Two-component regulatory systems (TCSs) are a major mechanism used by bacteria to sense and respond to their environments. Many of the same TCSs are used by biologically diverse organisms with different regulatory needs, suggesting that the functions of TCS must evolve. To explore this topic, we analysed the amino acid sequence divergence patterns of a large set of broadly conserved TCS across different branches of Enterobacteriaceae, a family of Gram-negative bacteria that includes biomedically important genera such as Salmonella, Escherichia, Klebsiella and others. Our analysis revealed trends in how TCS sequences change across different proteins or functional domains of the TCS, and across different lineages. Based on these trends, we identified individual TCS that exhibit atypical evolutionary patterns. We observed that the relative extent to which the sequence of a given TCS varies across different lineages is generally well conserved, unveiling a hierarchy of TCS sequence conservation with EnvZ/OmpR as the most conserved TCS. We provide evidence that, for the most divergent of the TCS analysed, PmrA/PmrB, different alleles were horizontally acquired by different branches of this family, and that different PmrA/PmrB sequence variants have highly divergent signal-sensing domains. Collectively, this study sheds light on how TCS evolve, and serves as a compendium for how the sequences of the TCS in this family have diverged over the course of evolution.

ABSTRACT AB 5 -type toxins are a diverse family of protein toxins composed of an enzymatic active (A) subunit and a pentameric delivery (B) subunit. Salmonella enterica serovar Typhi’s typhoid toxin features two A subunits, CdtB and PltA, in complex with the B subunit PltB. Recently, it was shown that S . Typhi encodes a horizontally acquired B subunit, PltC, that also assembles with PltA/CdtB to produce a second form of typhoid toxin. S . Typhi therefore produces two AB 5 toxins with the same A subunits but distinct B subunits, an evolutionary twist that is unique to typhoid toxin. Here, we show that, remarkably, the Salmonella bongori species independently evolved an analogous capacity to produce two typhoid toxins with distinct B subunits. S. bongori ’s alternate B subunit, PltD, is evolutionarily distant from both PltB and PltC and outcompetes PltB to form the predominant toxin. We show that, surprisingly, S. bongori elicits similar levels of CdtB-mediated intoxication as S . Typhi during infection of cultured human epithelial cells. This toxicity is exclusively due to the PltB toxin, and strains lacking pltD produce increased amounts of PltB toxin and exhibit increased toxicity compared to the wild type, suggesting that the acquisition of the PltD subunit potentially made S. bongori less virulent toward humans. Collectively, this study unveils a striking example of convergent evolution that highlights the importance of the poorly understood “two-toxin” paradigm for typhoid toxin biology and, more broadly, illustrates how the flexibility of A-B interactions has fueled the evolutionary diversification and expansion of AB 5 -type toxins. IMPORTANCE Typhoid toxin is an important Salmonella Typhi virulence factor and an attractive target for therapeutic interventions to combat typhoid fever. The recent discovery of a second version of this toxin has substantial implications for understanding S . Typhi pathogenesis and combating typhoid fever. In this study, we discover that a remarkably similar two-toxin paradigm evolved independently in Salmonella bongori , which strongly suggests that this is a critical aspect of typhoid toxin biology. We observe significant parallels between how the two toxins assemble and their capacity to intoxicate host cells during infection in S . Typhi and S. bongori , which provides clues to the biological significance of this unusual toxin arrangement. More broadly, AB5 toxins with diverse activities and mechanisms are essential virulence factors for numerous important bacterial pathogens. This study illustrates the capacity for novel A-B interactions to evolve and thus provides insight into how such a diverse arsenal of toxins might have emerged.

