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. H-NS:DNA nucleoprotein filaments inhibit transcription initiation by RNA polymerase. However, the discovery that genes silenced by H-NS can be activated by transcription originating from neighboring regions has suggested that elongating RNA polymerases can disassemble H-NS:DNA filaments. In this study, we present evidence that transcription-induced counter-silencing does not require transcription to reach the silenced gene; rather, it exerts its effect at a distance. Counter-silencing is suppressed by introducing a DNA gyrase binding site within the intervening segment, suggesting that the long-range effect results from transcription-driven positive DNA supercoils diffusing toward the silenced gene. We propose a model wherein H-NS:DNA complexes form in vivo on negatively supercoiled DNA, with H-NS bridging the two arms of the plectoneme. Rotational diffusion of positive supercoils generated by neighboring transcription will cause the H-NS-bound negatively-supercoiled plectoneme to "unroll" disrupting the H-NS bridges and releasing H-NS.

Analysis of Salmonella lineage-specific traits upon cell sorting.

Microbial cell individuality is receiving increasing interest in the scientific community. Individual cells within clonal populations exhibit noticeable phenotypic heterogeneity. The advent of fluorescent protein technology and advances in single-cell analysis has revealed phenotypic cell variant in bacterial populations. This heterogeneity is evident in a wide range of phenotypes, for example, individual cells display variable degrees of gene expression and survival under selective conditions and stresses, and can exhibit differing propensities to host interactions. Last few years, numerous cell sorting approaches have been employed for resolving the properties of bacterial subpopulations. This review provides an overview of applications of cell sorting to analyze Salmonella lineage-specific traits, including bacterial evolution studies, gene expression analysis, response to diverse cellular stresses and characterization of diverse bacterial phenotypic variants.

Pulmonary barotrauma in SCUBA diving-related fatalities: a histological and histomorphometric analysis.

Arterial gas embolism following pulmonary barotrauma occurs in 13-24% of cases of diving deaths. The study aimed to evaluate the usefulness of a histomorphometric digital analysis in the detection of air space over-distension due to pulmonary barotrauma. The study was performed on lung parenchyma specimens of 12 divers: six had died due to arterial gas embolism following pulmonary barotrauma (mean age at death of 54 years, range of 41-61 years), and six had drowned in saltwater without a diagnosis of pulmonary barotrauma (mean age at death of 54 years, range of 41-66 years) (positive controls). For negative controls, six cases of non-SCUBA divers (mean age of death of 42 years, range of 23-55 years) who died of intracerebral haemorrhage were evaluated. No significant differences were observed in the characteristics of the air spaces between control groups (positive and negative). However, differences were observed in the area occupied by air spaces and the percentage of air space area when we compared the case group to the controls (p < 0.01); and there was a slight difference in the maximum and minimum diameters of air space (p < 0.05). The mean area occupied by air spaces and the mean percentage of air space were the most useful for discriminating pulmonary barotrauma from other causes of death (100% sensitivity and 91.7% specificity). Based on our study, inclusion of an increased pattern of air spaces as a possible diagnostic criterion for pulmonary barotrauma would be useful in discerning the cause of diving death.

DNA Methylation in Prokaryotes.

The genomes of bacteria, archaea, and phage contain small amounts of C5-methylcytosine, N4-methylcytosine, and N6-methyladenine. Base methylation takes place after DNA replication and is catalyzed by DNA methyltransferases that recognize specific target sequences. Prokaryotic DNA methyltransferases can be classified into two main types: (1) belonging to restriction-modification systems and (2) solitary (or "orphan") enzymes that lack a restriction enzyme partner. All known roles of DNA methylation involve control of interactions between DNA-binding proteins and their cognate sites. Such roles include protection from DNA restriction, strand discrimination during mismatch repair, cell cycle control, and regulation of transcription. DNA methylation often affects the interaction of bacterial pathogens with their hosts, raising the possibility of epigenetic therapies for infectious diseases.

Genetic Mining of Newly Isolated Salmophages for Phage Therapy.

Salmonella enterica, 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 S. enterica 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 Salmonella biocontrol.

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

In Escherichia coli and Salmonella, 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 Salmonella pathogenicity island 1 (SPI-1) in cells depleted of Rho-cofactor NusG. Evidence from genetic experiments, semiquantitative 5' rapid amplification of complementary DNA ends sequencing (5' RACE-Seq), and chromatin immunoprecipitation sequencing (ChIP-Seq) shows that transcription originating from spurious antisense promoters, when not stopped by Rho, elongates into a H-NS-bound regulatory region of SPI-1, displacing H-NS and rendering the DNA accessible to the master regulator HilD. In turn, HilD's ability to activate its own transcription triggers a positive feedback loop that results in transcriptional activation of the entire SPI-1. Significantly, single-cell analyses revealed that this mechanism is largely responsible for the coexistence of two subpopulations of cells that either express or do not express SPI-1 genes. We propose that cell-to-cell differences produced by stochastic spurious transcription, combined with feedback loops that perpetuate the activated state, can generate bimodal gene expression patterns in bacterial populations.

