Organic Mixed-Valence Compounds and the Overhauser Effect in Insulating Solids.

Recent experiments have shown that the organic free radical 1,3-bisdiphenylene-2-phenylallyl (BDPA) can induce an Overhauser effect dynamic nuclear polarization in insulating solids, a feat previously considered not to be possible. Here, we establish that this peculiar ability of the BDPA radical stems from its mixed-valence nature and the ensuing intramolecular charge transfer. Using state-of-the-art DMRGSCF calculations, we confirm the class II mixed-valence nature of BDPA with the characteristic double-well potential energy surface, and we investigate the mechanism of the consequent electron hopping. A two-component vibronic Hamiltonian is then employed to compute the rate of electron hopping from a quantum dynamical time-propagation of the density matrix. The predicted hyperfine coupling oscillations indeed fall within the frequency range required for an Overhauser effect. The paradigm of mixed-valence compounds as a mining source opens many possibilities for the development and fine tuning of novel polarizing agents.

Microbial Production of Natural and Unnatural Monolignols with Escherichia coli.

Phenylpropanoids and phenylpropanoid-derived plant polyphenols find numerous applications in the food and pharmaceutical industries. In recent years, several microbial platform organisms have been engineered towards producing such compounds. However, for the most part, microbial (poly)phenol production is inspired by nature, so naturally occurring compounds have predominantly been produced to date. Here we have taken advantage of the promiscuity of the enzymes involved in phenylpropanoid synthesis and exploited the versatility of an engineered Escherichia coli strain harboring a synthetic monolignol pathway to convert supplemented natural and unnatural phenylpropenoic acids into their corresponding monolignols. The performed biotransformations showed that this strain is able to catalyze the stepwise reduction of chemically interesting unnatural phenylpropenoic acids such as 3,4,5-trimethoxycinnamic acid, 5-bromoferulic acid, 2-nitroferulic acid, and a "bicyclic" p-coumaric acid derivative, in addition to six naturally occurring phenylpropenoic acids.

Chemoenzymatic Total Synthesis of the Proposed Structures of Putaminoxins B and D.

Different enzymatic and nonenzymatic approaches were tested and compared to afford enantiopure homoallylic and allylic alcohols as building blocks in a total synthesis showcase. Thereby, highly enantioselective alcohol dehydrogenases and the P450 BM3 monooxygenase variant A74G L188Q were compared to classical asymmetric reagent-controlled allyl additions. Thus, the first total syntheses of the proposed structures for putaminoxins B/D and their respective enantiomers were accomplished. Detailed spectroscopic analysis of the newly synthesized compounds unraveled a discrepancy with respect to the reported structures of putaminoxins B/D. Furthermore, it was demonstrated that total synthesis is generally required for unequivocal assignment of configuration, because purely comparative NMR studies and judgment by analogy can lead to false predictions.

Effect of new alleles of the histidine kinase gene ciaH on the activity of the response regulator CiaR in Streptococcus pneumoniae R6.

The two-component regulatory system CiaRH of Streptococcus pneumoniae affects ß-lactam susceptibility, autolysis, bacteriocin production, competence development, host colonization and virulence. The system was discovered in a screen for S. pneumoniae R6 mutants resistant to the ß-lactam antibiotic cefotaxime. A mutation in the histidine kinase gene ciaH led to this phenotype by enhancing CiaR-mediated gene expression. Additional mutations in ciaH have been described in other spontaneous ß-lactam-resistant mutants of S. pneumoniae R6, but their influence on CiaR-mediated gene regulation has not been determined. Likewise, altered ciaH alleles are present in clinical S. pneumoniae isolates, none of which had been characterized. These novel ciaH variants were introduced into S. pneumoniae R6 to measure their ability to activate CiaR-dependent regulation. The ciaH alleles from spontaneous mutants obtained in the laboratory increased the activity of CiaR-dependent promoters between four- and 26-fold, while variants from clinical strains were less effective, with a threefold activation at most. Accordingly, phenotypes associated with a hyperactive CiaRH system, ß-lactam resistance, and prevention of competence development, were far more pronounced in the laboratory mutants. Amino acid changes affecting CiaH function were positioned throughout the protein. Five of the most activating changes are located close to the conserved histidine and one in the extracytoplasmic sensor domain. The characterization of new alleles of ciaH expands the spectrum of CiaH variants, which may help to elucidate signal transduction of this important regulatory system. Our study also demonstrates that ciaH alleles overstimulating CiaR regulon expression are present in clinical isolates of S. pneumoniae.

Identification of genes for small non-coding RNAs that belong to the regulon of the two-component regulatory system CiaRH in Streptococcus.

BACKGROUND: Post-transcriptional regulation by small RNAs (sRNAs) in bacteria is now recognized as a wide-spread regulatory mechanism modulating a variety of physiological responses including virulence. In Streptococcus pneumoniae, an important human pathogen, the first sRNAs to be described were found in the regulon of the CiaRH two-component regulatory system. Five of these sRNAs were detected and designated csRNAs for cia-dependent small RNAs. CiaRH pleiotropically affects ß-lactam resistance, autolysis, virulence, and competence development by yet to be defined molecular mechanisms. Since CiaRH is highly conserved among streptococci, it is of interest to determine if csRNAs are also included in the CiaRH regulon in this group of organisms consisting of commensal as well as pathogenic species. Knowledge on the participation of csRNAs in CiaRH-dependent regulatory events will be the key to define the physiological role of this important control system. RESULTS: Genes for csRNAs were predicted in streptococcal genomes and data base entries other than S. pneumoniae by searching for CiaR-activated promoters located in intergenic regions that are followed by a transcriptional terminator. 61 different candidate genes were obtained specifying csRNAs ranging in size from 51 to 202 nt. Comparing these genes among each other revealed 40 different csRNA types. All streptococcal genomes harbored csRNA genes, their numbers varying between two and six. To validate these predictions, S. mitis, S. oralis, and S. sanguinis were subjected to csRNA-specific northern blot analysis. In addition, a csRNA gene from S. thermophilus plasmid pST0 introduced into S. pneumoniae was also tested. Each of the csRNAs was detected on these blots and showed the anticipated sizes. Thus, the method applied here is able to predict csRNAs with high precision. CONCLUSIONS: The results of this study strongly suggest that genes for small non-coding RNAs, csRNAs, are part of the regulon of the two-component regulatory system CiaRH in all streptococci.

