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

Régulation du gène yifK par le petit ARN multi-cible GcvB chez Salmonella

GcvB est un ARN bactérien conservé de 200 nucléotides, qui régule négativement l expression de plusieurs gènes impliqués dans l import et la biosynthèse des acides aminés. Bien que le rôle physiologique de GcvB ne soit pas complètement élucidé, il contribuerait vraisemblablement à équilibrer les ressources nutritionnelles en conditions de croissance rapide. GcvB inhibe la traduction des ARNm cibles en s appariant avec des séquences à l intérieur ou en amont du site de liaison du ribosome. Dans cette étude, la caractérisation d un nouveau locus régulé par GcvB a permis de dévoiler des aspects singuliers du mode de fonctionnement de cet ARN régulateur. Nous avons découvert que GcvB réprime yifK - un gène très conservé, codant pour un transporteur d acides aminés putatif - en ciblant un élément activateur de la traduction sur l ARNm. Deux motifs ACA dans la séquence cible sont les déterminants principaux de la fonction activatrice. Le remplacement de l un ou l autre avec des triplets aléatoires, provoque une diminution de 10 fois du niveau d expression de yifK, quelque soit l allèle de GcvB (délétion ou changement de séquence permettant la reconnaissance de la cible mutante). Il apparait ainsi que l efficacité de GcvB à réguler négativement sa cible serait liée a sa capacité d antagoniser l élément activateur. Lorsque l activateur est éliminé, l action de GcvB n est plus un facteur limitant pour l expression de yifK. Dans son ensemble, cette étude apporte une meilleure compréhension de la fonction de GcvB et révèle un nouvel aspect du processus d initiation de la traduction. En plus du contrôle par GcvB, le locus yifK est régulé au niveau transcriptionnel par Lrp (leucine-responsive regulatory protein) et par HdfR (YifA) un régulateur transcriptionnel peu connu qui requerrait le produit du gène adjacent orienté de façon divergente, yifE, pour son expression ou activité. Enfin, la transcription initiée au niveau du promoteur yifK s étend dans l opéron d ARNt argX-hisR-leuT-proM adjacent, donnant lieu à un transcrit primaire qui est à la fois un lARNm et un précurseur des ARNt. Cet ARN chimère est rapidement maturé par l ARNase E.GcvB is a conserved 200 nucleotide RNA that downregulates several genes involved in amino acid uptake or biosynthesis in bacteria. The physiological role of GcvB action is not entirely clear, but it is likely aimed at balancing of nutritional resources under fast growth conditions. GcvB inhibits translation of target messenger RNAs by pairing with sequences inside or upstream of ribosome binding sites. In the present study, characterization of a novel GcvB-regulated locus revealed some unique features in the mode of functioning of this regulatory RNA. We found that GcvB represses yifK - a highly conserved locus encoding a putative amino acid transporter - by targeting a translational enhancer element. Two ACA motifs within the target sequence are the main determinants of the enhancer activity. Replacing either of these motifs with random triplets caused up to a 10-fold decrease in yifK expression regardless of the GcvB allele (deleted or suitably modified to recognize the mutated target). It thus appears that GcvB effectiveness as a regulator results from countering the enhancer activity. When the enhancer is removed, GcvB action no longer constitutes a rate-limiting factor for yifK expression. Overall, this study is relevant not only to a better understanding of GcvB function but it also provides insight into an elusive aspect of the translation initiation process. Besides the GcvB control, the yifK locus is regulated at the transcriptional level by the leucine responsive regulator Lrp, and by HdfR (YifA) a poorly known transcriptional regulator, that appears to require the product of the adjacent, divergently oriented gene, yifE, for expression or activity. Transcription initiating at the yifK promoter extends into the adjacent argX-hisR-leuT-proM tRNA operon yielding an unusual primary transcript which both a messenger RNA and a tRNA precursor. This chimeric RNA si rapidly processed by RNAse E.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

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

Recombineering applications for the mutational analysis of bacterial RNA-binding proteins and their sites of action.

International audienceGenetics remains a powerful tool to study structure-function relationships in proteins and RNA. Structural elements important for the biological activity of these molecules can be dissected through the isolation of mutations and analysis of their effects on the mechanism under study. In suitable model organisms, this approach can greatly benefit from the ability to introduce mutations directly in the chromosomal context in ways that do not perturb neighboring sequences. Methods for performing such "markerless" site-directed chromosomal mutagenesis in bacteria have been developed in recent years. One such technique, used routinely in our laboratory, is described here

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