controlling the timing of this shift or the extent of solvent formation. Analysis of the available C. acetobutylicum genome sequences revealed the presence of eight putative RRNPP-type quorum-sensing systems, here designated qssA to qssH, each consisting of an RRNPP-type regulator gene followed by a small open reading frame encoding a putative signalling peptide precursor. The identified regulator and signal peptide precursor genes were designated qsrA to qsrH and qspA to qspH, respectively. Triplicate regulator mutants were generated in strain ATCC 824 for each of the eight systems and screened for phenotypic changes. The qsrB mutants showed increased solvent formation during early solventogenesis and hence the QssB system was selected for further characterization. Overexpression of qsrB severely reduced solvent and endospore formation and this effect could be overcome by adding short synthetic peptides to the culture medium representing a specific region of the QspB signalling peptide precursor. In addition, overexpression of qspB increased the production of acetone and butanol and the initial (48 h) titre of heat-resistant endospores. Together, these findings establish a role for QssB quorum sensing in the regulation of early solventogenesis and sporulation in C. acetobutylicum .

H/D Isotope Effects on 1H-NMR Chemical Shifts in Cyclic Heterodimers and Heterotrimers of Phosphinic and Phosphoric Acids

Hydrogen-bonded heterocomplexes formed by POOH-containing acids (diphenylphosphoric 1, dimethylphosphoric 2, diphenylphosphinic 3, and dimethylphosphinic 4) are studied by the low-temperature (100 K) 1H-NMR and 31P-NMR using liquefied gases CDF3/CDF2Cl as a solvent. Formation of cyclic dimers and cyclic trimers consisting of molecules of two different acids is confirmed by the analysis of vicinal H/D isotope effects (changes in the bridging proton chemical shift, δH, after the deuteration of a neighboring H-bond). Acids 1 and 4 (or 1 and 3) form heterotrimers with very strong (short) H-bonds (δH ca. 17 ppm). While in the case of all heterotrimers the H-bonds are cyclically arranged head-to-tail, ···O=P–O–H···O=P–O–H···, and thus their cooperative coupling is expected, the signs of vicinal H/D isotope effects indicate an effective anticooperativity, presumably due to steric factors: when one of the H-bonds is elongated upon deuteration, the structure of the heterotrimer adjusts by shortening the neighboring hydrogen bonds. We also demonstrate the formation of cyclic tetramers: in the case of acids 1 and 4 the structure has alternating molecules of 1 and 4 in the cycle, while in case of acids 1 and 3 the cycle has two molecules of 1 followed by two molecules of 3.

The Entropy of Deep Eutectic Solvent Formation

The standard entropies S298°E of deep eutectic solvents (DESs), which are liquid binary mixtures of a hydrogen bond acceptor component and a hydrogen bod donor one, are calculated from their molecular volumes, derived from their densities or crystal structures. These values are compared with those of the components—pro-rated according to the DES composition—to obtain the standard entropies of DES formation ΔfS. These quantities are positive, due to the increased number and kinds of hydrogen bonds present in the DESs relative to those in the components. The ΔfS values are also compared with the freezing point depressions of the DESs ΔfusT/K, but no general conclusions on their mutual relationship could be drawn.

Small and Low but Potent: the Complex Regulatory Role of the Small RNA SolB in Solventogenesis inClostridium acetobutylicum

ABSTRACTThe recently revivedClostridium acetobutylicum-based acetone-butanol-ethanol (ABE) fermentation is widely celebrated and studied for its impact on industrial biotechnology.C. acetobutylicumhas been studied and engineered extensively, yet critical areas of the molecular basis for how solvent formation is regulated remain unresolved. The core solventogenic genes (adhE1/aad,ctfA,ctfB, andadc) are carried on the sol locus of the pSOL1 megaplasmid, whose loss leads to asporogenous, “degenerate” cells. The sol locus includes a noncoding small RNA (sRNA), SolB, whose role is presumed to be critical for solventogenesis but has eluded resolution. In the present study, SolB overexpression downregulated the sol-locus genes at the transcript level, resulting in attenuated protein expression and a solvent-deficient phenotype, thus suggesting that SolB affects expression of all sol-locus transcripts and seemingly validating its hypothesized role as a repressor. However, deletion ofsolBresulted in a total loss of acetone production and severe attenuation of butanol formation, with complex effects on sol-locus genes and proteins: it had a small impact onadcmRNA or its corresponding protein (acetoacetate decarboxylase) expression level, somewhat reducedadhE1andctfA-ctfBmRNA levels, and abolished thectfA-ctfB-encoded coenzyme A transferase (CoAT) activity. Computational predictions support a model whereby SolB expressed at low levels enables the stabilization and translation of sol-locus transcripts to facilitate tuning of the production of various solvents depending on the prevailing culture conditions. A key predicted SolB target is the ribosome binding site (RBS) of thectfAtranscript, and this was verified by expressing variants of thectfA-ctfBgenes to demonstrate the importance of SolB for acetone formation.IMPORTANCESmall noncoding RNAs regulate many important metabolic and developmental programs in prokaryotes, but their role in anaerobes has been explored minimally. Regulation of solvent formation in the important industrial organismC. acetobutylicumremains incompletely understood. While the genes for solvent formation and their promoters are known, the means by which this organism tunes the ratios of key solvents, notably the butanol/acetone ratio to balance its electron resources, remains unknown. Significantly, the roles of several coding and noncoding genes in the sol locus in tuning the solvent formation ratios have not been explored. Here we show that the small RNA SolB fine-tunes the expression of solvents, with acetone formation being a key target, by regulating the translation of the acetone formation rate-limiting enzyme, the coenzyme A transferase (CoAT). It is notable that SolB expressed at very low levels enables CoAT translation, while at high, nonphysiological expression levels, it leads to degradation of the corresponding transcript.