Environmental stress perception activates structural remodeling of extant Streptococcus mutans biofilms.

Transcription regulators from the LexA-like Protein Superfamily control a highly diverse assortment of genetic pathways in response to environmental stress. All characterized members of this family modulate their functionality and stability via a strict coordination with the coprotease function of RecA. Using the LexA-like protein IrvR from Streptococcus mutans, we demonstrate an exception to the RecA paradigm and illustrate how this evolutionary innovation has been coopted to diversify the stress responsiveness of S. mutans biofilms. Using a combination of genetics and biophysical measurements, we demonstrate how non-SOS stresses and SOS stresses each trigger separate regulatory mechanisms that stimulate production of a surface lectin responsible for remodeling the viscoelastic properties of extant biofilms during episodes of environmental stress. These studies demonstrate how changes in the external environment or even anti-biofilm therapeutic agents can activate biofilm-specific adaptive mechanisms responsible for bolstering the integrity of established biofilm communities. Such changes in biofilm community structure are likely to play central roles in the notorious recalcitrance of biofilm infections.

A tetracycline-inducible integrative expression system for Streptococcus pneumoniae.

Inducible gene expression systems are very useful to analyze cellular processes. The ability to switch the expression state of genes of interest may even be crucial if essential traits or genetic instability are involved. An integrative plasmid, pTEX2, was designed using the (anhydro)tetracycline-inducible promoter Pxyl/tet from staphylococcal plasmid pRAB11 to control gene expression in Streptococcus pneumoniae. The system was evaluated by expressing genes of the two-component regulatory system ciaRH of S. pneumoniae. With full induction of Pxyl/tet, wild-type levels of the response regulator CiaR were obtained, while the uninduced basal expression was low. Hyperactive variants of the kinase gene ciaH normally causing pronounced genetic instability could be handled without any problems upon cloning into pTEX2. Therefore, the expression system is well suited to express physiological levels of proteins in S. pneumoniae and also to aid regulatory studies.

Activity of the response regulator CiaR in mutants of Streptococcus pneumoniae R6 altered in acetyl phosphate production.

The two-component regulatory system (TCS) CiaRH of Streptococcus pneumoniae is implicated in competence, ß-lactam resistance, maintenance of cell integrity, bacteriocin production, host colonization, and virulence. Depending on the growth conditions, CiaR can be highly active in the absence of its cognate kinase CiaH, although phosphorylation of CiaR is required for DNA binding and gene regulation. To test the possibility that acetyl phosphate (AcP) could be the alternative phosphodonor, genes involved in pyruvate metabolism were disrupted to alter cellular levels of acetyl phosphate. Inactivating the genes of pyruvate oxidase SpxB, phosphotransacetylase Pta, and acetate kinase AckA, resulted in very low AcP levels and in strongly reduced CiaR-mediated gene expression in CiaH-deficient strains. Therefore, alternative phosphorylation of CiaR appears to proceed via AcP. The AcP effect on CiaR is not detected in strains with CiaH. Attempts to obtain elevated AcP by preventing its degradation by acetate kinase AckA, were not successful in CiaH-deficient strains with a functional SpxB, the most important enzyme for AcP production in S. pneumoniae. The ciaH-spxB-ackA mutant producing intermediate amounts of AcP could be constructed and showed a promoter activation, which was much higher than expected. Since activation was dependent on AcP, it can apparently be used more efficiently for CiaR phosphorylation in the absence of AckA. Therefore, high AcP levels in the absence of CiaH and AckA may cause extreme overexpression of the CiaR regulon leading to synthetic lethality. AckA is also involved in a regulatory response, which is mediated by CiaH. Addition of acetate to the growth medium switch CiaH from kinase to phosphatase. This switch is lost in the absence of AckA indicating metabolism of acetate is required, which starts with the production of AcP by AckA. Therefore, AckA plays a special regulatory role in the control of the CiaRH TCS.

Activity of the two-component regulatory system CiaRH in Streptococcus pneumoniae R6.

The two-component regulatory system CiaRH of Streptococcus pneumoniae affects a variety of processes such as competence development, autolysis, bacteriocin production, host colonization, and virulence. While the targets of the regulator CiaR are known, the role of phosphorylation in CiaR regulation has not been defined. To address this issue, the presumed phosphorylation site of CiaR, aspartic acid at position 51, was replaced by alanine. The mutant CiaRD51A protein was no longer able to activate CiaR-dependent promoters, strongly suggesting that the phosphorylated form of CiaR is active in regulation. However, depending on the growth medium, inactivation of the kinase gene ciaH resulted in a subtle increase of CiaR-dependent promoter activities or in a strong reduction. Therefore, CiaH may act as a kinase or phosphatase and CiaR is apparently able to obtain its phosphate independently of CiaH. On the other hand, promoter measurements in cells with an intact CiaRH system demonstrated a high, nearly constitutive, expression level of the CiaR regulon independent from the growth medium. Thus, in contrast to many other two-component regulatory systems, CiaRH has apparently evolved to maintain high levels of gene expression under a variety of conditions rather than responding strongly to a signal.