Novichkov, P. S. | Search bioRxiv

bioRxiv2019

A dual functioning small RNA/Riboswitch controls the expression of the methionine biosynthesis regulator SahR in Desulfovibrio vulgaris Hildenborough

Riboswitches are cis-acting RNA regulatory elements that control expression of a downstream gene(s) by directly binding to a specific metabolite. Here we report a S-adenosylmethionine (SAM)-I riboswitch in the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough (DvH) that plays an additional regulatory role as a trans small noncoding RNA (sRNA) targeting the methionine biosynthesis cycle transcriptional regulator SahR. Sequence and expression analyses indicated that DseA (Desulfovibrio SAM element A) is located upstream of a small hypothetical protein DVU1170 and that the two are co-transcribed. Multiple techniques were used to verify the riboswitch activity of DseA and its activity as a transcriptional terminator in response to SAM. While determining a potential role for DseA in the methionine biosynthesis pathway, a mRNA target encoding SahR was identified. Subsequent electrophoretic mobility shift assays confirmed the ability of DseA to bind the sahR transcript and qRT-PCR analysis of a DseA deletion strain suggested a negative regulatory role. This study presents the first regulatory role for a newly discovered sRNA in Desulfovibrio. Additionally, this study suggests that DseA acts not only as a riboswitch, but also as a trans regulatory molecule.\n\nIMPORTANCESulfate-reducing bacteria (SRB) are important contributors to global geochemical cycles while also causing major issues for the petroleum and oil industry due to biocorrosion and souring of oil wells. Despite their significance, gene regulatory networks and pathways remain poorly understood in SRB. Here, we report a trans acting small noncoding RNA that plays a dual role as a SAM sensing riboswitch that controls the expression of a small hypothetical protein. Our findings provide important insights into the regulatory repertoire of sulfate-reducing bacteria.

microbiology↗

bioRxiv2018

Dub-seq: dual-barcoded shotgun expression library sequencing for high-throughput characterization of functional traits

A major challenge in genomics is the knowledge gap between sequence and its encoded function. Gain-of-function methods based on gene overexpression are attractive avenues for phenotype-based functional screens, but are not easily applied in high-throughput across many experimental conditions. Here, we present Dual Barcoded Shotgun Expression Library Sequencing (Dub-seq), a method that greatly increases the throughput of genome-wide overexpression assays. In Dub-seq, a shotgun expression library is cloned between dual random DNA barcodes and the precise breakpoints of DNA fragments are associated to the barcode sequences prior to performing assays. To assess the fitness of individual strains carrying these plasmids, we use DNA barcode sequencing (BarSeq), which is amenable to large-scale sample multiplexing. As a demonstration of this approach, we constructed a Dub-seq library with total Escherichia coli genomic DNA, performed 155 genome-wide fitness assays in 52 experimental conditions, and identified 813 genes with high-confidence overexpression phenotypes across 4,151 genes assayed. We show that Dub-seq data is reproducible, accurately recapitulates known biology, and identifies hundreds of novel gain-of-function phenotypes for E. coli genes, a subset of which we verified with assays of individual strains. Dub-seq provides complementary information to loss-of-function approaches such as transposon site sequencing or CRISPRi and will facilitate rapid and systematic functional characterization of microbial genomes.\n\nImportanceMeasuring the phenotypic consequences of overexpressing genes is a classic genetic approach for understanding protein function; for identifying drug targets, antibiotic and metal resistance mechanisms; and for optimizing strains for metabolic engineering. In microorganisms, these gain-of-function assays are typically done using laborious protocols with individually archived strains or in low-throughput following qualitative selection for a phenotype of interest, such as antibiotic resistance. However, many microbial genes are poorly characterized and the importance of a given gene may only be apparent under certain conditions. Therefore, more scalable approaches for gain-of-function assays are needed. Here, we present Dual Barcoded Shotgun Expression Library Sequencing (Dub-seq), a strategy that couples systematic gene overexpression with DNA barcode sequencing for large-scale interrogation of gene fitness under many experimental conditions at low cost. Dub-seq can be applied to many microorganisms and is a valuable new tool for large-scale gene function characterization.

microbiology↗