Epitope tagging of chromosomal genes in <i>Salmonella</i>

We have developed a simple and efficient procedure for adding an epitope-encoding tail to one or more genes of interest in the bacterial chromosome. The procedure is a modification of the gene replacement method of Datsenko and Wanner [Datsenko, K. A. &amp; Wanner, B. L. (2000) Proc. Natl. Acad. Sci. USA 97, 6640–6645]. A DNA module that begins with the epitope-encoding sequence and includes a selectable marker is amplified by PCR with primers that carry extensions (as short as 36 nt) homologous to the last portion of the targeted gene and to a region downstream from it. Transformation of a strain expressing bacteriophage ʎ red functions yields recombinants carrying the targeted gene fused to the epitope-encoding sequence. The resulting C-terminal-tagged protein can be identified by standard immuno-detection techniques. In an initial application of the method, we have added the sequences encoding the FLAG and 3xFLAG and influenza virus hemagglutinin epitopes to various genes of Salmonella enterica serovar Typhimurium, including putative and established pathogenic determinants present in prophage genomes. Epitope fusion proteins were detected in bacteria growing in vitro, tissue culture cells, and infected mouse tissues. This work identified a prophage locus specifically expressed in bacteria growing intracellularly. The procedure described here should be applicable to a wide variety of Gram-negative bacteria and is particularly suited for the study of intracellular pathogens

Insertion hot spot for horizontally acquired DNA within a bidirectional small-RNA locus in Salmonella enterica

In Escherichia coli and Salmonella enterica, RyeA and RyeB RNAs are encoded on opposite DNA strands at the same locus. We present evidence indicating that the last 23 bp of the ryeB gene, corresponding to an internal portion of the ryeA gene, served repeatedly as the integration site for exogenous DNA during Salmonella evolution and still act as an attachment site for present-day bacteriophages. Interestingly, ryeA sequence and expression are modified upon lysogenization.Ministerio de Educación y Ciencia BIO2004-3455-CO2-0

The tripartite capsid gene of Salmonella phage Gifsy-2 yields a capsid assembly pathway engaging features from HK97 and λ

AbstractPhage Gifsy-2, a lambdoid phage infecting Salmonella, has an unusually large composite gene coding for its major capsid protein (mcp) at the C-terminal end, a ClpP-like protease at the N-terminus, and a ∼200 residue central domain of unknown function but which may have a scaffolding role. This combination of functions on a single coding region is more extensive than those observed in other phages such as HK97 (scaffold–capsid fusion) and λ (protease–scaffold fusion). To study the structural phenotype of the unique Gifsy-2 capsid gene, we have purified Gifsy-2 particles and visualized capsids and procapsids by cryoelectron microscopy, determining structures to resolutions up to 12Å. The capsids have lambdoid T=7 geometry and are well modeled with the atomic structures of HK97 mcp and phage λ gpD decoration protein. Thus, the unique Gifsy-2 capsid protein gene yields a capsid maturation pathway engaging features from both phages HK97 and λ

Prophage Gifsy-1 Induction in Salmonella enterica Serovar Typhimurium Reduces Persister Cell Formation after Ciprofloxacin Exposure

Persister cells are drug-tolerant bacteria capable of surviving antibiotic treatment despite the absence of heritable resistance mechanisms. It is generally thought that persister cells survive antibiotic exposure through the implementation of stress responses and/or energy-sparing strategies. Exposure to DNA gyrase-targeting antibiotics could be particularly detrimental for bacteria that carry prophages integrated in their genomes. Gyrase inhibitors are known to induce prophages to switch from their dormant lysogenic state into the lytic cycle, causing the lysis of their bacterial host. However, the influence of resident prophages on the formation of persister cells has only been recently appreciated. Here, we evaluated the effect of endogenous prophage carriage on the generation of bacterial persistence during Salmonella enterica serovar Typhimurium exposure to both gyrase-targeting antibiotics and other classes of bactericidal antibiotics. Results from the analysis of strain variants harboring different prophage combinations revealed that prophages play a major role in limiting the formation of persister cells during exposure to DNA-damaging antibiotics. In particular, we present evidence that prophage Gifsy-1 (and its encoded lysis proteins) are major factors limiting persister cell formation upon ciprofloxacin exposure. Resident prophages also appear to have a significant impact on the initial drug susceptibility, resulting in an alteration of the characteristic biphasic killing curve of persister cells into a triphasic curve. In contrast, a prophage-free derivative of S. Typhimurium showed no difference in the killing kinetics for β-lactam or aminoglycoside antibiotics. Our study demonstrates that induction of prophages increased the susceptibility toward DNA gyrase inhibitors in S. Typhimurium, suggesting that prophages have the potential for enhancing antibiotic efficacy

