Evolution of a bistable genetic system in fluctuating and nonfluctuating environments.

Epigenetic mechanisms can generate bacterial lineages capable of spontaneously switching between distinct phenotypes. Currently, mathematical models and simulations propose epigenetic switches as a mechanism of adaptation to deal with fluctuating environments. However, bacterial evolution experiments for testing these predictions are lacking. Here, we exploit an epigenetic switch in Salmonella enterica, the opvAB operon, to show clear evidence that OpvAB bistability persists in changing environments but not in stable conditions. Epigenetic control of transcription in the opvAB operon produces OpvABOFF (phage-sensitive) and OpvABON (phage-resistant) cells in a reversible manner and may be interpreted as an example of bet-hedging to preadapt Salmonella populations to the encounter with phages. Our experimental observations and computational simulations illustrate the adaptive value of epigenetic variation as an evolutionary strategy for mutation avoidance in fluctuating environments. In addition, our study provides experimental support to game theory models predicting that phenotypic heterogeneity is advantageous in changing and unpredictable environments.

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.

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.

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.

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.

Std fimbriae-fucose interaction increases Salmonella-induced intestinal inflammation and prolongs colonization.

Expression of ABO and Lewis histo-blood group antigens by the gastrointestinal epithelium is governed by an α-1,2-fucosyltransferase enzyme encoded by the Fut2 gene. Alterations in mucin glycosylation have been associated with susceptibility to various bacterial and viral infections. Salmonella enterica serovar Typhimurium is a food-borne pathogen and a major cause of gastroenteritis. In order to determine the role of Fut2-dependent glycans in Salmonella-triggered intestinal inflammation, Fut2+/+ and Fut2-/- mice were orally infected with S. Typhimurium and bacterial colonization and intestinal inflammation were analyzed. Bacterial load in the intestine of Fut2-/- mice was significantly lower compared to Fut2+/+ mice. Analysis of histopathological changes revealed significantly lower levels of intestinal inflammation in Fut2-/- mice compared to Fut2+/+ mice and measurement of lipocalin-2 level in feces corroborated histopathological findings. Salmonella express fimbriae that assist in adherence of bacteria to host cells thereby facilitating their invasion. The std fimbrial operon of S. Typhimurium encodes the π-class Std fimbriae which bind terminal α(1,2)-fucose residues. An isogenic mutant of S. Typhimurium lacking Std fimbriae colonized Fut2+/+ and Fut2-/- mice to similar levels and resulted in similar intestinal inflammation. In vitro adhesion assays revealed that bacteria possessing Std fimbriae adhered significantly more to fucosylated cell lines or primary epithelial cells in comparison to cells lacking α(1,2)-fucose. Overall, these results indicate that Salmonella-triggered intestinal inflammation and colonization are dependent on Std-fucose interaction.

Regulation of bistability in the std fimbrial operon of Salmonella enterica by DNA adenine methylation and transcription factors HdfR, StdE and StdF.

Bistable expression of the Salmonella enterica std operon is controlled by an AND logic gate involving three transcriptional activators: the LysR-type factor HdfR and the StdE and StdF regulators encoded by the std operon itself. StdE activates transcription of the hdfR gene, and StdF activates std transcription together with HdfR. Binding of HdfR upstream of the std promoter is hindered by methylation of GATC sites located within the upstream activating sequence (UAS). Epigenetic control by Dam methylation thus antagonizes formation of the StdE-StdF-HdfR loop and tilts the std switch toward the StdOFF state. In turn, HdfR binding hinders methylation of the UAS, permitting activation of the StdE-StdF-HdfR loop and concomitant formation of StdON cells. Bistability is thus the outcome of competition between DNA adenine methylation and the StdE-StdF-HdfR activator loop.

Formation of phenotypic lineages in Salmonella enterica by a pleiotropic fimbrial switch.

The std locus of Salmonella enterica, an operon acquired by horizontal transfer, encodes fimbriae that permit adhesion to epithelial cells in the large intestine. Expression of the std operon is bistable, yielding a major subpopulation of StdOFF cells (99.7%) and a minor subpopulation of StdON cells (0.3%). In addition to fimbrial proteins, the std operon encodes two proteins, StdE and StdF, that have DNA binding capacity and control transcription of loci involved in flagellar synthesis, chemotaxis, virulence, conjugal transfer, biofilm formation, and other cellular functions. As a consequence of StdEF pleiotropic transcriptional control, StdON and StdOFF subpopulations may differ not only in the presence or absence of Std fimbriae but also in additional phenotypic traits. Separation of StdOFF and StdON lineages by cell sorting confirms the occurrence of lineage-specific features. Formation of StdOFF and StdON lineages may thus be viewed as a rudimentary bacterial differentiation program.