The virulence properties of numerous important bacterial pathogens have been shaped by the horizontal acquisition of an AB5-type toxin. A major obstacle to the fruitful horizontal transfer of an AB5-type toxin amongst bacteria that infect divergent host species is that these toxins bind specific glycan receptors on target host cells to mediate toxin uptake, and glycosylation patterns vary substantially between different host species. In this study, we explore the evolutionary and functional connections between the related typhoid toxins that are encoded by S. enterica serovar Typhi, a human-adapted pathogen, and by S. bongori , which infects cold-blooded animals. We uncover genetic fingerprints that indicate S . Typhi’s typhoid toxin islet evolved from a larger virulence islet similar to S. bongori ’s, and show that S . Typhi’s glycan-binding PltB subunit is more similar to S. bongori PltB than it is to many diverse PltB sequence variants found in S. enterica . We further show that, surprisingly, S . Typhi strains that encode the S. bongori typhoid toxin islet intoxicate infected human epithelial cells with substantially greater potency than wild-type S . Typhi. The results of this study therefore suggest that the unexpected potency of the S. bongori typhoid toxin toward mammalian cells paved the way for sequence variants derived from this species to be acquired by - and effectively integrated into the virulence program of - the human-adapted pathogen S . Typhi. ### Competing Interest Statement The authors have declared no competing interest.

S. Typhi, the cause of typhoid fever, is a bacterial pathogen of substantial global importance. Typhoid toxin is a secreted AB-type toxin that is a key S. Typhi virulence factor encoded within a 5-gene genetic islet. Four genes in this islet have well-defined roles in typhoid toxin biology, however the function of the fifth gene is unknown. Here, we investigate the function of this gene, which we name ttaP. We show that ttaP is co-transcribed with the typhoid toxin subunit cdtB, and we perform genomic analyses that indicate that TtaP is very highly conserved in typhoid toxin islets found in diverse salmonellae. We show that TtaP is a distant homolog of group XIV secreted phospholipase A2 (PLA2) enzymes, and experimentally demonstrate that TtaP is a bona fide PLA2. Sequence and structural analyses indicate that TtaP differs substantially from characterized PLA2s, and thus represents a novel class of PLA2. Secretion assays revealed that TtaP is neither co-secreted with typhoid toxin, nor is it required for toxin secretion. Although TtaP is a phospholipase that remains associated with the S. Typhi cell, assays that probed for altered cell envelope integrity failed to identify any differences between wild-type S. Typhi and a ttaP deletion strain. Collectively, this study identifies a biochemical activity for the lone uncharacterized typhoid toxin islet gene and lays the groundwork for exploring how this gene factors into S. Typhi pathogenesis. This study further identifies a novel class of PLA2, enzymes that have a wide range of industrial applications.

AB5-type toxins are a group of secreted protein toxins that are central virulence factors for bacterial pathogens such as Shigella dysenteriae, Vibrio cholerae, Bordetella pertussis, and certain lineages of pathogenic Escherichia coli and Salmonella enterica. AB5 toxins are composed of an active (A) subunit that manipulates host cell biology in complex with a pentameric binding/delivery (B) subunit that mediates the toxin's entry into host cells and its subsequent intracellular trafficking. Broadly speaking, all known AB5-type toxins adopt similar structural architectures and employ similar mechanisms of binding, entering and trafficking within host cells. Despite this, there is a remarkable amount of diversity amongst AB5-type toxins; this includes different toxin families with unrelated activities, as well as variation within families that can have profound functional consequences. In this review, we discuss the diversity that exists amongst characterized AB5-type toxins, with an emphasis on the genetic and functional variability within AB5 toxin families, how this may have evolved, and its impact on human disease.

