Salmonella Type III Secretion Effector SrfJ: A Glucosylceramidase Affecting the Lipidome and the Transcriptome of Mammalian Host Cells.

Type III secretion systems are found in many Gram-negative pathogens and symbionts of animals and plants. Salmonella enterica has two type III secretion systems associated with virulence, one involved in the invasion of host cells and another involved in maintaining an appropriate intracellular niche. SrfJ is an effector of the second type III secretion system. In this study, we explored the biochemical function of SrfJ and the consequences for mammalian host cells of the expression of this S. enterica effector. Our experiments suggest that SrfJ is a glucosylceramidase that alters the lipidome and the transcriptome of host cells, both when expressed alone in epithelial cells and when translocated into macrophages in the context of Salmonella infection. We were able to identify seventeen lipids with higher levels and six lipids with lower levels in the presence of SrfJ. Analysis of the forty-five genes, the expression of which is significantly altered by SrfJ with a fold-change threshold of two, suggests that this effector may be involved in protecting Salmonella from host immune defenses.

DNA Breaks-Mediated Fitness Cost Reveals RNase HI as a New Target for Selectively Eliminating Antibiotic-Resistant Bacteria.

Antibiotic resistance often generates defects in bacterial growth called fitness cost. Understanding the causes of this cost is of paramount importance, as it is one of the main determinants of the prevalence of resistances upon reducing antibiotics use. Here we show that the fitness costs of antibiotic resistance mutations that affect transcription and translation in Escherichia coli strongly correlate with DNA breaks, which are generated via transcription-translation uncoupling, increased formation of RNA-DNA hybrids (R-loops), and elevated replication-transcription conflicts. We also demonstrated that the mechanisms generating DNA breaks are repeatedly targeted by compensatory evolution, and that DNA breaks and the cost of resistance can be increased by targeting the RNase HI, which specifically degrades R-loops. We further show that the DNA damage and thus the fitness cost caused by lack of RNase HI function drive resistant clones to extinction in populations with high initial frequency of resistance, both in laboratory conditions and in a mouse model of gut colonization. Thus, RNase HI provides a target specific against resistant bacteria, which we validate using a repurposed drug. In summary, we revealed key mechanisms underlying the fitness cost of antibiotic resistance mutations that can be exploited to specifically eliminate resistant bacteria.

Multidrug-resistant bacteria compensate for the epistasis between resistances.

Mutations conferring resistance to antibiotics are typically costly in the absence of the drug, but bacteria can reduce this cost by acquiring compensatory mutations. Thus, the rate of acquisition of compensatory mutations and their effects are key for the maintenance and dissemination of antibiotic resistances. While compensation for single resistances has been extensively studied, compensatory evolution of multiresistant bacteria remains unexplored. Importantly, since resistance mutations often interact epistatically, compensation of multiresistant bacteria may significantly differ from that of single-resistant strains. We used experimental evolution, next-generation sequencing, in silico simulations, and genome editing to compare the compensatory process of a streptomycin and rifampicin double-resistant Escherichia coli with those of single-resistant clones. We demonstrate that low-fitness double-resistant bacteria compensate faster than single-resistant strains due to the acquisition of compensatory mutations with larger effects. Strikingly, we identified mutations that only compensate for double resistance, being neutral or deleterious in sensitive or single-resistant backgrounds. Moreover, we show that their beneficial effects strongly decrease or disappear in conditions where the epistatic interaction between resistance alleles is absent, demonstrating that these mutations compensate for the epistasis. In summary, our data indicate that epistatic interactions between antibiotic resistances, leading to large fitness costs, possibly open alternative paths for rapid compensatory evolution, thereby potentially stabilizing costly multiple resistances in bacterial populations.

