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The stringent response is not required for HipA-mediated growth inhibition, and no HipA is sufficient to activate the stringent response in exponential phase cultures. (a) Growth curves of ΔspoT ectopically expressing each indicated HipA toxin performed as in Fig. 1a. (b) The indicated strains in exponential phase in M2G were subcultured to OD 660 = 0.02 in M2G supplemented with IPTG and theophylline to express HipA toxins. As a positive control, the wild-type NA1000 was subcultured in M2 medium lacking the carbon source to induce the stringent response. RNA was harvested after 3 hours of HipA expression or carbon starvation, and the indicated genes were subjected to RT-PCR analysis as described in Methods. Expression relative to an unstarved M2G control is reported, and values are the mean and standard deviation of three biological replicates, each used in two technical replicates.
Source publication
Antibiotic persistence is a transient phenotypic state during which a bacterium can withstand otherwise lethal antibiotic exposure or environmental stresses. In Escherichia coli, persistence is promoted by the HipBA toxin-antitoxin system. The HipA toxin functions as a serine/threonine kinase that inhibits cell growth, while the HipB antitoxin neut...
Contexts in source publication
Context 1
... three HipA toxins maintain the conserved active site residues of serine/threonine kinases ( Supplementary Fig. S2) 34 . We generated an aspartate-to-glutamine (D-Q) substitution in the active site of each HipA, modeled on a corresponding substitution used to create a kinase-dead E. coli HipA protein 35 . ...
Context 2
... the transcription of three genes that are highly upregulated and two genes that are significantly downregulated during the stringent response 39 . In contrast to incubation in minimal medium lacking a carbon source, the expression of individual HipA toxins did not produce the transcriptional changes characteristic of stringent response activation (Fig. 2b). However, RT-PCR measures bulk transcriptional changes occurring across the entire cell population, and stringent response activation in a small fraction of the cells (e.g., 10 −6 to 10 −3 ) would not be detected by this ...
Context 3
... further that not all phosphorylated substrates are important for HipA-mediated persistence. the stringent response is required for the accumulation of persister cells in stationary phase. Although ectopic HipA expression in exponential phase cells did not activate the stringent response and was not required for HipA-induced growth arrest (Fig. 2), the stringent response is important for persister cell formation in some bacteria 45 . We therefore asked if the (p)ppGpp synthase/hydrolase SpoT affects persistence Supplementary Fig. S7) or carbon starvation 38,46 . Biphasic killing was observed in stationary phase cultures of a ΔspoT strain grown in PYE medium when challenged with ...
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5-Aminolevulinic acid (5-ALA) is a precursor for the biosynthesis of tetrapyrrole compounds such as chlorophyll a, heme, and vitamin B12, which has been widely used in the fields of agriculture and medicine. 5-ALA can be biosynthesized through C4 pathway that use succinyl-CoA and glycine as precursor, or through C5 pathway that use glutamate as pre...
Citations
... The TA system, widely spreading in bacterial genomes, comprises a gene pair encoding a toxin and its corresponding antitoxin [52]. It is crucial in various biological processes, especially stress response and antimicrobial persistence [53]. Unexpectedly, no TA system was found in all strains of Cupriavidus (Table. ...
Background: Cupriavidusspecies have significant potential for environmental and industrial applications. Some have been recognized as opportunistic pathogens and are increasingly reported to be related to human infections. However, the taxonomic classification of Cupriavidus at the species level remains challenging. This may partly limit the understanding of pathogenesis in Cupriavidus.Here, we aim to clarify the Cupriavidus taxonomy and elucidate virulence-related genetic profiles using comparative genomic analysis.