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.

Transcriptional regulation of the Salmonella enterica std fimbrial operon by the RcsCDB system.

In Salmonella enterica serovar Typhimurium, the RcsCDB regulatory system controls the expression of genes involved in synthesis of colanic acid, formation of flagella and virulence. Here, we show that activation of the RcsCDB system downregulates expression of std, an operon that encodes fimbriae involved in Salmonella attachment to the mucus layer in the large intestine. Bioinformatic analysis predicts the existence of an RcsB-binding site located 180 bp upstream to the +1 transcription start site of the std promoter, and electrophoretic mobility shift assays confirm that RcsB binds the std promoter region in vitro. This study adds RcsB to the list of regulators of std transcription and provides an example of modulation of fimbriae synthesis by a signal transduction system.

Diving-related fatalities: multidisciplinary, experience-based investigation.

To describe the technical characteristics of fatal diving mishaps and to elucidate the causes of death using a sequence analysis and a multidisciplinary investigation of diving-related fatalities. All cases of diving deaths recorded on the coast of Girona (Spain) between January 2009 and May 2018 were analyzed. Most data were obtained from the police technical reports and the forensic pathology service. Each accident was analyzed in order to identify the trigger, disabling agent, disabling injury, and cause of death. During the study period 25 diving-related fatalities were recorded. Most of the victims were males aged 50-69 years, and 11 were experienced divers. Almost all victims were using open-circuit SCUBA to breathe with compressed air as their sole gas supply. None of the victims were diving alone. The most common identified triggers included exertion, panic, buoyancy problems, disorientation and confusion. The main factors identified as disabling agents were rapid ascent, a cardiac incident, panic and entrapment. Asphyxia, lung over expansion, and myocardial ischemia were the most frequent disabling injuries. Finally, drowning represented the main cause of death, followed by arterial gas embolism and natural causes or internal diseases. A differential diagnosis, performed in the setting of a multidisciplinary investigation, is essential for elucidating the cause of death in diving-related fatalities. The proposed sequence analysis allows to clarify underlying problems in these cases and to identify risk factors and unsafe behaviors in diving.

The UbiK protein is an accessory factor necessary for bacterial ubiquinone (UQ) biosynthesis and forms a complex with the UQ biogenesis factor UbiJ.

Ubiquinone (UQ), also referred to as coenzyme Q, is a widespread lipophilic molecule in both prokaryotes and eukaryotes in which it primarily acts as an electron carrier. Eleven proteins are known to participate in UQ biosynthesis in Escherichia coli, and we recently demonstrated that UQ biosynthesis requires additional, nonenzymatic factors, some of which are still unknown. Here, we report on the identification of a bacterial gene, yqiC, which is required for efficient UQ biosynthesis, and which we have renamed ubiK Using several methods, we demonstrated that the UbiK protein forms a complex with the C-terminal part of UbiJ, another UQ biogenesis factor we previously identified. We found that both proteins are likely to contribute to global UQ biosynthesis rather than to a specific biosynthetic step, because both ubiK and ubiJ mutants accumulated octaprenylphenol, an early intermediate of the UQ biosynthetic pathway. Interestingly, we found that both proteins are dispensable for UQ biosynthesis under anaerobiosis, even though they were expressed in the absence of oxygen. We also provide evidence that the UbiK-UbiJ complex interacts with palmitoleic acid, a major lipid in E. coli Last, in Salmonella enterica, ubiK was required for proliferation in macrophages and virulence in mice. We conclude that although the role of the UbiK-UbiJ complex remains unknown, our results support the hypothesis that UbiK is an accessory factor of Ubi enzymes and facilitates UQ biosynthesis by acting as an assembly factor, a targeting factor, or both.

Adaptation of Salmonella enterica to bile: essential role of AcrAB-mediated efflux.