Maintenance of Microbial Cooperation Mediated by Public Goods in Single- and Multiple-Trait Scenarios

Microbes often form densely populated communities, which favor competitive and cooperative interactions. Cooperation among bacteria often occurs through the production of metabolically costly molecules produced by certain individuals that become available to other neighboring individuals; such molecules are called public goods. This type of cooperation is susceptible to exploitation, since nonproducers of a public good can benefit from it while saving the cost of its production (cheating), gaining a fitness advantage over producers (cooperators). Thus, in mixed cultures, cheaters can increase in frequency in the population, relative to cooperators. Sometimes, and as predicted by simple game-theoretic arguments, such increases in the frequency of cheaters cause loss of the cooperative traits by exhaustion of the public goods, eventually leading to a collapse of the entire population. In other cases, however, both cooperators and cheaters remain in coexistence. This raises the question of how cooperation is maintained in microbial populations. Several strategies to prevent cheating have been studied in the context of a single trait and a unique environmental constraint. In this review, we describe current knowledge on the evolutionary stability of microbial cooperation and discuss recent discoveries describing the mechanisms operating in multiple-trait and multiple-constraint settings. We conclude with a consideration of the consequences of these complex interactions, and we briefly discuss the potential role of social interactions involving multiple traits and multiple environmental constraints in the evolution of specialization and division of labor in microbes.

Expression of IroN, the salmochelin siderophore receptor, requires mRNA activation by RyhB small RNA homologues.

The iroN gene of Salmonella enterica and uropathogenic Escherichia coli encodes the outer membrane receptor of Fe(3+) -bound salmochelin, a siderophore tailored to evade capture by the host's immune system. The iroN gene is under negative control of the Fur repressor and transcribed under iron limiting conditions. We show here that transcriptional de-repression is not sufficient to allow iroN expression, as this also requires activation by either of two partially homologous small RNAs (sRNAs), RyhB1 and RyhB2. The two sRNAs target the same sequence segment approximately in the middle of the 94-nucleotide 5' untranslated region (UTR) of iroN mRNA. Several lines of evidence suggest that base pair interaction stimulates iroN mRNA translation. Activation does not result from the disruption of a secondary structure masking the ribosome binding site; rather it involves sequences at the 5' end of iroN 5' UTR. In vitro 'toeprint' assays revealed that this upstream site binds the 30S ribosomal subunit provided that RyhB1 is paired with the mRNA. Altogether, our data suggest that RyhB1, and to lesser extent RyhB2, activate iroN mRNA translation by promoting entry of the ribosome at an upstream 'standby' site. These findings add yet an additional nuance to the polychromatic landscape of sRNA-mediated regulation.

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 (cat or kan) and the tetR repressor gene linked to a Ptet promoter-ccdB toxin gene fusion. In the absence of induction, the tetR gene product represses the Ptet promoter, preventing ccdB expression. The cassette is first inserted at the target site by selecting for chloramphenicol or kanamycin resistance. It is subsequently replaced by the sequence of interest by selecting for growth in the presence of anhydrotetracycline (AHTc), which inactivates the TetR repressor thereby causing CcdB-induced lethality. Unlike other CcdB-based counterselection schemes, which require specifically designed λ-Red delivery plasmids, the system described here uses the popular plasmid pKD46 as the source of λ-Red functions. This protocol allows a wide variety of modifications, including the intragenic insertion of fluorescent or epitope tags, gene replacements, deletions, and single base-pair substitutions, to be made. In addition, the procedure can be used to place the inducible Ptet promoter at a chosen position in the bacterial chromosome.

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. Deletions can be constructed by replacing a portion or the entirety of a target gene with an antibiotic-resistance cassette. In some commonly used template plasmids, the antibiotic-resistance gene can be coamplified with a pair of flanking FRT (Flp recombinase recognition target) sites that, following insertion of the fragment into the chromosome, allow excision of the antibiotic-resistance cassette via the activity of the site-specific Flp recombinase. The excision step leaves behind a "scar" sequence comprising an FRT site and flanking primer annealing sequences. Removal of the cassette minimizes undesired perturbations on the expression of neighboring genes. Even so, polarity effects can result from the occurrence of stop codons within, or downstream of, the scar sequence. These problems can be avoided by the appropriate choice of the template and by designing primers so that the reading frame of the target gene is maintained past the deletion end point. This protocol is optimized for use with Salmonella enterica and Escherichia coli.