Results:This study used eight Cupriavidus species to investigate the taxonomy of Cupriavidus. Of these, C. alkaliphilus, C. basilensis, C. gilardii, C. oxalaticus, C. necator, and C. taiwanensis are species complex, and the first four species are first reported as the species complex. More importantly, C. alkaliphilus and part of C. taiwanensis mixed, suggesting that some strains of these two species were probably mislabeled. The open pan-genome and prevalence of mobile genetic elements indicated high genetic variation in Cupriavidus. A total of 47 genes related to virulence factors were identified in Cupriavidus. Virulence factors in almost all Cupriavidus strains were related to the antimicrobial activity (acrB), biofilm (adeG, algU), stress survival (clpP, katA, sodB, ureA, ureB, and ureG), adherence (htpB,kdsA) and others (icl); the opportunistic pathogens, C. gilardii, and C. metallidurans, contained extra virulence genes (plc-2). Furthermore, six types of secretion systems (T1SS-T6SS) were identified in Cupriavidus. T2SS was conserved across all eight species; the other secretion systems presented diverse distribution. Interestingly, C. gilardii possesses two different T3SS clusters. Finally, emre, responsible for the efflux pump of aminoglycoside antibiotics, is a major antibiotic resistance gene of Cupriavidus strains. Other genes related to aminoglycosides, β-lactam, fosfomycin, and multidrug resistance are species-specific among opportunistic pathogens individually.
Conclusions: This study for the first time investigated the taxonomic classification ofCupriavidus andvirulence-related genetic profiles by comparative genomic analysis. Findings from this study lead us to propose updating the taxonomy of the six identified Cupriavidusspecies containing species complex. The virulence-related information, including virulence factors, secretion systems, and antibiotic resistance genes, provides insights into understanding the pathogenesis of Cupriavidus and may facilitate the diagnosis of infections caused by pathogenic Cupriavidus spp.
... The mechanisms underpinning persistence are still heavily debated, but persistence has been linked to a lack of growth and to toxin-antitoxin (TA) modules [11,18]. It has been proposed that TA pairs induce a state of dormancy and increase tolerance, and several TA systems have been linked to persistence [18][19][20]. However, some bacteria appear to form persisters dependent on ATP levels or metabolic genes instead of TA modules [21]. ...
Biofilms are responsible for most chronic infections and are highly resistant to antibiotic treatments. Previous studies have demonstrated that periodic dosing of antibiotics can help sensitize persistent subpopulations and reduce the overall dosage required for treatment. Because the dynamics and mechanisms of biofilm growth and the formation of persister cells are diverse and are affected by environmental conditions, it remains a challenge to design optimal periodic dosing regimens. Here, we develop a computational agent-based model to streamline this process and determine key parameters for effective treatment. We used our model to test a broad range of persistence switching dynamics and found that if periodic antibiotic dosing was tuned to biofilm dynamics, the dose required for effective treatment could be reduced by nearly 77%. The biofilm architecture and its response to antibiotics were found to depend on the dynamics of persister cells. Despite some differences in the response of biofilm governed by different persister switching rates, we found that a general optimized periodic treatment was still effective in significantly reducing the required antibiotic dose. As persistence becomes better quantified and understood, our model has the potential to act as a foundation for more effective strategies to target bacterial infections.
... It was previously reported that T6SS and HipA might contribute fitness advantages to the AHPND-causing V. parahaemolyticus over competing bacteria and in this way facilitating shrimp infection [53,54]. T6SS systems are complex systems that inject so-called 'anti-bacterial' and 'anti-eukaryotic' effector proteins into target cells, targeting both, eukaryotic hosts and bacterial competitors [55,56], while the serine/threonine protein kinase HipA is a toxin that causes inhibition of cell growth [57]. In contrast to previous reports, our results did not reveal functions related to antibiotic transport and galactose metabolic process associated with Group 1 [29]. ...