Adaptation to bile is the ability to endure the lethal effects of bile salts after growth on sublethal concentrations. Surveys of adaptation to bile in Salmonella enterica ser. Tyhimurium reveal that active efflux is essential for adaptation while other bacterial functions involved in bile resistance are not. Among S. enterica mutants lacking one or more efflux systems, only strains lacking AcrAB are unable to adapt, thus revealing an essential role for AcrAB. Transcription of the acrAB operon is upregulated in the presence of a sublethal concentration of sodium deoxycholate (DOC) while other efflux loci are either weakly upregulated or irresponsive. Upregulation of acrAB transcription is strong during exponential growth, and weak in stationary cultures. Single cell analysis of ethidium bromide accumulation indicates that DOC-induced AcrAB-mediated efflux occurs in both exponential and stationary cultures. Upregulation of acrAB expression may thus be crucial at early stages of adaptation, while sustained AcrAB activity may be sufficient to confer bile resistance in nondividing cells.

OxyR-dependent formation of DNA methylation patterns in OpvABOFF and OpvABON cell lineages of Salmonella enterica.

Phase variation of the Salmonella enterica opvAB operon generates a bacterial lineage with standard lipopolysaccharide structure (OpvAB(OFF)) and a lineage with shorter O-antigen chains (OpvAB(ON)). Regulation of OpvAB lineage formation is transcriptional, and is controlled by the LysR-type factor OxyR and by DNA adenine methylation. The opvAB regulatory region contains four sites for OxyR binding (OBSA-D), and four methylatable GATC motifs (GATC1-4). OpvAB(OFF) and OpvAB(ON) cell lineages display opposite DNA methylation patterns in the opvAB regulatory region: (i) in the OpvAB(OFF) state, GATC1 and GATC3 are non-methylated, whereas GATC2 and GATC4 are methylated; (ii) in the OpvAB(ON) state, GATC2 and GATC4 are non-methylated, whereas GATC1 and GATC3 are methylated. We provide evidence that such DNA methylation patterns are generated by OxyR binding. The higher stability of the OpvAB(OFF) lineage may be caused by binding of OxyR to sites that are identical to the consensus (OBSA and OBSc), while the sites bound by OxyR in OpvAB(ON) cells (OBSB and OBSD) are not. In support of this view, amelioration of either OBSB or OBSD locks the system in the ON state. We also show that the GATC-binding protein SeqA and the nucleoid protein HU are ancillary factors in opvAB control.

Small RNA-based feedforward loop with AND-gate logic regulates extrachromosomal DNA transfer in Salmonella.

Horizontal gene transfer via plasmid conjugation is a major driving force in microbial evolution but constitutes a complex process that requires synchronization with the physiological state of the host bacteria. Although several host transcription factors are known to regulate plasmid-borne transfer genes, RNA-based regulatory circuits for host-plasmid communication remain unknown. We describe a posttranscriptional mechanism whereby the Hfq-dependent small RNA, RprA, inhibits transfer of pSLT, the virulence plasmid of Salmonella enterica. RprA employs two separate seed-pairing domains to activate the mRNAs of both the sigma-factor σ(S) and the RicI protein, a previously uncharacterized membrane protein here shown to inhibit conjugation. Transcription of ricI requires σ(S) and, together, RprA and σ(S) orchestrate a coherent feedforward loop with AND-gate logic to tightly control the activation of RicI synthesis. RicI interacts with the conjugation apparatus protein TraV and limits plasmid transfer under membrane-damaging conditions. To our knowledge, this study reports the first small RNA-controlled feedforward loop relying on posttranscriptional activation of two independent targets and an unexpected role of the conserved RprA small RNA in controlling extrachromosomal DNA transfer.

Epigenetic Control of Salmonella enterica O-Antigen Chain Length: A Tradeoff between Virulence and Bacteriophage Resistance.

The Salmonella enterica opvAB operon is a horizontally-acquired locus that undergoes phase variation under Dam methylation control. The OpvA and OpvB proteins form intertwining ribbons in the inner membrane. Synthesis of OpvA and OpvB alters lipopolysaccharide O-antigen chain length and confers resistance to bacteriophages 9NA (Siphoviridae), Det7 (Myoviridae), and P22 (Podoviridae). These phages use the O-antigen as receptor. Because opvAB undergoes phase variation, S. enterica cultures contain subpopulations of opvABOFF and opvABON cells. In the presence of a bacteriophage that uses the O-antigen as receptor, the opvABOFF subpopulation is killed and the opvABON subpopulation is selected. Acquisition of phage resistance by phase variation of O-antigen chain length requires a payoff: opvAB expression reduces Salmonella virulence. However, phase variation permits resuscitation of the opvABOFF subpopulation as soon as phage challenge ceases. Phenotypic heterogeneity generated by opvAB phase variation thus preadapts Salmonella to survive phage challenge with a fitness cost that is transient only.