Typhoid toxin is an A2B5 protein toxin and an important virulence factor for the human-adapted bacterial pathogen Salmonella enterica serovar Typhi, the causative agent of typhoid fever. Typhoid toxin contains two enzymatic subunits, PltA and CdtB, which dock onto a pentameric delivery platform composed of the protein PltB. It was recently reported that the same enzymatic subunits can assemble with a different delivery platform composed of the protein PltC, forming a distinct version of typhoid toxin. However, the differences in structure and receptor specificity between the PltC and PltB typhoid toxins remain unknown. Here, we determined atomic-level structures of the pentameric PltC subunit, the fully assembled PltC typhoid toxin, and the PltC pentamers in complex with glycan receptors. Biochemical and structural analyses indicate that PltB and PltC are unable to form heteromeric delivery complexes due to electrostatic repulsion at the subunit interface and thus form separate toxins only. We further observed that, despite low sequence similarity between PltB and PltC, they interact with PltA in a similar manner but that PltC exhibits stronger electrostatic interactions with PltA, enabling it to outcompete PltB in toxin assembly. The ligand-bound atomic structures of PltC show an additional glycan binding site not found in PltB and glycan array analysis indicates that PltB and PltC exhibit significant differences in glycan binding specificity. Collectively, this study offers atomic-level insights into how S. Typhi produces two distinct versions of typhoid toxin, thereby generating functional diversity in this key virulence factor. IMPORTANCE Typhoid fever is a devastating disease that kills more than 115,000 people every year and is caused by Salmonella Typhi. Typhoid toxin, exclusively produced by S. Typhi, was demonstrated to be responsible for the pathogenesis of typhoid fever. Typhoid toxin consists of a pentameric delivery B subunit to transport the catalytic A subunits into the host cell through binding of the glycan receptors. Recent study shows that S. Typhi encodes two homologous delivery B subunits that are able to associate with the same active subunits to produce alternative toxins with distinct functional characteristics. Here, we show that the two delivery subunits can form only homopentameric delivery platforms that compete to associate with typhoid toxin's active subunits and that the two resulting toxins have distinct glycan-binding properties that confer distinct functional traits. These findings highlight the unique assembly and functional diversification of typhoid toxins.

Salmonella enterica is a diverse species of bacterial pathogens comprised of >2,500 serovars with variable host ranges and virulence properties. Accumulating evidence indicates that two AB5-type toxins, typhoid toxin and ArtAB toxin, contribute to the more severe virulence properties of the Salmonella strains that encode them. It was recently discovered that there are two distinct types of artAB-like genetic elements in Salmonella: those that encode ArtAB toxins (artAB elements) and those in which the artA gene is degraded and the ArtB homolog, dubbed PltC, serves as an alternative delivery subunit for typhoid toxin (pltC elements). Here, we take a multifaceted approach to explore the evolutionary diversification of artAB-like genetic elements in Salmonella. We identify 7 subtypes of ArtAB toxins and 4 different PltC sequence groups that are distributed throughout the Salmonella genus. Both artAB and pltC are encoded within numerous diverse prophages, indicating a central role for phages in their evolutionary diversification. Genetic and structural analyses revealed features that distinguish pltC elements from artAB and identified evolutionary adaptations that enable PltC to efficiently engage typhoid toxin A subunits. For both pltC and artAB, we find that the sequences of the B subunits are especially variable, particularly amongst amino acid residues that fine tune the chemical environment of their glycan binding pockets. This study provides a framework to delineate the remarkably complex collection of Salmonella artAB/pltC-like genetic elements and provides a window into the mechanisms of evolution for AB5-type toxins.

Typhoid fever is a major global health problem and is the result of systemic infections caused by the human-adapted bacterial pathogen Salmonella enterica serovar Typhi (S. Typhi). The pathology underlying S. Typhi infections significantly differ from infections caused by broad host range serovars of the same species, which are a common cause of gastroenteritis. Accordingly, identifying S. Typhi genetic factors that impart functionality absent from broad host range serovars offers insights into its unique biology. Here, we used an in-silico approach to explore the function of an uncharacterized 14-gene S. Typhi genomic islet. Our results indicated that this islet was specific to the S. enterica species, where it was encoded by the Typhi and Paratyphi A serovars, but was generally absent from non-typhoidal serovars. Evidence was gathered using comparative genomics and sequence analysis tools, and indicated that this islet was comprised of Type VI secretion system (T6SS) and contact-dependent growth inhibition (CDI) genes, the majority of which appeared to encode orphan immunity proteins that protected against the activities of effectors and toxins absent from the S. Typhi genome. We herein propose that this islet represents an immune system that protects S. Typhi against competing bacteria within the human gut.

