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An improved approach was developed for the genetic manipulation of the Grampositive extremophile Halalkalibacterium halodurans (formerly called Bacillus halodurans). We describe an allelic replacement method originally developed for Staphylococcus aureus that allows the deletion, mutation, or insertion of genes without leaving markers or other genetic scars. In addition, a protocol for rapid in vitro plasmid methylation and transformation is presented. The combined methods allow the routine genetic manipulation of H. halodurans from initial transformation to the desired strain in 8 days. These methods improve H. halodurans as a model organism for the study of extremophiles. [ABSTRACT FROM AUTHOR]

Nearly all representatives of experimentally validated riboswitch classes in bacteria control the expression of genes for the transport or synthesis of key metabolic compounds. Recent findings have revealed that some riboswitches also regulate genes involved in physiological changes, virulence, and stress responses. Many novel RNA motifs are being identified by using bioinformatics algorithms that search for conserved sequence and structural features located in intergenic regions. Some of these RNAs are likely to function as riboswitches for metabolites or signaling compounds, and confirmation of this function would reveal the basis of the genetic control of new regulons. Herein we describe the analysis of the ydaO riboswitch candidate, which represents one of the most widespread candidates remaining to be validated. These RNAs are common in Gram-positive bacteria, and their genomic associations with diverse genes suggest that they sense a compound that signals broader physiological changes. We determined that the ydaO motif exhibits sequence- and structure-dependent gene control, and reporter assays indicate that its natural ligand is present even when cells are grown in defined media. A transposon-mediated knockout screen resulted in mutants with a dysregulated expression of genes controlled by the RNA motif. The mutations disrupt genes that drastically modulate energy-generating pathways, suggesting that the intracellular concentration of the ligand sensed by the ydaO motif is altered under these stress conditions.

Riboswitches rely on structured aptamer domains to selectively sense their target ligands and regulate gene expression. However, some riboswitch aptamers in bacteria carry mutations in their otherwise strictly conserved binding pockets that change ligand specificities. The aptamer domain of a riboswitch class originally found to selectively sense guanine forms a three-stem junction that has since been observed to exploit numerous alterations in its ligand-binding pocket. These rare variants have modified their ligand specificities to sense other purines or purine derivatives, including adenine, 2'-deoxyguanosine (three classes), and xanthine. Herein, we report the characteristics of a rare variant that is narrowly distributed in the Paenibacillaceae family of bacteria. Known representatives are always associated with genes encoding 8-oxoguanine deaminase. As predicted from this gene association, these variant riboswitches tightly bind 8-oxoguanine (8-oxoG), strongly discriminate against other purine derivatives, and function as genetic "ON" switches. Following exposure of cells to certain oxidative stresses, a representative 8-oxoG riboswitch activates gene expression, likely caused by the accumulation of 8-oxoG due to oxidative damage to G nucleobases in DNA, RNA, and the nucleotide pool. Furthermore, an engineered version of the variant aptamer was prepared that exhibits specificity for 8-oxoadenine, further demonstrating that RNA aptamers can acquire mutations that expand their ability to detect and respond to oxidative damage. [ABSTRACT FROM AUTHOR]