Hustmyer, C. M. | bioRxiv Science

bioRxiv2020

Hybrid analysis reveals how DNA sequence governs genomic location and DNA contacts of bacterial chromatin H-NS filaments

Gene silencing in bacteria is mediated by chromatin proteins, of which Escherichia coli H-NS is a paradigmatic example. H-NS forms nucleoprotein filaments with either one or two DNA duplexes. However, the structures, arrangements of DNA-binding domains (DBDs), and positions of DBD-DNA contacts in linear and bridged filaments are uncertain. To characterize the contacts that silence transcription by RNA polymerase, we combined {middle dot}OH footprinting, molecular dynamics, statistical modeling, and DBD mapping using a chemical nuclease (Fe2+-EDTA) tethered to the DBDs (TEN-map). We find that H-NS DBDs contact DNA at indistinguishable locations in bridged or linear filaments and that the DBDs vary in orientation and position with ~10-bp average spacing. Our results support a hemi-sequestration model of linear-to-bridged H-NS switching in which linear filaments able to inhibit only transcription initiation switch to bridged filaments able to inhibit both initiation and elongation using the same irregularly spaced DNA contact sites. HighlightsO_LITethered-nuclease mapping (TEN-map) of H-NS DNA-binding domains detects DNA contacts C_LIO_LIBridged and linear H-NS filaments use the same DNA contact sites C_LIO_LIH-NS-DNA contacts are unevenly spaced with ~10 bp average separation C_LIO_LIAT-steps, minor groove width, and electrostatic potential best predict contact sites C_LI

molecular biology↗

bioRxiv2017

Promoter boundaries for the luxCDABE and betIBA-proXWV operons in Vibrio harveyi defined by the method RAIL: Rapid Arbitrary PCR Insertion Libraries

Experimental studies of transcriptional regulation in bacteria require the ability to precisely measure changes in gene expression, often accomplished through the use of reporter genes. However, the boundaries of promoter sequences required for transcription are often unknown, thus complicating construction of reporters and genetic analysis of transcriptional regulation. Here, we analyze reporter libraries to define the promoter boundaries of the luxCDABE bioluminescence operon and the betIBA-proXWV osmotic stress operon in Vibrio harveyi. We describe a new method called RAIL (Rapid Arbitrary PCR Insertion Libraries) that combines the power of arbitrary PCR and isothermal DNA assembly to rapidly clone promoter fragments of various lengths upstream of reporter genes to generate large libraries. To demonstrate the versatility and efficiency of RAIL, we analyzed the promoters driving expression of the luxCDABE and betIBA-proXWV operons and created libraries of DNA fragments from these loci fused to fluorescent reporters. Using flow cytometry sorting and deep sequencing, we identified the DNA regions necessary and sufficient for maximum gene expression for each promoter. These analyses uncovered previously unknown regulatory sequences and validated known transcription factor binding sites. We applied this high-throughput method to gfp, mCherry, and lacZ reporters and multiple promoters in V. harveyi. We anticipate that the RAIL method will be easily applicable to other model systems for genetic, molecular, and cell biological applications.\n\nImportanceGene reporter constructs have long been essential tools for studying gene regulation in bacteria, particularly following the recent advent of fluorescent gene reporters. We developed a new method that enables efficient construction of promoter fusions to reporter genes to study gene regulation. We demonstrate the versatility of this technique in the model bacterium Vibrio harveyi by constructing promoter libraries for three bacterial promoters using three reporter genes. These libraries can be used to determine the DNA sequences required for gene expression, revealing regulatory elements in promoters. This method is applicable to various model systems and reporter genes for assaying gene expression.

molecular biology↗