Structure-function dissection of Myxococcus xanthus CarD N-terminal domain, a defining member of the CarD-CdnL-TRCF family of RNA polymerase interacting proteins

© 2015 Bernal-Bernal et al. Two prototypes of the large CarD-CdnL-TRCF family of bacterial RNA polymerase (RNAP)-binding proteins, Myxococcus xanthus CarD and CdnL, have distinct functions whose molecular basis remain elusive. CarD, a global regulator linked to the action of several extracytoplasmic function (ECF) σ-factors, binds to the RNAP β subunit (RNAP-β) and to protein CarG via an N-terminal domain, CarDNt, and to DNA via an intrinsically unfolded C-terminal domain resembling eukaryotic high-mobility-group A (HMGA) proteins. CdnL, a CarDNt-like protein that is essential for cell viability, is implicated in σA-dependent rRNA promoter activation and interacts with RNAP-β but not with CarG. While the HMGA-like domain of CarD by itself is inactive, we find that CarDNt has low but observable ability to activate ECF σ-dependent promoters in vivo, indicating that the C-terminal DNA-binding domain is required to maximize activity. Our structure-function dissection of CarDNt reveals an N-terminal, five-stranded β-sheet Tudor-like domain, CarD1-72, whose structure and contacts with RNAP-β mimic those of CdnL. Intriguingly, and in marked contrast to CdnL, CarD mutations that disrupt its interaction with RNAP-β did not annul activity. Our data suggest that the CarDNt C-terminal segment, CarD61-179, may be structurally distinct from its CdnL counterpart, and that it houses at least two distinct and crucial function determinants: (a) CarG-binding, which is specific to CarD; and (b) a basic residue stretch, which is also conserved and functionally required in CdnL. This study highlights the evolution of shared and divergent interactions in similar protein modules that enable the distinct activities of two related members of a functionally important and widespread bacterial protein family.Peer Reviewe

Plasticity in oligomerization, operator architecture, and DNA binding in the mode of action of a bacterial B12-based photoreceptor.

Newly discovered bacterial photoreceptors called CarH sense light by using 5′-deoxyadenosylcobalamin (AdoCbl). They repress their own expression and that of genes for carotenoid synthesis by binding in the dark to operator DNA as AdoCbl-bound tetramers, whose light-induced disassembly relieves repression. High-resolution structures of Thermus thermophilus CarHTt have provided snapshots of the dark and light states and have revealed a unique DNA-binding mode whereby only three of four DNA-binding domains contact an operator comprising three tandem direct repeats. To gain further insights into CarH photoreceptors and employing biochemical, spectroscopic, mutational, and computational analyses, here we investigated CarHBm from Bacillus megaterium. We found that apoCarHBm, unlike monomeric apoCarHTt, is an oligomeric molten globule that forms DNA-binding tetramers in the dark only upon AdoCbl binding, which requires a conserved W-X9-EH motif. Light relieved DNA binding by disrupting CarHBm tetramers to dimers, rather than to monomers as with CarHTt. CarHBm operators resembled that of CarHTt, but were larger by one repeat and overlapped with the −35 or −10 promoter elements. This design persisted in a six-repeat, multipartite operator we discovered upstream of a gene encoding an Spx global redox-response regulator whose photoregulated expression links photooxidative and general redox responses in B. megaterium. Interestingly, CarHBm recognized the smaller CarHTt operator, revealing an adaptability possibly related to the linker bridging the DNA- and AdoCbl-binding domains. Our findings highlight a remarkable plasticity in the mode of action of B12-based CarH photoreceptors, important for their biological functions and development as optogenetic tools

A bacterial antirepressor with SH3 domain topology mimics operator DNA in sequestering the repressor DNA recognition helix

Direct targeting of critical DNA-binding elements of a repressor by its cognate antirepressor is an effective means to sequester the repressor and remove a transcription initiation block. Structural descriptions for this, though often proposed for bacterial and phage repressor–antirepressor systems, are unavailable. Here, we describe the structural and functional basis of how the Myxococcus xanthus CarS antirepressor recognizes and neutralizes its cognate repressors to turn on a photo-inducible promoter. CarA and CarH repress the carB operon in the dark. CarS, produced in the light, physically interacts with the MerR-type winged-helix DNA-binding domain of these repressors leading to activation of carB. The NMR structure of CarS1, a functional CarS variant, reveals a five-stranded, antiparallel β-sheet fold resembling SH3 domains, protein–protein interaction modules prevalent in eukaryotes but rare in prokaryotes. NMR studies and analysis of site-directed mutants in vivo and in vitro unveil a solvent-exposed hydrophobic pocket lined by acidic residues in CarS, where the CarA DNA recognition helix docks with high affinity in an atypical ligand-recognition mode for SH3 domains. Our findings uncover an unprecedented use of the SH3 domain-like fold for protein–protein recognition whereby an antirepressor mimics operator DNA in sequestering the repressor DNA recognition helix to activate transcription