Bacterial toxins with an AB 5 architecture consist of an active (A) subunit inserted into a ring-like platform comprised of five delivery (B) subunits. Salmonella Typhi, the cause of typhoid fever, produces an unusual A 2 B 5 toxin known as typhoid toxin. Here, we report that upon infection of human cells, S . Typhi produces two forms of typhoid toxin that have distinct delivery components but share common active subunits. The two typhoid toxins exhibit different trafficking properties, elicit different effects when administered to laboratory animals, and are expressed using different regulatory mechanisms and in response to distinct metabolic cues. Collectively, these results indicate that the evolution of two typhoid toxin variants has conferred functional versatility to this virulence factor. More broadly, this study reveals a new paradigm in toxin biology and suggests that the evolutionary expansion of AB 5 toxins was likely fueled by the plasticity inherent to their structural design coupled to the functional versatility afforded by the combination of homologous toxin components.

Salmonella Typhi is the cause of typhoid fever, a major global health concern. An essential virulence factor of this pathogen is typhoid toxin. In contrast to most AB-type toxins, typhoid toxin is exclusively expressed by intracellular bacteria. The regulatory networks that ensure this unique gene expression pattern are unknown. Here, we developed FAST-INSeq, a genome-wide screening approach to identify S. Typhi genes required for typhoid toxin expression within infected cells. We find that typhoid toxin expression is controlled by a silencing and counter-silencing mechanism through the opposing actions of the PhoP/PhoQ two-component regulatory system and the histone-like protein H-NS. The screen also identified bacterial mutants that alter the proportion of intracellular S. Typhi that reside within an intravacuolar environment, which was essential for toxin expression. Collectively, these data describe a regulatory mechanism that allows a bacterial pathogen to exclusively express a virulence factor when located within a specific intracellular compartment.

Typhoid toxin is a unique A2B5 exotoxin and an important virulence factor for Salmonella Typhi, the cause of typhoid fever. In the decade since its initial discovery, great strides have been made in deciphering the unusual biological program of this toxin, which is fundamentally different from related toxins in many ways. Purified typhoid toxin administered to laboratory animals causes many of the symptoms of typhoid fever, suggesting that typhoid toxin is a central factor in this disease. Further advances in understanding the biology of this toxin will help guide the development of badly needed diagnostics and therapeutic interventions that target this toxin to detect, prevent or treat typhoid fever.

A riboswitch is an RNA element that detects the level of a specific metabolite within the cell and regulates the expression of co-transcribed genes. By fusing a riboswitch to a reporter protein in a carefully designed and tested construct, this ability can be exploited to create an intracellular sensor that detects the level of a particular small molecule within live bacterial cells. There is a great deal of flexibility in the design of such a sensor and factors such as the molecule to be detected and the downstream experiments in which the sensor will be applied should guide the specific blueprint of the final construct. The completed sensor plasmid needs to be rigorously tested with appropriate controls to ensure that its dynamic range, signal strength, sensitivity and specificity are suitable for its intended applications. In this chapter, methods for the design, assessment and use of riboswitch sensors are provided along with those for one example application for which riboswitch sensors are ideally suited.

Riboswitches have a number of characteristics that make them ideal regulatory elements for a wide range of synthetic biology applications. To maximize their utility, methods are required to create custom riboswitches de novo or to modify existing riboswitches to suit specific experimental needs. This chapter describes such a method, which exploits fluorescence-activated cell sorting (FACS) to quickly and efficiently sort through large libraries of riboswitch-like sequences to identify those with the desired activity. Suggestions for the experimental setup are provided, along with detailed protocols for testing and optimizing FACS conditions FACS selection steps, and follow-up assays to identify and characterize individual riboswitches.

