The Transcriptional Program of Phage K Is Affected by a Host Mutation That Confers Phage K Resistance.

To better understand host-phage interactions and the genetic bases of phage resistance in a model system relevant to potential phage therapy, we isolated several spontaneous mutants of the USA300 clinical isolate NRS384 that were resistant to phage K. Six of these had a single missense mutation in the host gene, which encodes the RNA polymerase β' subunit. To examine the hypothesis that mutations in the host RNA polymerase affect the transcription of phage genes, we performed RNA-seq analysis on total RNA samples collected from NRS384 wild-type (WT) and mutant cultures infected with phage K, at different timepoints after infection.

A single rare σ70 variant establishes a unique gene expression pattern in the E. coli pathobiont LF82.

LF82, an adherent-invasive Escherichia coli (AIEC) pathobiont, is associated with Crohn's disease, an inflammatory bowel disease of unknown etiology. Although AIEC phenotypes differ from those of 'commensal' or pathogenic E. coli, work has failed to identify genetic features accounting for these differences.

The pathobiont LF82 encodes a unique variant of σ that results in specific gene expression changes and altered phenotypes.

LF82, an adherent invasive pathobiont, is associated with ileal Crohn's disease, an inflammatory bowel disease of unknown etiology. Although LF82 contains no virulence genes, it carries several genetic differences, including single nucleotide polymorphisms (SNPs), that distinguish it from nonpathogenic . We have identified and investigated an extremely rare SNP that is within the highly conserved gene, encoding σ, the primary sigma factor for RNA polymerase.

Conformational change of the response regulator BvgA accompanies its activation of the virulence gene .

The BvgAS two-component system regulates virulence gene expression in . Although precise three-dimensional structural information is not available for the response regulator BvgA, its sequence conservation with NarL and previous studies have indicated that it is composed of 3 domains: an -terminal domain (NTD) containing the phosphorylation site, a linker, and a DNA-binding C-terminal domain (CTD). Previous work has determined how BvgA dimers interact with the promoter (P ) of , the gene encoding the virulence adhesin filamentous hemagglutinin.

The Vibrio cholerae master regulator for the activation of biofilm biogenesis genes, VpsR, senses both cyclic di-GMP and phosphate.

Vibrio cholerae biofilm formation/maintenance is controlled by myriad factors; chief among these are the regulator VpsR and cyclic di-guanosine monophosphate (c-di-GMP). VpsR has strong sequence similarity to enhancer binding proteins (EBPs) that activate RNA polymerase containing sigma factor σ54. However, we have previously shown that transcription from promoters within the biofilm biogenesis/maintenance pathways uses VpsR, c-di-GMP and RNA polymerase containing the primary sigma factor (σ70).

Identification of BvgA-Dependent and BvgA-Independent Small RNAs (sRNAs) in Bordetella pertussis Using the Prokaryotic sRNA Prediction Toolkit ANNOgesic.

Noncoding small RNAs (sRNAs) are crucial for the posttranscriptional regulation of gene expression in all organisms and are known to be involved in the regulation of bacterial virulence. In the human pathogen Bordetella pertussis, which causes whooping cough, virulence is controlled primarily by the master two-component system BvgA (response regulator)/BvgS (sensor kinase). In this system, BvgA is phosphorylated (Bvg mode) or nonphosphorylated (Bvg mode), with global transcriptional differences between the two.

A phage-encoded nucleoid associated protein compacts both host and phage DNA and derepresses H-NS silencing.

Nucleoid Associated Proteins (NAPs) organize the bacterial chromosome within the nucleoid. The interaction of the NAP H-NS with DNA also represses specific host and xenogeneic genes. Previously, we showed that the bacteriophage T4 early protein MotB binds to DNA, co-purifies with H-NS/DNA, and improves phage fitness.

Combining Gel Retardation and Footprinting to Determine Protein-DNA Interactions of Specific and/or Less Stable Complexes.

DNA footprinting is a classic technique to investigate protein-DNA interactions. However, traditional footprinting protocols can be unsuccessful or difficult to interpret if the binding of the protein to the DNA is weak, the protein has a fast off-rate, or if several different protein-DNA complexes are formed. Our protocol differs from traditional footprinting protocols, because it provides a method to isolate the protein-DNA complex from a native gel after treatment with the footprinting agent, thus removing the bound DNA from the free DNA or other protein-DNA complexes.

VpsR Directly Activates Transcription of Multiple Biofilm Genes in Vibrio cholerae.

biofilm biogenesis, which is important for survival, dissemination, and persistence, requires multiple genes in the polysaccharides () operons I and II as well as the cluster of ribomatrix () genes. Transcriptional control of these genes is a complex process that requires several activators/repressors and the ubiquitous signaling molecule, cyclic di-GMP (c-di-GMP). Previously, we demonstrated that VpsR directly activates RNA polymerase containing σ (σ-RNAP) at the promoter (P ), which precedes the -II operon, in a c-di-GMP-dependent manner by stimulating formation of the transcriptionally active, open complex.

A Novel Bvg-Repressed Promoter Causes -Like Transcription of but Does Not Result in the Production of Serotype 3 Fimbriae in Bvg Mode Bordetella pertussis.