Background
Environmental monitoring of bacterial pathogens is critical for disease control in coastal marine ecosystems to maintain animal welfare and ecosystem function and to prevent significant economic losses. This requires accurate taxonomic identification of environmental bacterial pathogens, which often cannot be achieved by commonly used genetic markers (e.g., 16S rRNA gene), and an understanding of their pathogenic potential based on the information encoded in their genomes. The decreasing costs of whole genome sequencing (WGS), combined with newly developed bioinformatics tools, now make it possible to unravel the full potential of environmental pathogens, beyond traditional microbiological approaches. However, obtaining a high-quality bacterial genome, requires initial cultivation in an axenic culture, which is a bottleneck in environmental microbiology due to cross-contamination in the laboratory or isolation of non-axenic strains.
Results
We applied WGS to determine the pathogenic potential of two Vibrio isolates from coastal seawater. During the analysis, we identified cross-contamination of one of the isolates and decided to use this dataset to evaluate the possibility of bioinformatic contaminant removal and recovery of bacterial genomes from a contaminated culture. Despite the contamination, using an appropriate bioinformatics workflow, we were able to obtain high quality and highly identical genomes (Average Nucleotide Identity value 99.98%) of one of the Vibrio isolates from both the axenic and the contaminated culture. Using the assembled genome, we were able to determine that this isolate belongs to a sub-lineage of Vibrio campbellii associated with several diseases in marine organisms. We also found that the genome of the isolate contains a novel Vibrio plasmid associated with bacterial defense mechanisms and horizontal gene transfer, which may offer a competitive advantage to this putative pathogen.
Conclusions
Our study shows that, using state-of-the-art bioinformatics tools and a sufficient sequencing effort, it is possible to obtain high quality genomes of the bacteria of interest and perform in-depth genomic analyses even in the case of a contaminated culture. With the new isolate and its complete genome, we are providing new insights into the genomic characteristics and functional potential of this sub-lineage of V. campbellii. The approach described here also highlights the possibility of recovering complete bacterial genomes in the case of non-axenic cultures or obligatory co-cultures.
... We called this phenomenon "persistence" because it shares the essential attribute of antibiotic persistence (39): that bacteria can temporarily survive the insult without a specific mechanism of resistance. Just as toxin/antitoxin-mediated antibiotic persis tence (40) is not a specific defense against antibiotics (but rather a mechanism for stochastic dormancy), capsule-mediated bacteriophage persistence is not a specific defense against bacteriophage but a response to mechanical touch that may serve Research Article mBio other purposes as well. For example, the Rcs pathway promotes persistent Salmonella infections (41), which could rely on the mechanical sensitivity of this pathway in the gut. ...
Although the relationship between bacteria and lytic bacteriophage is fundamentally antagonistic, these microbes not only coexist but thrive side by side in myriad ecological environments. The mechanisms by which coexistence is achieved, however, are not fully understood. By examining Escherichia coli and bacteriophage T7 population dynamics at the single-cell and single-virion level using a novel microfluidics assay, we observed bacteria growing “persistently” when perfused with high-titer bacteriophage. Bacteriophage persistence occurred at a frequency five orders of magnitude higher than is expected from the natural selection of bacteriophage-resistant mutants. Rather, the frequency of persistence was correlated with the degree to which the bacteria were mechanically compressed by the microfluidic perfusion chamber. Using a combination of mutagenesis and fluorescent imaging techniques, we discovered that compression induces persistence by activating the Rcs phosphorelay pathway, which results in the synthesis of extracellular capsule that sterically blocks bacteriophage adsorption. Other forms of mechanical perturbation also promoted Rcs activity and persistence. These findings have important implications for our understanding of microbial ecology in many important environments, including the gut and the soil, where bacteria grow in confinement.
IMPORTANCE
Bacteria and bacteriophage form one of the most important predator-prey relationships on earth, yet how the long-term stability of this ecological interaction is achieved is unclear. Here, we demonstrate that Escherichia coli can rapidly grow during bacteriophage predation if they are doing so in spatially confined environments. This discovery revises our understanding of bacteria-bacteriophage population dynamics in many real-world environments where bacteria grow in confinement, such as the gut and the soil. Additionally, this result has clear implications for the potential of bacteriophage therapy and the role of mechanosensation during bacterial pathogenesis.