Many bacteria contain an ortholog of the Ro autoantigen, a ring-shaped protein that binds noncoding RNAs (ncRNAs) called Y RNAs. In the only studied bacterium, Deinococcus radiodurans, the Ro ortholog Rsr functions in heat-stress-induced ribosomal RNA (rRNA) maturation and starvation-induced rRNA decay. However, the mechanism by which this conserved protein and its associated ncRNAs act has been obscure. We report that Rsr and the exoribonuclease polynucleotide phosphorylase (PNPase) form an RNA degradation machine that is scaffolded by Y RNA. Single-particle electron microscopy, followed by docking of atomic models into the reconstruction, suggests that Rsr channels single-stranded RNA into the PNPase cavity. Biochemical assays reveal that Rsr and Y RNA adapt PNPase for effective degradation of structured RNAs. A Ro ortholog and ncRNA also associate with PNPase in Salmonella Typhimurium. Our studies identify another ribonucleoprotein machine and demonstrate that ncRNA, by tethering a protein cofactor, can alter the substrate specificity of an enzyme.

The study of biological transporters can be hampered by a dearth of methodology for tracking their activity within cells. Here, we present a means of monitoring the function of transport machinery within bacteria, exploiting a genetically encoded riboswitch-based sensor to detect the accumulation of the substrate in the cytoplasm. This method was used to investigate the model ABC transporter BtuC2D2F, which permits vitamin B12 uptake in Escherichia coli. We exploited the wealth of structural data available for this transporter to probe the functional and mechanistic importance of key residues of the substrate binding protein BtuF that are predicted to support its interaction with its substrate or with the BtuC channel-forming subunits. Our results reveal molecular interaction requirements for substrate binding proteins and demonstrate the utility of riboswitch-based sensors in the study of biological transport.

Small molecules play crucial roles in every major cellular process. Despite this, detecting their levels within cells remains a significant challenge. Here, we describe intracellular sensors of coenzyme B-12 that make use of the exquisite molecular detection capabilities of a naturally occurring riboswitch. These probes sensitively detect their target using colorimetric, fluorescent, or luminescent reporters. To assess their utility in the study of biological systems, the sensors were applied to examine the synthesis and the import of coenzyme B-12. The sensors were able to monitor the effects of genetic deletions, recombinant expression of foreign genes, and varied growth conditions on both of these processes. These results indicate that riboswitch-based sensors can provide valuable information on intracellular small molecule concentrations that can be employed in the study of related cellular processes.

Recently, aptamers began to emerge as therapeutics. In this issue of Chemistry & Biology, Famulok and colleagues [1] present a twist on this concept, using an enzyme-inhibiting aptamer as a tool to screen for small molecules that block HIV replication.

UV light induces the expression of a wide variety of genes. At present, it is unclear how cells sense the extent of DNA damage and alter the expression of UV-induced genes appropriately. UV light induces DNA damage that blocks transcription, and the probability that a gene sustains transcription-blocking DNA damage is proportional to locus size and dose of UV light. Using colon carcinoma cells that express a temperature-sensitive variant of p53 and undergo p53-dependent apoptosis after UV irradiation, we found that the number of p53-induced genes identified by oligonucleotide microarray analysis decreased in a UV dose-dependent manner. This was associated with a statistically significant shift in the spectrum of p53-induced genes toward compact genes with fewer and smaller introns. Genes encoding proapoptotic proteins involved in the initiation of the mitochondrial apoptotic cascade were prominent among the compact p53 target genes, whereas genes encoding negative regulators of p53 and the mitochondrial apoptotic pathway were significantly larger. We propose that the shift in spectrum of UV-responsive gene expression caused by passive effects of UV lesions on transcription acts as a molecular dosimeter, ensuring the elimination of cells sustaining irreparable transcription-blocking DNA damage.