Waddington's Landscapes in the Bacterial World.

Conrad Waddington's epigenetic landscape, a visual metaphor for the development of multicellular organisms, is appropriate to depict the formation of phenotypic variants of bacterial cells. Examples of bacterial differentiation that result in morphological change have been known for decades. In addition, bacterial populations contain phenotypic cell variants that lack morphological change, and the advent of fluorescent protein technology and single-cell analysis has unveiled scores of examples. Cell-specific gene expression patterns can have a random origin or arise as a programmed event. When phenotypic cell-to-cell differences are heritable, bacterial lineages are formed. The mechanisms that transmit epigenetic states to daughter cells can have strikingly different levels of complexity, from the propagation of simple feedback loops to the formation of complex DNA methylation patterns. Game theory predicts that phenotypic heterogeneity can facilitate bacterial adaptation to hostile or unpredictable environments, serving either as a division of labor or as a bet hedging that anticipates future challenges. Experimental observation confirms the existence of both types of strategies in the bacterial world.

Genome-Wide Identification and Expression Analysis of SOS Response Genes in Salmonella enterica Serovar Typhimurium.

A bioinformatic search for LexA boxes, combined with transcriptomic detection of loci responsive to DNA damage, identified 48 members of the SOS regulon in the genome of Salmonella enterica serovar Typhimurium. Single cell analysis using fluorescent fusions revealed that heterogeneous expression is a common trait of SOS response genes, with formation of SOSOFF and SOSON subpopulations. Phenotypic cell variants formed in the absence of external DNA damage show gene expression patterns that are mainly determined by the position and the heterology index of the LexA box. SOS induction upon DNA damage produces SOSOFF and SOSON subpopulations that contain live and dead cells. The nature and concentration of the DNA damaging agent and the time of exposure are major factors that influence the population structure upon SOS induction. An analogy can thus be drawn between the SOS response and other bacterial stress responses that produce phenotypic cell variants.

Evidence for Involvement of the Salmonella enterica Z-Ring Assembly Factors ZapA and ZapB in Resistance to Bile.

Genes annotated as ygfE and yiiU in the genome of Salmonella enterica serovar Typhimurium encode proteins homologous to Escherichia coli cell division factors ZapA and ZapB, respectively. ZapA- and ZapB- mutants of S. enterica are bile-sensitive. The amount of zapB mRNA increases in the presence of a sublethal concentration of sodium deoxycholate (DOC) while zapA mRNA remains unaffected. Increased zapB mRNA level in the presence of DOC is not caused by upregulation of zapB transcription but by increased stability of zapB mRNA. This increase is suppressed by an hfq mutation, suggesting the involvement of a small regulatory RNA. We provide evidence that such sRNA is MicA. The ZapB protein is degraded in the presence of DOC, and degradation appears to involve the Lon protease. We propose that increased stability of zapB mRNA in the presence of DOC may counter degradation of bile-damaged ZapB, thereby providing sufficient level of functional ZapB protein to permit Z-ring assembly in the presence of bile.

Muertes en el buceo: un modelo de investigación multidisciplinar / Diving deaths: A multidisciplinary research model

El buceo es una de las actividades subacuáticas más practicadas en el litoral español. Los sistemas más comunes utilizados son el buceo con escafandra autónoma con circuito abierto, cerrado o semicerrado. Aunque hoy en día el buceo con escafandra autónoma se considera una práctica segura, cada año se describen nuevas muertes relacionadas con esta actividad. El ahogamiento es la principal causa de muerte, pero se han descrito otras, como la embolia gaseosa arterial, la enfermedad por descompresión, la enfermedad natural asociada y los traumatismos. Las autopsias de muertes relacionadas con el buceo son un gran desafío para cualquier patólogo forense, y es aconsejable poseer conocimientos sobre fisiopatología del buceo y experiencia en la práctica de técnicas de autopsia específicas. Colaborar con un equipo multidisciplinar proporciona información, evidencias y hallazgos patológicos suficientes para una investigación altamente calificada de los casos y poder resolver las principales cuestiones médico-legales