Diagnosis of arterial gas embolism in SCUBA diving: modification suggestion of autopsy techniques and experience in eight cases.

The purpose of this study was to suggest modifications of autopsy techniques in order to improve post-mortem diagnosis of arterial gas embolism (AGE) based on multidisciplinary investigation of SCUBA diving fatalities. Five adult human cadavers from the voluntary donation program of the Human Anatomy Laboratory, and eight judicial autopsied bodies of SCUBA divers from the Forensic Pathology Service were assessed. Before performing any autopsies, we accessed the diving plan and the divers' profiles for each case. We then introduced a new dissection procedure that included identification, isolation, and manipulation of carotid, vertebral and thoracic arterial systems. The dissected vascular structures that allowed optimall isolation of the systemic arterial circulation were identified and ligated. In three of the eight judicial cases, we had a strongly suggestive history of arterial gas embolism following pulmonary barotrauma (PBt/AGE). In these cases, the additional arterial dissection allowed us to clearly diagnose AGE in one of them. The autopsy of the rest of the cases showed other causes of death such as asphyxia by drowning and heart attack. In all cases we were able to reject decompression sickness, and in some of them we showed the presence of artefacts secondary to decomposition and resuscitation maneuvers. These results allow us to suggest a specific autopsy technique divided into four steps, aimed at confirming or excluding some evidence of dysbaric disorders according to a re-enactment of the incident. We have demonstrated the presence of large volumes of intravascular air, which is typical of PBt/AGE.

Contribution of phenotypic heterogeneity to adaptive antibiotic resistance.

Antibiotic-resistant isolates of Salmonella enterica were selected on plates containing lethal concentrations of rifampicin, kanamycin, and nalidixic acid. The stability of the resistance phenotype was scored after nonselective growth. Rifampicin-resistant (Rif(r)) isolates were stable, suggesting that they had arisen by mutation. Mutations in the rpoB gene were detected indeed in Rif(r) mutants. In contrast, a fraction of kanamycin-resistant (Km(r)) and nalidixic acid-resistant (Nal(r)) isolates showed reduced resistance after nonselective growth, suggesting that mechanisms other than mutation had contributed to bacterial survival upon lethal selection. Single-cell analysis revealed heterogeneity in expression of the porin gene ompC, and subpopulation separation provided evidence that reduced ompC expression confers adaptive resistance to kanamycin. In the case of Nal(r) isolates, mutations in the gyrA gene were present in most nalidixic acid-resistant isolates. However, the efflux pump inhibitor Phe-Arg-ß-naphtylamide (PAßN) reduced the level of resistance in Nal(r) mutants, indicating that active efflux contributes to the overall level of nalidixic acid resistance. Heterogeneous efflux pump activity was detected in single cells and colonies, and a correlation between high efflux and increased resistance to nalidixic acid was found. These observations suggest that fluctuations in the expression and the activity of critical functions of the bacterial cell, alone or combined with mutations, can contribute to adaptive resistance to antibiotics.

Pseudomonas syringae Differentiates into Phenotypically Distinct Subpopulations During Colonization of a Plant Host.

Bacterial microcolonies with heterogeneous sizes are formed during colonization of Phaseolus vulgaris by Pseudomonas syringae. Heterogeneous expression of structural and regulatory components of the P. syringae type III secretion system (T3SS), essential for colonization of the host apoplast and disease development, is likewise detected within the plant apoplast. T3SS expression is bistable in the homogeneous environment of nutrient-limited T3SS-inducing medium, suggesting that subpopulation formation is not a response to different environmental cues. T3SS bistability is reversible, indicating a non-genetic origin, and the T3SSHIGH and T3SSLOW subpopulations show differences in virulence. T3SS bistability requires the transcriptional activator HrpL, the double negative regulatory loop established by HrpV and HrpG, and may be enhanced through a positive feedback loop involving HrpA, the main component of the T3SS pilus. To our knowledge, this is the first example of phenotypic heterogeneity in the expression of virulence determinants during colonization of a non-mammalian host.