Rho-dependent transcriptional switches regulate the bacterial response to cold shock.

Binding of the bacterial Rho helicase to nascent transcripts triggers Rho-dependent transcription termination (RDTT) in response to cellular signals that modulate mRNA structure and accessibility of Rho utilization (Rut) sites. Despite the impact of temperature on RNA structure, RDTT was never linked to the bacterial response to temperature shifts. We show that Rho is a central player in the cold-shock response (CSR), challenging the current view that CSR is primarily a posttranscriptional program.

Transcription-driven DNA supercoiling counteracts H-NS-mediated gene silencing in bacterial chromatin.

In all living cells, genomic DNA is compacted through interactions with dedicated proteins and/or the formation of plectonemic coils. In bacteria, DNA compaction is achieved dynamically, coordinated with dense and constantly changing transcriptional activity. H-NS, a major bacterial nucleoid structuring protein, is of special interest due to its interplay with RNA polymerase.

Dynamics of macrophage polarization support persistence in a whole living organism.

Numerous intracellular bacterial pathogens interfere with macrophage function, including macrophage polarization, to establish a niche and persist. However, the spatiotemporal dynamics of macrophage polarization during infection within host remain to be investigated. Here, we implement a model of persistent Typhimurium infection in zebrafish, which allows visualization of polarized macrophages and bacteria in real time at high resolution.

Generating Libraries of Random or Gene Fusions in the Bacterial Chromosome: Screening for Differentially Expressed Fusions.

Transposable elements engineered to generate random gene fusions in the bacterial chromosome are valuable tools in the study of gene expression. In this protocol, we describe the use of a new series of transposons designed to obtain random fusions to either the operon or the gene for superfolder green fluorescent protein (sfGFP). Transposition is achieved through the activity of the hyperactive variant of Tn5 transposase (Tnp) whose gene is positioned in with respect to the transposable module and under the control of the anyhydrotetracycline (AHTc)-inducible P promoter.

Use of Transposable Reporters in the Analysis of Bacterial Regulatory Networks.

Transposable elements are genetic entities that have the capacity to promote their own translocation from one site to another within a genome. Initially discovered in by Barbara McClintock at the Cold Spring Harbor Laboratory, transposable elements have been found to populate the genomes of all forms of life. In bacteria, the discovery of transposons significantly enhanced genetic analyses; they have been widely used to make insertion mutants and have inspired elegant strategies for strain construction and in vivo genome engineering.

Mapping Transposon Insertion Sites by Inverse Polymerase Chain Reaction and Sanger Sequencing.

Inverse polymerase chain reaction (PCR) is a method designed to amplify a segment of DNA for which only a portion of the sequence is known. The method consists of circularizing the DNA fragment by self-ligation and performing PCR with primers annealing inside the known sequence but pointing away from each other (hence the technique is also called "inside-out PCR"). Here we describe how inverse PCR can be used to identify the site of transposon insertion in the bacterial chromosome.

Construction of Single-Copy Fluorescent Protein Fusions by One-Step Recombineering.

We describe a simple recombineering-based procedure for generating single-copy gene fusions to superfolder GFP (sfGFP) and monomeric Cherry (mCherry). The open reading frame (orf) for either protein is inserted at the targeted chromosomal location by λ Red recombination using an adjacent drug-resistance cassette ( or ) for selection. The drug-resistance gene is flanked by flippase (Flp) recognition target (FRT) sites in direct orientation, which allows removal of the cassette by Flp-mediated site-specific recombination once the construct is obtained, if desired.

Converting an FRT-Tagged Gene into a Fluorescent Protein Gene Fusion by Flp-Mediated Site-Specific Recombination.

This protocol uses conditional plasmids carrying the open reading frame (orf) of either superfolder green fluorescent protein (sfGFP) or monomeric Cherry (mCherry) fused to a flippase (Flp) recognition target (FRT) site. In cells expressing the Flp enzyme, site-specific recombination between the plasmid-borne FRT and an FRT "scar" in a target gene in the bacterial chromosome results in chromosomal integration of the plasmid with the concomitant in-frame fusion of the target gene to the fluorescent protein orf. This event can be positively selected using an antibiotic-resistance marker ( or ) present on the plasmid.

DNA Recombineering Applications.

The ability to manipulate the bacterial genome is an obligatory premise for the study of gene function and regulation in bacterial cells. The λ red recombineering technique allows modification of chromosomal sequences with base-pair precision without the need of intermediate molecular cloning steps. Initially conceived to construct insertion mutants, the technique lends itself to a wide variety of applications including the creation of point mutants, seamless deletions, reporter, and epitope tag fusions and chromosomal rearrangements.

Recombineering 101: Making an in-Frame Deletion Mutant.

DNA recombineering uses phage λ Red recombination functions to promote integration of DNA fragments generated by polymerase chain reaction (PCR) into the bacterial chromosome. The PCR primers are designed to have the last 18-22 nt anneal on either side of the donor DNA and to carry 40- to 50-nt 5' extensions homologous to the sequences flanking the chosen insertion site. The simplest application of the method results in knockout mutants of nonessential genes.

Scarless DNA Recombineering.

The method described here allows editing of the bacterial genome without leaving any secondary changes (scars) behind. This method uses a tripartite selectable and counterselectable cassette comprising an antibiotic-resistance gene ( or ) and the repressor gene linked to a P promoter- toxin gene fusion. In the absence of induction, the gene product represses the P promoter, preventing expression.

