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 beta-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.

A vitamin B12-based system for conditional expression reveals dksA to be an essential gene in Myxococcus xanthus.

Myxococcus xanthus is a prokaryotic model system for the study of multicellular development and the response to blue light. The previous analyses of these processes and the characterization of new genes would benefit from a robust system for controlled gene expression, which has been elusive so far for this bacterium. Here, we describe a system for conditional expression of genes in M. xanthus based on our recent finding that vitamin B12 and CarH, a MerR-type transcriptional repressor, together downregulate a photoinducible promoter. Using this system, we confirmed that M. xanthus rpoN, encoding sigma(54), is an essential gene, as reported earlier. We then tested it with ftsZ and dksA. In most bacteria, ftsZ is vital due to its role in cell division, whereas null mutants of dksA, whose product regulates the stringent response via transcriptional control of rRNA and amino acid biosynthesis promoters, are viable but cause pleiotropic effects. As with rpoN, it was impossible to delete endogenous ftsZ or dksA in M. xanthus except in a merodiploid background carrying another functional copy, which indicates that these are essential genes. B12-based conditional expression of ftsZ was insufficient to provide the high intracellular FtsZ levels required. With dksA, as with rpoN, cells were viable under permissive but not restrictive conditions, and depletion of DksA or sigma(54) produced filamentous, aberrantly dividing cells. dksA thus joins rpoN in a growing list of genes dispensable in many bacteria but essential in M. xanthus.

Analysis of solvent tolerance in Pseudomonas putida DOT-T1E based on its genome sequence and a collection of mutants.

Pseudomonas putida strains are prevalent in a variety of pristine and polluted environments. The genome of the solvent-tolerant P. putida strain DOT-T1E which thrives in the presence of high concentrations of monoaromatic hydrocarbons, contains a circular 6.3 Mbp chromosome and a 133 kbp plasmid. Omics information has been used to identify the genes and proteins involved in solvent tolerance in this bacterium. This strain uses a multifactorial response that involves fine-tuning of lipid fluidity, activation of a general stress-response system, enhanced energy generation, and induction of specific efflux pumps that extrude solvents to the medium. Local and global transcriptional regulators participate in a complex network of metabolic functions, acting as the decision makers in the response to solvents.

Physiological and transcriptomic characterization of a fliA mutant of Pseudomonas putida KT2440.

Pseudomonas putida KT2440 encodes 23 alternative sigma factors. The fliA gene, which encodes σ(28) , is in a cluster with other genes involved in flagella biosynthesis and chemotaxis. Reverse transcriptase-PCR revealed that this cluster is comprised of four independent transcriptional units: flhAF, fleNfliA, cheYZA and cheBmotAB. We generated a nonpolar fliA mutant by homologous recombination and tested its motility, adhesion to biotic and abiotic surfaces, and responses to various stress conditions. The mutant strain was nonmotile and exhibited decreased capacity to bind to corn seeds, although its ability to colonize the rhizosphere of plants was unaffected. The mutant was also affected in binding to abiotic surfaces and its ability to form biofilms decreased by almost threefold. In the fliA mutant background expression of 25 genes was affected: two genes were upregulated and 23 genes were downregulated. In addition to a number of motility and chemotaxis genes, the fliA gene product is also necessary for the expression of some genes potentially involved in amino acid utilization or stress responses; however, we were unable to assign specific phenotypes linked to these genes since the fliA mutant used the same range of amino acids as the parental strain, and was as tolerant as the wild type to stress imposed by heat, antibiotics, NaCl, sodium dodecyl sulfate, H2 O2 and benzoate. Based on the sequence alignment of promoters recognized by FliA and genome in silico analysis, we propose that P. putidaσ(28) recognizes a TCAAG-t-N12 -GCCGATA consensus sequence located between -34 and -8 and that this sequence is preferentially associated with an AT-rich upstream region.

Structural basis for operator and antirepressor recognition by Myxococcus xanthus CarA repressor.

Blue light induces carotenogenesis in Myxococcus xanthus. The carB operon encodes all but one of the structural genes involved, and its expression is regulated by the CarA-CarS repressor-antirepressor pair. In the dark, CarA-operator binding represses carB. CarS, produced on illumination, interacts physically with CarA to dismantle the CarA-operator complex and activate carB. Both operator and CarS bind to the autonomously folded N-terminal domain of CarA, CarA(Nter), which in excess represses carB. Here, we report the NMR structure of CarA(Nter), and map residues that interact with operator and CarS by NMR chemical shift perturbations, and in vivo and in vitro analyses of site-directed mutants. We show CarA(Nter) adopts the winged-helix topology of MerR-family DNA-binding domains, and conserves the majority of the helix-turn-helix and wing contacts with DNA. Tellingly, helix alpha2 in CarA, a key element in operator DNA recognition, is also critical for interaction with CarS, implying that the CarA-CarS protein-protein and the CarA-operator protein-DNA interfaces overlap. Thus, binding of CarA to operator and to antirepressor are mutually exclusive, and CarA may discern structural features in the acidic CarS protein that resemble operator DNA. Repressor inactivation by occluding the DNA-binding region may be a recurrent mechanism of action for acidic antirepressors.