Diving is one of the most practiced underwater activities on the Spanish coast. The most common methods used in diving activities are SCUBA (self-contained underwater breathing apparatus), CCR (closed circuit rebreather) and SCR (semi-closed circuit rebreather). Although recreational diving is safe overall, diving accidents are potentially serious and even fatal. Despite all the precautions taken by divers, fatalities related to this activity are reported every year. Drowning is the main cause of death, but others have been described, such as arterial gas embolism, decompression sickness, natural pathology, and trauma. Autopsies of SCUBA diving-related deaths are a big challenge for any forensic pathologist. It is advisable to have some knowledge about diving physiopathology and experience in the practice of special autopsy techniques. Collaboration with a multidisciplinary team provides enough information, evidence and pathological findings for highly-qualified investigation of the incidents to be able to prevent similar incidents in the future

Redox controls RecA protein activity via reversible oxidation of its methionine residues.

Reactive oxygen species (ROS) cause damage to DNA and proteins. Here, we report that the RecA recombinase is itself oxidized by ROS. Genetic and biochemical analyses revealed that oxidation of RecA altered its DNA repair and DNA recombination activities. Mass spectrometry analysis showed that exposure to ROS converted four out of nine Met residues of RecA to methionine sulfoxide. Mimicking oxidation of Met35 by changing it for Gln caused complete loss of function, whereas mimicking oxidation of Met164 resulted in constitutive SOS activation and loss of recombination activity. Yet, all ROS-induced alterations of RecA activity were suppressed by methionine sulfoxide reductases MsrA and MsrB. These findings indicate that under oxidative stress MsrA/B is needed for RecA homeostasis control. The implication is that, besides damaging DNA structure directly, ROS prevent repair of DNA damage by hampering RecA activity.

Single Cell Analysis of Bistable Expression of Pathogenicity Island 1 and the Flagellar Regulon in Salmonella enterica.

Bistable expression of the Salmonella enterica pathogenicity island 1 (SPI-1) and the flagellar network (Flag) has been described previously. In this study, simultaneous monitoring of OFF and ON states in SPI-1 and in the flagellar regulon reveals independent switching, with concomitant formation of four subpopulations: SPI-1OFF FlagOFF, SPI-1OFF FlagON, SPI-1ON FlagOFF, and SPI-1ON FlagON. Invasion assays upon cell sorting show that none of the four subpopulations is highly invasive, thus raising the possibility that FlagOFF cells might contribute to optimal invasion as previously proposed for SPI-1OFF cells. Time lapse microscopy observation indicates that expression of the flagellar regulon contributes to the growth impairment previously described in SPI-1ON cells. As a consequence, growth resumption in SPI-1ON FlagON cells requires switching to both SPI-1OFF and FlagOFF states.

Copy Number Heterogeneity in the Virulence Plasmid of Salmonella enterica.

Quantitative PCR analysis shows that the virulence plasmid of Salmonella enterica serovar Typhimurium (pSLT) is a low-copy-number plasmid, with 1-2 copies per chromosome. However, fluorescence microscopy observation of pSLT labeled with a lacO fluorescent tag reveals cell-to-cell differences in the number of foci, which ranges from 1 to 8. As each focus must correspond to ≥1 plasmid copy, the number of foci can be expected to indicate the minimal number of pSLT copies per cell. A correlation is found between the number of foci and the bacterial cell volume. In contrast, heterogeneity in the number of loci appears to be independent of the cell volume and may have stochastic origin. As a consequence of copy number heterogeneity, expression of a pSLT-bone reporter gene shows high levels of cell-to-cell variation, especially in actively dividing cultures. These observations support the notion that low-copy-number plasmids can be a source of gene expression noise in bacterial populations.

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

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.

Epigenetic biosensors for bacteriophage detection and phage receptor discrimination.

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 OpvABON 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 OpvABON 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.

Corrigendum: Copy Number Heterogeneity in the Virulence Plasmid of Salmonella enterica.

[This corrects the article DOI: 10.3389/fmicb.2020.599931.].

The bacterial epigenome.

In all domains of life, genomes contain epigenetic information superimposed over the nucleotide sequence. Epigenetic signals control DNA-protein interactions and can cause phenotypic change in the absence of mutation. A nearly universal mechanism of epigenetic signalling is DNA methylation. In bacteria, DNA methylation has roles in genome defence, chromosome replication and segregation, nucleoid organization, cell cycle control, DNA repair and regulation of transcription. In many bacterial species, DNA methylation controls reversible switching (phase variation) of gene expression, a phenomenon that generates phenotypic cell variants. The formation of epigenetic lineages enables the adaptation of bacterial populations to harsh or changing environments and modulates the interaction of pathogens with their eukaryotic hosts.

Publisher Correction: 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.