... HipT Ec was previously shown to phosphorylate tryptophan tRNA-ligase (TrpS) in order to arrest cellular growth (13), whereas the canonical target of HipA Ec is glutamyl tRNA-synthetase (GltX) (46,47). TrpS and lysine tRNA-ligase have also been reported as substrates for the HipBA systems in Caulobacter crescentus (15,48). In E. coli, co-expression of TrpS or GltX with HipT Ec or HipA Ec , respectively, rescues the growth inhibitory effect of the toxins. ...
Toxin-antitoxin (TA) systems are abundant genetic modules in bacterial chromosomes and on mobile elements. They are often patchily distributed, as a consequence of horizontal exchange, and the breadth of their functionality in bacterial physiology remains unknown. Here, we characterize a TA system in Legionella pneumophila that is highly conserved across Legionella species. This system is distantly related to Escherichia coli HipBST, and we demonstrate that it is a functional tripartite TA system (denoted HipBST Lp ). We identify HipBST Lp homologs in diverse taxa, yet in the Gammaproteobacteria, these are almost exclusively found in Legionella species. Notably, the toxin HipT Lp was previously reported to be a pathogenic effector protein that is translocated by L. pneumophila into its eukaryotic hosts. Contrary to this, we find no signal of HipT Lp translocation beyond untranslocated control levels and make several observations consistent with a canonical role as a bacterial toxin. We present structural and biochemical insights into the regulation and neutralization of HipBST Lp , and identify key variations between this system and HipBST Ec . Finally, we show that the target of HipT Lp is likely not conserved with any characterized HipA or HipT toxin. This work serves as a useful comparison of a TA system across bacterial species and illustrates the molecular diversity that exists within a single TA family.
IMPORTANCE
Toxin-antitoxin (TA) systems are parasitic genetic elements found in almost all bacterial genomes. They are exchanged horizontally between cells and are typically poorly conserved across closely related strains and species. Here, we report the characterization of a tripartite TA system in the bacterial pathogen Legionella pneumophila that is highly conserved across Legionella species genomes. This system (denoted HipBST Lp ) is a distant homolog of the recently discovered split-HipA system in Escherichia coli (HipBST Ec ). We present bioinformatic, molecular, and structural analyses of the divergence between these two systems and the functionality of this newly described TA system family. Furthermore, we provide evidence to refute previous claims that the toxin in this system (HipT Lp ) possesses bifunctionality as an L. pneumophila virulence protein. Overall, this work expands our understanding of the split-HipA system architecture and illustrates the potential for undiscovered biology in these abundant genetic elements.
... Additionally, hipBA, but not relBE, vapBC or mazEF, was suggested to rely on SR, albeit the conclusion cannot be generalized to antibiotics other than ampicillin and ciprofloxacin [31,32]. Notably, in Caulobacter crescentus, although the SR regulator SpoT is required for HipA-mediated persistence in the stationary phase, persister cells can arise in the absence of hipBA or SpoT [33], highlighting the possibility of different persister-producing routes in C. cresentus. ...
... MqsR overexpression induces persistence probably without (p)ppGpp, and while ΔmqsR or ΔmqsRA shows reduced persistence, ΔmqsR's phenotype varies with inoculum age [30,32,33]. Interestingly, ΔmqsR's phenotype was observed in E. coli K12 BW25113 but not MG1655 [38,39], suggesting the potential impact of genotype on persistence. ...
Bacteria withstand antibiotic onslaughts by employing a variety of strategies, one of which is persistence. Persistence occurs in a bacterial population where a subpopulation of cells (persisters) survives antibiotic treatment and can regrow in a drug-free environment. Persisters may cause the recalcitrance of infectious diseases and can be a stepping stone to antibiotic resistance, so understanding persistence mechanisms is critical for therapeutic applications. However, current understanding of persistence is pervaded by paradoxes that stymie research progress, and many aspects of this cellular state remain elusive. In this review, we summarize the putative persister mechanisms, including toxin–antitoxin modules, quorum sensing, indole signalling and epigenetics, as well as the reasons behind the inconsistent body of evidence. We highlight present limitations in the field and underscore a clinical context that is frequently neglected, in the hope of supporting future researchers in examining clinically important persister mechanisms.