Bacteriophage Crosstalk: Coordination of Prophage Induction by Trans-Acting Antirepressors

Many species of bacteria harbor multiple prophages in their genomes. Prophages often carry genes that confer a selective advantage to the bacterium, typically during host colonization. Prophages can convert to infectious viruses through a process known as induction, which is relevant to the spread of bacterial virulence genes. The paradigm of prophage induction, as set by the phage Lambda model, sees the process initiated by the RecA-stimulated self-proteolysis of the phage repressor. Here we show that a large family of lambdoid prophages found in Salmonella genomes employs an alternative induction strategy. The repressors of these phages are not cleaved upon induction; rather, they are inactivated by the binding of small antirepressor proteins. Formation of the complex causes the repressor to dissociate from DNA. The antirepressor genes lie outside the immunity region and are under direct control of the LexA repressor, thus plugging prophage induction directly into the SOS response. GfoA and GfhA, the antirepressors of Salmonella prophages Gifsy-1 and Gifsy-3, each target both of these phages' repressors, GfoR and GfhR, even though the latter proteins recognize different operator sites and the two phages are heteroimmune. In contrast, the Gifsy-2 phage repressor, GtgR, is insensitive to GfoA and GfhA, but is inactivated by an antirepressor from the unrelated Fels-1 prophage (FsoA). This response is all the more surprising as FsoA is under the control of the Fels-1 repressor, not LexA, and plays no apparent role in Fels-1 induction, which occurs via a Lambda CI-like repressor cleavage mechanism. The ability of antirepressors to recognize non-cognate repressors allows coordination of induction of multiple prophages in polylysogenic strains. Identification of non-cleavable gfoR/gtgR homologues in a large variety of bacterial genomes (including most Escherichia coli genomes in the DNA database) suggests that antirepression-mediated induction is far more common than previously recognized

Competing endogenous RNAs: a target-centric view of small RNA regulation in bacteria

International audienceMany bacterial regulatory small RNAs (sRNAs) have several mRNA targets, which places them at the centre of regulatory networks that help bacteria to adapt to environmental changes. However, different mRNA targets of any given sRNA compete with each other for binding to the sRNA; thus, depending on relative abundances and sRNA affinity, competition for regulatory sRNAs can mediate cross-regulation between bacterial mRNAs. This 'target-centric' perspective of sRNA regulation is reminiscent of the competing endogenous RNA (ceRNA) hypothesis, which posits that competition for a limited pool of microRNAs (miRNAs) in higher eukaryotes mediates cross-regulation of mRNAs. In this Opinion article, we discuss evidence that a similar network of RNA crosstalk operates in bacteria, and that this network also includes crosstalk between sRNAs and competition for RNA-binding proteins

Resuscitation of a Defective Prophage in Salmonella Cocultures

Widely studied Salmonella enterica serovar Typhimurium strains ATCC 14028s and SL1344 harbor a cryptic ST64B prophage unable to produce infectious virions. We found that coculturing either strain with an isogenic sibling lacking the prophage leads to the appearance of active forms of the virus. Active phage originates from reversion of a +1 frameshift mutation at a monotonous G:C run in a presumptive tail assembly pseudogene

Terminator still moving forward: expanding roles for Rho factor

International audienceRho factor is a molecular motor that translocates along nascent RNA and acts on the transcription elongation complex to promote termination. Besides contributing to transcriptional punctuation of the bacterial genome, Rho can act intragenically under conditions that perturb coupling of translation and transcription. Recent advances have shed new light onto several aspects of Rho function, including the translocation mechanism, the avoidance of potential conflicts between DNA replication and transcription, suppression of pervasive antisense transcription and recruitment in riboswitch and small RNA-dependent regulation. Altogether, these findings further highlight the relevance of Rho factor, both as a multi-task housekeeper and gene regulator

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.Agence Nationale de la Recherche ANR-15-CE11-0024-0