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. Here, we introduce some of the most common implementations of the method.

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 (kan or cat) present on the plasmid. This method is slightly more laborious than generating the fusion directly by recombineering and has the limitation that the selectable marker is no longer removable. However, it has the advantage that it can be more readily integrated in mutational studies, allowing conversion of in-frame deletions resulting from Flp-mediated excision of a drug-resistance cassette (e.g., all those of the "Keio collection") into fluorescent protein fusions. Furthermore, in studies that require that the amino-terminal moiety of the hybrid protein keeps its biological activity, presence of the FRT "linker" sequence at the fusion junction makes it less likely for the fluorescent domain to sterically interfere with the folding of the amino-terminal domain.

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 (kan or cat) 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. The method is specifically designed for the construction of translational fusions producing hybrid proteins with a fluorescent carboxyl-terminal domain. The fluorescent protein-encoding sequence can be placed at any codon position of the target gene's mRNA where the fusion produces a reliable reporter for gene expression. Internal and carboxyl-terminal fusions to sfGFP are suitable for studying protein localization in bacterial subcellular compartments.

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. This protocol, implemented here with a class of transposons generating reporter gene fusions, involves (i) preparing genomic DNA from the strain harboring the unknown insertion, (ii) cleaving the genomic DNA with a restriction enzyme, (iii) performing a ligation reaction under conditions favoring circularization of the DNA fragments, and (iv) performing inverse PCRs with inside-out primers annealing near either or both termini of the transposon. This last step results in the amplification of the chromosomal sequences immediately adjacent to the transposon, which can then be identified by Sanger sequencing. The protocol can be performed in parallel on several strains providing an effective and economic way for rapidly identifying multiple transposon insertion sites.

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 Zea mays 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. In one application, transposons have been modified to include a reporter gene engineered in such a way that the reporter can become fused to a chromosomal gene upon inserting randomly in the bacterial chromosome. Screening this type of transposon library for expression of the reporter under different conditions allows identifying fusions that respond coordinately to a specific treatment or stress condition. Characterization of these fusions provides a genome-wide snapshot of the organization of a bacterial regulatory network.

Generating Libraries of Random lacZY or GFP 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 lacZY 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 cis with respect to the transposable module and under the control of the anyhydrotetracycline (AHTc)-inducible Ptet promoter. The transposable module comprises a promoter-less lacZY operon or the sfGFP gene, with or without the lacZ or sfGFP ribosome-binding site, plus a kan gene for selection. The transposon-transposase unit is harbored on an R6K-based suicide plasmid. The plasmid is introduced into recipient cells by electro-transformation and the synthesis of Tn5 Tnp is induced transiently by including AHTc in the recovery medium. Cells are then plated on kanamycin-supplemented medium (without AHTc) where the plasmid DNA is lost and only cells in which transposition has occurred can form colonies. Fusions are detected by screening for colony color on lactose indicator plates (lacZ transposition) or monitoring green fluorescence (sfGFP transposition). Depending on whether the reporter gene carries or lacks the ribosome binding sequence, the fusions obtained will either be transcriptional or translational. Parallel screening of colonies grown in the absence and presence of a drug (or condition) eliciting a global regulatory response allows identification of fusions specifically activated or repressed as part of such response.

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 Escherichia coli and Salmonella enterica We begin by reviewing the aspects of the biology of these two species that are most relevant for their manipulation in the laboratory.

Quick Transformation with Plasmid DNA.

Genomic engineering of Escherichia coli and Salmonella 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 E. coli or Salmonella with plasmid DNA that uses as little as 2 mL of culture.