In , two serologically distinct fimbriae, FIM2 and FIM3, undergo on/off phase variation independently of each other via variation in the lengths of C stretches in the promoters for their major subunit genes, and These two promoters are also part of the BvgAS virulence regulon and therefore, if in an on configuration, are activated by phosporylated BvgA (BvgA~P) under normal growth conditions (Bvg mode) but not in the Bvg mode, inducible by growth in medium containing MgSO or other compounds, termed modulators. In the Tohama I strain (FIM2 FIM3), the promoter is in the off state. However, a high level of transcription of the gene is observed in the Bvg mode.

VpsR and cyclic di-GMP together drive transcription initiation to activate biofilm formation in Vibrio cholerae.

The small molecule cyclic di-GMP (c-di-GMP) is known to affect bacterial gene expression in myriad ways. In Vibrio cholerae in vivo, the presence of c-di-GMP together with the response regulator VpsR results in transcription from PvpsL, a promoter of biofilm biosynthesis genes. VpsR shares homology with enhancer binding proteins that activate σ54-RNA polymerase (RNAP), but it lacks conserved residues needed to bind to σ54-RNAP and to hydrolyze adenosine triphosphate, and PvpsL transcription does not require σ54 in vivo.

The Bacteriophage T4 MotB Protein, a DNA-Binding Protein, Improves Phage Fitness.

The lytic bacteriophage T4 employs multiple phage-encoded early proteins to takeover the host. However, the functions of many of these proteins are not known. In this study, we have characterized the T4 early gene , located in a dispensable region of the T4 genome.

The phage T4 MotA transcription factor contains a novel DNA binding motif that specifically recognizes modified DNA.

During infection, bacteriophage T4 produces the MotA transcription factor that redirects the host RNA polymerase to the expression of T4 middle genes. The C-terminal 'double-wing' domain of MotA binds specifically to the MotA box motif of middle T4 promoters. We report the crystal structure of this complex, which reveals a new mode of protein-DNA interaction.

The BvgAS Regulon of .

Nearly all virulence factors in are activated by a master two-component system, BvgAS, composed of the sensor kinase BvgS and the response regulator BvgA. When BvgS is active, BvgA is phosphorylated (BvgA~P), and virulence-activated genes (s) are expressed [Bvg(+) mode]. When BvgS is inactive and BvgA is not phosphorylated, virulence-repressed genes (s) are induced [Bvg(-) mode].

A 3D puzzle approach to building protein-DNA structures.

Despite recent advances in structural analysis, it is still challenging to obtain a high-resolution structure for a complex of RNA polymerase, transcriptional factors, and DNA. However, using biochemical constraints, 3D printed models of available structures, and computer modeling, one can build biologically relevant models of such supramolecular complexes.

Visualizing the phage T4 activated transcription complex of DNA and E. coli RNA polymerase.

The ability of RNA polymerase (RNAP) to select the right promoter sequence at the right time is fundamental to the control of gene expression in all organisms. However, there is only one crystallized structure of a complete activator/RNAP/DNA complex. In a process called σ appropriation, bacteriophage T4 activates a class of phage promoters using an activator (MotA) and a co-activator (AsiA), which function through interactions with the σ(70) subunit of RNAP.

Bordetella pertussis fim3 gene regulation by BvgA: phosphorylation controls the formation of inactive vs. active transcription complexes.

Two-component systems [sensor kinase/response regulator (RR)] are major tools used by microorganisms to adapt to environmental conditions. RR phosphorylation is typically required for gene activation, but few studies have addressed how and if phosphorylation affects specific steps during transcription initiation. We characterized transcription complexes made with RNA polymerase and the Bordetella pertussis RR, BvgA, in its nonphosphorylated or phosphorylated (BvgA∼P) state at P(fim3), the promoter for the virulence gene fim3 (fimbrial subunit), using gel retardation, potassium permanganate and DNase I footprinting, cleavage reactions with protein conjugated with iron bromoacetamidobenzyl-EDTA, and in vitro transcription.

Strong inhibition of fimbrial 3 subunit gene transcription by a novel downstream repressive element in Bordetella pertussis.

The Bvg-regulated promoters for the fimbrial subunit genes fim2 and fim3 of Bordetella pertussis behave differently from each other both in vivo and in vitro. In vivo Pfim2 is significantly stronger than Pfim3 , even though predictions based on the DNA sequences of BvgA-binding motifs and core promoter elements would indicate the opposite. In vitro Pfim3 demonstrated robust BvgA∼P-dependent transcriptional activation, while none was seen with Pfim2 .

Architecture of the bacteriophage T4 activator MotA/promoter DNA interaction during sigma appropriation.

Gene expression can be regulated through factors that direct RNA polymerase to the correct promoter sequence at the correct time. Bacteriophage T4 controls its development in this way using phage proteins that interact with host RNA polymerase. Using a process called σ appropriation, the T4 co-activator AsiA structurally remodels the σ(70) subunit of host RNA polymerase, while a T4 activator, MotA, engages the C terminus of σ(70) and binds to a DNA promoter element, the MotA box.

Transcription regulation at the core: similarities among bacterial, archaeal, and eukaryotic RNA polymerases.

Multisubunit RNA polymerases are complex protein machines that require a specificity factor for the recognition of a specific transcription start site. Although bacterial σ factors are thought to be quite different from the specificity factors employed in higher organisms, a comparison of the σ/RNA polymerase structures with recent structures of eukaryotic Pol II together with TFIIB highlights significant functional similarities. Other work reveals that both bacterial and eukaryotic promoters are composed of modular elements that are used in different combinations.