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ABSTRACT LuxR-type transcription factors are the master regulators of quorum sensing in vibrios. LuxR proteins are unique members of the TetR superfamily of transcription factors because they activate and repress large regulons of genes. Here, we used chromatin immunoprecipitation and nucleotide sequencing (ChIP-seq) to identify LuxR binding sites in the Vibrio harveyi genome. Bioinformatics analyses showed that the LuxR consensus binding site at repressed promoters is a symmetric palindrome, whereas at activated promoters it is asymmetric and contains only half of the palindrome. Using a genetic screen, we isolated LuxR mutants that separated activation and repression functions at representative promoters. These LuxR mutants exhibit sequence-specific DNA binding defects that restrict activation or repression activity to subsets of target promoters. Altering the LuxR DNA binding site sequence to one more closely resembling the ideal LuxR consensus motif can restore in vivo function to a LuxR mutant. This study provides a mechanistic understanding of how a single protein can recognize a variety of binding sites to differentially regulate gene expression. IMPORTANCE Bacteria use the cell-cell communication process called quorum sensing to regulate collective behaviors. In vibrios, LuxR-type transcription factors control the quorum-sensing gene expression cascade. LuxR-type proteins are structural homologs of TetR-type transcription factors. LuxR proteins were assumed to function analogously to TetR proteins, which typically bind to a single conserved binding site to repress transcription of one or two genes. We find here that unlike TetR proteins, LuxR acts a global regulator, directly binding upstream of and controlling more than 100 genes. Again unlike TetR, LuxR functions as both an activator and a repressor, and these two activities can be separated by mutagenesis. Finally, the consensus binding motifs driving LuxR-activated and -repressed genes are distinct. This work shows that LuxR, although structurally similar to TetR, has evolved unique features enabling it to differentially control a large regulon of genes in response to quorum-sensing cues.

BACKGROUND:Methanomicrobiales is the least studied order of methanogens. While these organisms appear to be more closely related to the Methanosarcinales in ribosomal-based phylogenetic analyses, they are metabolically more similar to Class I methanogens. METHODOLOGY/PRINCIPAL FINDINGS:In order to improve our understanding of this lineage, we have completely sequenced the genomes of two members of this order, Methanocorpusculum labreanum Z and Methanoculleus marisnigri JR1, and compared them with the genome of a third, Methanospirillum hungatei JF-1. Similar to Class I methanogens, Methanomicrobiales use a partial reductive citric acid cycle for 2-oxoglutarate biosynthesis, and they have the Eha energy-converting hydrogenase. In common with Methanosarcinales, Methanomicrobiales possess the Ech hydrogenase and at least some of them may couple formylmethanofuran formation and heterodisulfide reduction to transmembrane ion gradients. Uniquely, M. labreanum and M. hungatei contain hydrogenases similar to the Pyrococcus furiosus Mbh hydrogenase, and all three Methanomicrobiales have anti-sigma factor and anti-anti-sigma factor regulatory proteins not found in other methanogens. Phylogenetic analysis based on seven core proteins of methanogenesis and cofactor biosynthesis places the Methanomicrobiales equidistant from Class I methanogens and Methanosarcinales. CONCLUSIONS/SIGNIFICANCE:Our results indicate that Methanomicrobiales, rather than being similar to Class I methanogens or Methanomicrobiales, share some features of both and have some unique properties. We find that there are three distinct classes of methanogens: the Class I methanogens, the Methanomicrobiales (Class II), and the Methanosarcinales (Class III).

We report here the sequencing and analysis of the genome of the thermophilic bacterium Carboxydothermus hydrogenoformans Z-2901. This species is a model for studies of hydrogenogens, which are diverse bacteria and archaea that grow anaerobically utilizing carbon monoxide (CO) as their sole carbon source and water as an electron acceptor, producing carbon dioxide and hydrogen as waste products. Organisms that make use of CO do so through carbon monoxide dehydrogenase complexes. Remarkably, analysis of the genome of C. hydrogenoformans reveals the presence of at least five highly differentiated anaerobic carbon monoxide dehydrogenase complexes, which may in part explain how this species is able to grow so much more rapidly on CO than many other species. Analysis of the genome also has provided many general insights into the metabolism of this organism which should make it easier to use it as a source of biologically produced hydrogen gas. One surprising finding is the presence of many genes previously found only in sporulating species in the Firmicutes Phylum. Although this species is also a Firmicutes, it was not known to sporulate previously. Here we show that it does sporulate and because it is missing many of the genes involved in sporulation in other species, this organism may serve as a "minimal" model for sporulation studies. In addition, using phylogenetic profile analysis, we have identified many uncharacterized gene families found in all known sporulating Firmicutes, but not in any non-sporulating bacteria, including a sigma factor not known to be involved in sporulation previously.