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. Streaking, a technique to disperse bacterial cells on the surface of the agar, serves the purpose of isolating individual colonies. A colony originates from a single cell and is a nearly pure culture. On rich LB medium after 24 h of incubation at 37°C, a colony of Salmonella contains ∼5 × 108 cells (about 29 generations). Streaking is also required in experiments that themselves generate single colonies as a result of selection (e.g., when constructing strains or introducing plasmids). Except in the few instances in which the selection efficiently kills all counter-selected bacteria, colonies growing on the selective plates are contaminated, sometimes heavily, with cells from the bacterial lawn. "Purifying" the colonies arising in such experiments by streaking on selective plates is therefore a mandatory step. Here, we show how this can be conveniently done using simple toothpicks. We also briefly describe the steps involved in inoculating liquid cultures, spreading plates, and replica plating.

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. This is not the case for chromosomal DNA, which, upon neutralization, aggregates with denatured proteins through hydrophobic interactions. As a result, plasmid DNA remains in solution and can be easily separated from most of the other macromolecules that coprecipitate. For the subsequent purification step, one can use the silica membrane technology integrated in many commercial kits. This technology exploits the ability of DNA to bind to silica in the presence of chaotropic salts. DNA is retained by a silica-based column, whereas most of the polysaccharides and proteins flow through. After wash steps to eliminate residual contaminants and salts, DNA is selectively eluted under low-salt conditions. A kit-free but relatively more cumbersome alternative to this procedure is the traditional phenol-chloroform extraction method followed by ethanol precipitation. Both methods are detailed here.

Working with Phage P22.

Transduction experiments in Escherichia coli and Salmonella are usually performed with virulent phage variants. A widely used P1 mutant, called P1 vir, 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 int-201 This phage has a high transducing (HT) frequency due to a mutant nuclease with lower specificity for the pac sequence. As a result, ∼50% of the P22 HT phage heads carry random transducing fragments of chromosomal DNA. The int mutation reduces the formation of stable lysogens. The basic steps in handling the P22 HT105/1 int-201 phage and in performing transduction experiments in Salmonella are described here.

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). Polysaccharides and proteins do not bind well to the column and flow through. Their residual traces, along with the guanidine salts, are removed during alcohol-based wash steps. The DNA is then selectively eluted under low-salt conditions. This method is quick and easy and yields genomic DNA suitable for most downstream applications. The second procedure, implemented for many years in our laboratory before the appearance of commercial kits, is based on the ability of the cationic detergent cetyl trimethyl ammonium bromide (CTAB) to complex with polysaccharides and proteins, producing an emulsion that can be removed by chloroform-isoamyl alcohol extraction. This procedure is therefore especially suited to working with Gram-negative bacteria, which typically produce large amounts of polysaccharides. Its main advantage, besides cost-effectiveness, is the high yield of the DNA obtained; its main disadvantage is that the workflow is relatively cumbersome.

Recognition of heptameric seed sequence underlies multi-target regulation by RybB small RNA in Salmonella enterica.

Prokaryotic regulatory small RNAs act by a conserved mechanism and yet display a stunning structural variability. In the present study, we used mutational analysis to dissect the functional anatomy of RybB, a σ(E)-dependent sRNA that regulates the synthesis of major porins in Escherichia coli and Salmonella. Mutations in the chromosomal rybB locus that altered the expression of an ompC-lac fusion were identified. Some of the mutations cluster within a seven-nucleotide segment at the 5' end of the sRNA and affect its ability to pair with a sequence 40 nucleotides upstream from ompC translation start site. Other mutations map near the 3' end of RybB, destabilizing the sRNA or altering its binding to Hfq. The 5' end of RybB is also involved in ompD regulation. In this case, the sRNA can choose between two mutually exclusive pairing sites within the translated portion of the mRNA. Some of the RybB 5' end mutations affect the choice between the two sites, resulting in regulatory responses that diverge from those observed in ompC. Further analysis of RybB target specificity identified chiP (ybfM), a gene encoding an inducible chitoporin, as an additional member of the RybB regulon. Altogether, our results indicate that an heptameric 'seed' sequence is sufficient to confer susceptibility to RybB regulation.