Preparing Bacterial Genomic DNA.

We describe two alternative procedures for purifying bacterial chromosomal DNA. The first procedure incorporates the use of a commercial kit based on silica membrane technology. This approach relies on the selective binding of DNA to a silica-based column in the presence of chaotropic salts (guanidine salts).

Working with Phage P22.

Transduction experiments in and are usually performed with virulent phage variants. A widely used P1 mutant, called P1 , carries one or more uncharacterized mutations that prevent formation of lysogens. In the case of P22, by far the most frequently used variant is named P22 HT105/1 This phage has a high transducing (HT) frequency due to a mutant nuclease with lower specificity for the sequence.

Preparing Plasmid DNA from Bacteria.

The most common method for isolating plasmid DNA is derived from an alkaline lysis procedure. The procedure exploits the differential partitioning of plasmid and chromosomal DNA when denatured by alkali and subsequently renatured by neutralization of the medium. The circular covalently closed nature of plasmid DNA allows the denatured DNA strands to quickly find each other and reanneal during the renaturation step.

Basic Bacteriological Routines.

In experimental bacteriology, bacteria are generally manipulated, stored, and shipped in the form of cultures. Depending on various factors, including strain genotype, storage and shipping methods, and manipulator skills, the culture may contain genetic variants or simply contaminants. It is therefore important to begin an experiment by streaking the culture on an agar plate.

Quick Transformation with Plasmid DNA.

Genomic engineering of and often requires introducing plasmids into strains obtained during the intermediate stages of the process. Such strains are typically transformed only once, making the preparation of large batches of competent cells for storage purposes unnecessary. Here, we describe a simple scaled-down procedure for transforming or with plasmid DNA that uses as little as 2 mL of culture.

Working with Bacteria, Phage, and Plasmids.

Methods for the in vivo manipulation of bacterial genomes have improved greatly in recent years because of the discovery of new mechanisms and the gigantic leap forward in DNA-sequencing technology. Many cutting-edge approaches still rely on a variety of technical routines, the correct implementation of which is critical for the success of an experiment. Here, we introduce some of these procedures as used for and We begin by reviewing the aspects of the biology of these two species that are most relevant for their manipulation in the laboratory.

Pervasive transcription enhances the accessibility of H-NS-silenced promoters and generates bistability in virulence gene expression.

In and many genes silenced by the nucleoid structuring protein H-NS are activated upon inhibiting Rho-dependent transcription termination. This response is poorly understood and difficult to reconcile with the view that H-NS acts mainly by blocking transcription initiation. Here we have analyzed the basis for the up-regulation of H-NS-silenced pathogenicity island 1 (SPI-1) in cells depleted of Rho-cofactor NusG.

6S RNA-Dependent Susceptibility to RNA Polymerase Inhibitors.

Bacterial small RNAs (sRNAs) contribute to a variety of regulatory mechanisms that modulate a wide range of pathways, including metabolism, virulence, and antibiotic resistance. We investigated the involvement of sRNAs in rifampicin resistance in the opportunistic pathogen Staphylococcus aureus. Using a competition assay with an sRNA mutant library, we identified 6S RNA as being required for protection against low concentrations of rifampicin, an RNA polymerase (RNAP) inhibitor.

Genome archaeology of two laboratory Salmonella enterica enterica sv Typhimurium.

The Salmonella research community has used strains and bacteriophages over decades, exchanging useful new isolates among laboratories for the study of cell surface antigens, metabolic pathways and restriction-modification (RM) studies. Here we present the sequences of two laboratory Salmonella strains (STK005, an isolate of LB5000; and its descendant ER3625). In the ancestry of LB5000, segments of ∼15 and ∼42 kb were introduced from Salmonella enterica sv Abony 803 into S.

Regulatory interplay between small RNAs and transcription termination factor Rho.

The largest and best studied group of regulatory small RNAs (sRNAs) in bacteria act by modulating translation or turnover of messenger RNAs (mRNAs) through base-pairing interactions that typically take place near the 5' end of the mRNA. This allows the sRNA to bind the complementary target sequence while the remainder of the mRNA is still being made, creating conditions whereby the action of the sRNA can extend to transcriptional steps, most notably transcription termination. Increasing evidence corroborates the existence of a functional interplay between sRNAs and termination factor Rho.

NusG prevents transcriptional invasion of H-NS-silenced genes.

Evolutionarily conserved NusG protein enhances bacterial RNA polymerase processivity but can also promote transcription termination by binding to, and stimulating the activity of, Rho factor. Rho terminates transcription upon anchoring to cytidine-rich motifs, the so-called Rho utilization sites (Rut) in nascent RNA. Both NusG and Rho have been implicated in the silencing of horizontally-acquired A/T-rich DNA by nucleoid structuring protein H-NS.

Sponges and Predators in the Small RNA World.

Most noncoding small RNAs (sRNAs) that regulate gene expression do so by base-pairing with mRNAs, affecting their translation and/or stability. Regulators as evolutionarily distant as the -encoded sRNAs of bacteria and the microRNAs (miRNAs) of higher eukaryotes share the property of targeting short sequence segments that occur in multiple copies in bacterial and eukaryotic transcriptomes. This target promiscuity has major implications for sRNA function.