VARIACIÓN DE FASE EN MYXOCOCCUS XANTHKUS: EFECTO DE AGENTES MUTAGÉNICOS

This paper includes quantitative data on a phase vanation phenomenon in the bactenum Myxococcus Xanrhus which affects colonial pigmentation. The spontaneous or mutagen induced frequencies of phase vanation, both in wild type and colour mutant strains, can be explained by assuming that phase variation in M. xanrhus is geneticaily controlled and is also sensitive to physiological circumstances. The mutagen nitrosoguanidine induces a transitory unstability of the cellular phase, whereas UV light has not any effect on phase vanation.Este trabajo aborda un análisis cuantitativo de un fenómeno de variación de fase, que afecta a la pigmentación de las colonias en la bacteria Myxococcus xanrhus. El estudio de la frecuencia de variación de fase en estirpes silvestres y mutantes en la pigmentación colonial, tanto espontánea como inducida por agentes mutagénicos, revela que dicho fenómeno está controlado genéticamente y es sensible a cambios fisiológicos. El agente mutagénico nitrosoguanidina provoca una inestabilidad transitoria de la fase celular. Mientras que la luz ultravioleta no tiene efecto sobre la vanación de fase en M. xanrhus

Anaerobic bacteria need their vitamin B12 to digest estrogen

3 pags., 1 fig.This work is supported by grants PGC2018-094635-B-C21 and PGC2018-094635-B-C22 from the Agencia Estatal de Investigacion of Spain and the European Regional Development Fund and grant 20992/PI/18 from Fundacion Seneca of Murcia, Spain.Peer reviewe

Germinación de Myxococcus xanthus / Monserrat Elías Arnanz ; director Fco. J. Murillo Araujo

Tesis-Universidad de Murcia.Consulte la tesis en: BCA. GENERAL. ARCHIVO UNIVERSITARIO. D 29.Consulte la tesis en: BCA. GENERAL. ARCHIVO UNIVERSITARIO. T.M.-369

Functional interactions between a phage histone-like protein and a transcriptional factor in regulation of ø29 early-late transcriptional switch

Protein p6 is a nonspecific DNA-binding protein occurring in high abundance in phage φ29-infected cells. Here, we demonstrate a novel role for this versatile histone-like protein: its involvement in regulating the viral switch between early and late transcription. p6 performs this role by exhibiting a reciprocal functional interaction with the regulatory protein p4, also phage encoded, which is required for repression of the early A2b and A2c promoters and activation of the late A3 promoter. On the one hand, p6 promotes p4-mediated repression of the A2b promoter and activation of the A3 promoter by enhancing binding of p4 to its recognition site at PA3; on the other, p4 promotes p6-mediated repression of the A2c promoter by favoring the formation of a stable p6–nucleoprotein complex that interferes with RNA polymerase binding to PA2c. We propose that the observed interplay between proteins p6 and p4 is based on their DNA architectural properties.This investigation was funded by research grants 2R01 GM27242-19 from the National Institutes of Health, PB93-0173 from the Dirección General de Investigación Científica y Técnica, and an Institutional grant from Fundación Ramón Areces. M.E.-A. was supported by the Ministerio de Educación y Cultura (Spain).Peer reviewe

B12-based photoreceptors: from structure and function to applications in optogenetics and synthetic biology

Vitamin B12-based photoreceptor proteins sense ultraviolet (UV), blue or green light using 5´-deoxyadenosylcobalamin (AdoCbl). The prototype of this widespread bacterial photoreceptor family, CarH, controls light-dependent gene expression in photoprotective cellular responses. It represses transcription in the dark by binding to operator DNA as an AdoCbl-bound tetramer, whose disruption by light relieves operator binding to allow transcription. Structures of the "dark" (free and DNA-bound) and "light" CarH states and studies on the unusual AdoCbl photochemistry have provided fundamental insights into these photoreceptors. We highlight these, the plasticity within a conserved mode of action among CarH homologs, their distribution, and their promising applications in optogenetics and synthetic biology