No abstract

Combining production of antibacterial compounds and uptake of DNA material released by dead cells, competence is one of the most efficient survival strategies in streptococci. Yet, this powerful tactic is energy consuming and reprograms the metabolism to such an extent that cell proliferation is transiently impaired.

Competence allows bacteria to internalize exogenous DNA fragments for the acquisition of new phenotypes such as antibiotic resistance or virulence traits. In most streptococci, competence is regulated by ComRS signaling, a system based on the mature ComS pheromone (XIP), which is internalized to activate the (R)RNPP-type ComR sensor by triggering dimerization and DNA binding. Cross-talk analyses demonstrated major differences of selectivity between ComRS systems and raised questions concerning the mechanism of pheromone-sensor recognition and coevolution. Here, we decipher the molecular determinants of selectivity of the closely related ComRS systems fromStreptococcus thermophilusandStreptococcus vestibularis. Despite high similarity, we show that the divergence in ComR-XIP interaction does not allow reciprocal activation. We perform the structural analysis of the ComRS system fromS. vestibularis.Comparison with its ortholog fromS. thermophilusreveals an activation mechanism based on a toggle switch involving the recruitment of a key loop by the XIP C terminus. Together with a broad mutational analysis, we identify essential residues directly involved in peptide binding. Notably, we generate a ComR mutant that displays a fully reversed selectivity toward the heterologous pheromone with only five point mutations, as well as other ComR variants featuring XIP bispecificity and/or neofunctionalization for hybrid XIP peptides. We also reveal that a single XIP mutation relaxes the strictness of ComR activation, suggesting fast adaptability of molecular communication phenotypes. Overall, this study is paving the way toward the rational design or directed evolution of artificial ComRS systems for a range of biotechnological and biomedical applications.

Previous genetic studies in Sphingomonas macrogolitabida strain TFA have established that expression of genes involved in tetralin biodegradation (thn genes) requires the function of the LysR type activator ThnR and also ThnY. Sequence comparison indicated that ThnY is homologous to bacterial oxygenase-coupled NAD(P)H-dependent ferredoxin reductases. However, ThnY showed substitutions in highly conserved positions of the pyridine nucleotide binding domain of these ferredoxin reductases. ThnY expression is co-regulated with all other genes required for tetralin biodegradation, and presumably thnY is part of the thnCA3A4RY operon. ThnY has been purified, and its biochemical and functional properties were characterized. ThnY was found to be a monomeric orange-brown iron-sulfur flavoprotein (estimated mass of 37,000 Da) containing one non-covalently attached flavin adenine dinucleotide and one plant type ferredoxin 2Fe-2S cluster. It can be efficiently reduced by dithionite, but reduction by pyridine nucleotides was very poor. Consistently, ThnY-dependent reduction of cytochrome c, ferricyanide, or 2,6-dichlorophenolindophenol using NAD(P)H as the electron donor was undetectable or very weak. The addition of ThnY to electrophoretic mobility shift assays containing ThnR and a probe bearing two thn divergent promoters resulted in a 3-fold increase in protein-DNA complex formation affinity, which indicates that ThnY directly promotes thn transcription activation by ThnR.Tetralin (1,2,3,4-tetrahydronaphthalene) is a bicyclic molecule composed of an aromatic and an alicyclic moiety, which is found at low concentrations in different crude oils, and it is also industrially produced for its use as an organic solvent. Biodegradation of tetralin has been characterized most extensively in Sphingomonas macrogolitabida strain TFA. The genes required for tetralin biodegradation (thn genes) have been sequenced, and the functions of the encoded proteins have been characterized to elucidate the biodegradation pathway (1-4) that yields pyruvate and pimelate, which is subsequently metabolized via ␤-oxidation (5). Rieske type oxygenases are multicomponent enzymes known to initiate the oxidative biodegradation of various environmentally hazardous aromatic compounds in bacteria, such as crude oil components, polycyclic aromatic hydrocarbons, and heterocyclic aromatic compounds, in a reaction requiring oxygen and an external electron supply (6). In this reaction, the electrons are transferred from NAD(P)H via flavin and [2Fe-2S] redox centers to the terminal oxygenase component that has the catalytic active site. Tetralin dioxygenase is encoded by the thnA1 and thnA2 genes, whereas the ferredoxin and the ferredoxin reductase components of the electron transfer system are encoded by thnA3 and thnA4, respectively. According to the number of components and the nature of the redox centers of the electron transport chain, bacterial oxygenases have been classified into three main classes, classes I, II, and III (7-9). The tetralin dioxygenase s...