Bacteriophage Removal from Infected Salmonella Cultures

Bacteriophages, or simply phages, play a vital role in microbial environments, impacting bacterial populations and shaping their evolution and ...

Universidad de Sevilla

We describe a portable epigenetic switch based on opvAB, a Salmonella enterica operon that undergoes bistable expression under DNA methylation control. A DNA fragment containing the opvAB promoter and the opvAB upstream regulatory region confers bistability to heterologous genes, yielding OFF and ON subpopulations. Bistable expression under opvAB control is reproducible in Escherichia coli, showing that the opvAB switch can be functional in a heterologous host. Subpopulations of different sizes can be produced at will using engineered opvAB variants. Controlled formation of antibiotic-resistant and antibiotic-susceptible subpopulations may allow use of the opvAB switch in the study of bacterial heteroresistance to antibiotics.

A portable epigenetic switch for bistable gene expression in bacteria.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Publisher Correction: A portable epigenetic switch for bistable gene expression in bacteria.

Environmental monitoring of bacteria using phage-based biosensors has been widely developed for many different species. However, there are only a few available methods to detect specific bacteriophages in raw environmental samples. In this work, we developed a simple and efficient assay to rapidly monitor the phage content of a given sample. The assay is based on the bistable expression of the Salmonella enterica opvAB operon. Under regular growth conditions, opvAB is only expressed by a small fraction of the bacterial subpopulation. In the OpvAB subpopulation, synthesis of the OpvA and OpvB products shortens the O-antigen and confers resistance to phages that use LPS as a receptor. As a consequence, the OpvAB subpopulation is selected in the presence of such phages. Using an opvAB::gfp fusion, we could monitor LPS-binding phages in various media, including raw water samples. To enlarge our phage-biosensor panoply, we also developed biosensors able to detect LPS, as well as protein-binding coliphages. Moreover, the combination of these tools allowed to identify the bacterial receptor triggering phage infection. The epigenetic opvAB::gfp biosensor thus comes in different flavours to detect a wide range of bacteriophages and identify the type of receptor they recognize.

Epigenetic biosensors for bacteriophage detection and phage receptor discrimination.

Expression of Salmonella enterica loci harboring undermethylated GATC sites at promoters or regulatory regions was monitored by single cell analysis. Cell-to-cell differences in expression were detected in ten such loci (carA, dgoR, holA, nanA, ssaN, STM1290, STM3276, STM5308, gtr and opvAB), with concomitant formation of ON and OFF subpopulations. The ON and OFF subpopulation sizes varied depending on the growth conditions, suggesting that the population structure can be modulated by environmental control. All the loci under study except STM5308 displayed altered patterns of expression in strains lacking or overproducing Dam methylase, thereby confirming control by Dam methylation. Bioinformatic analysis identified potential binding sites for transcription factors OxyR, CRP and Fur, and analysis of expression in mutant backgrounds confirmed transcriptional control by one or more of such factors. Surveys of gene expression in pairwise combinations of Dam methylation-dependent loci revealed independent switching, thus predicting the formation of a high number of cell variants. This study expands the list of S. enterica loci under transcriptional control by Dam methylation, and underscores the relevance of the DNA adenine methylome as a source of phenotypic heterogeneity.

Contribution of DNA adenine methylation to gene expression heterogeneity in Salmonella enterica.

Advances in technologies that permit high-resolution analysis of events in single cells have revealed that phenotypic heterogeneity is a widespread phenomenon in bacteria. Flow cytometry has the potential to describe the distribution of cellular properties within a population of bacterial cells and has yielded invaluable information about the ability of isogenic cells to diversify into phenotypic subpopulations. This review will discuss several single-cell approaches that have recently been applied to define phenotypic heterogeneity in populations of Salmonella enterica.

Identifying Bacterial Lineages in Salmonella by Flow Cytometry.

, a Gram-negative zoonotic bacterium, is mainly a food-borne pathogen and the main cause of diarrhea in humans worldwide. The main reservoirs are found in poultry farms, but they are also found in wild birds. The development of antibiotic resistance in  species raises concerns about the future of efficient therapies against this pathogen and revives the interest in bacteriophages as a useful therapy against bacterial infections. Here, we aimed to decipher and functionally annotate 10 new Salmonella phage genomes isolated in Spain in the light of phage therapy. We designed a bioinformatic pipeline using available building blocks to de novo assemble genomes and perform syntaxic annotation. We then used genome-wide analyses for taxonomic annotation enabled by vContact2 and VICTOR. We were also particularly interested in improving functional annotation using remote homologies detection and comparisons with the recently published phage-specific PHROG protein database. Finally, we searched for useful functions for phage therapy, such as systems encoded by the phage to circumvent cellular defenses with a particular focus on anti-CRISPR proteins. We, thus, were able to genetically characterize nine virulent phages and one temperate phage and identify putative functions relevant to the formulation of phage cocktails for  biocontrol.

David R. Olivenza

Departamento de Genética,
Facultad de Biología,
Departamento de Genética, Facultad de Biología

Universidad de Sevilla

Bacteriophage Removal from Infected Salmonella Cultures

David R. Olivenza

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Departamento de Genética,Facultad de Biología,Departamento de Genética, Facultad de Biología

Universidad de Sevilla

Bacteriophage Removal from Infected Salmonella Cultures

Departamento de Genética,
Facultad de Biología,
Departamento de Genética, Facultad de Biología