... No reuse allowed without permission. Caulobacter crescentus (62,63), however the primary targets of these systems remain uncertain. In E. ...
Toxin-antitoxin (TA) systems are abundant genetic modules in bacterial chromosomes and on mobile elements. They are often patchily distributed and their physiological functions remain poorly understood. Here, we characterize a TA system in Legionella pneumophila that is highly conserved across Legionella species. This system is distantly related to Escherichia coli HipBST and we demonstrate that it is a functional tripartite TA system (denoted HipBST Lp ). We identify HipBST Lp homologs in diverse taxa, yet in the Gammaproteobacteria these are almost exclusively found in Legionella species. Notably, the toxin HipT Lp was previously reported to be a pathogenic effector protein that is translocated by L. pneumophila into its eukaryotic hosts. Contrary to this, we find no signal of HipT Lp translocation beyond untranslocated control levels and make several observations consistent with a canonical role as a bacterial toxin. We present structural and biochemical insights into the regulation and neutralization of HipBST Lp , and identify key variations between this system and HipBST Ec . Finally, we show that the target of HipT Lp is likely not conserved with any characterized HipA or HipT toxin. This work serves as a unique comparison of a TA system across bacterial species and illustrates the molecular diversity that exists within a single TA family.
... B. ambifaria T16 contains TA modules (ParE/RelE, HipAB and VapBC) which possess different molecular activities. The RelE superfamily (RelE,YefM/YoeB) are ribosome-dependent mRNA endonucleases, whereas HipA functions as a serine/threonine kinase that phosphorylate different substrates (Huang et al. 2020). Moreover, VapC ribonucleases cleave tRNA and rRNA at specific positions, inhibiting protein synthesis or differentially suppressing gene expression (Harms et al. 2018). ...
Burkholderia ambifaria T16 is a bacterium isolated from the rhizosphere of barley plants that showed a remarkable antifungal activity. This strain was also able to degrade fusaric acid (5-Butylpyridine-2-carboxylic acid) and detoxify this mycotoxin in inoculated barley seedlings. Genes and enzymes responsible for fusaric acid degradation have an important biotechnological potential in the control of fungal diseases caused by fusaric acid producers, or in the biodegradation/bio catalysis processes of pyridine derivatives. In this study, the complete genome of B. ambifaria T16 was sequenced and analyzed to identify genes involved in survival and competition in the rhizosphere, plant growth promotion, fungal growth inhibition, and degradation of aromatic compounds. The genomic analysis revealed the presence of several operons for the biosynthesis of antimicrobial compounds, such as pyrrolnitrin, ornibactin, occidiofungin and the membrane-associated AFC-BC11. These compounds were also detected in bacterial culture supernatants by mass spectrometry analysis. In addition, this strain has multiple genes contributing to its plant growth-promoting profile, including those for acetoin, 2,3-butanediol and indole-3-acetic acid production, siderophores biosynthesis, and solubilisation of organic and inorganic phosphate. A pan-genomic analysis demonstrated that the genome of strain T16 possesses large gene clusters that are absent in the genomes of B. ambifaria reference strains. According to predictions, most of these clusters would be involved in aromatic compounds degradation. One genomic region, encoding flavin-dependent monooxygenases of unknown function, is proposed as a candidate responsible for fusaric acid degradation.
... TA system is a pair of genes encoding a toxin and its cognate anti-toxin, and it helps bacteria withstand lethal antibiotic exposure or environmental stresses [63]. We identified seven TA systems in A. faecis and three in A. lanthieri ( Table 5). ...
... Also, hipBA TA systems are present in both species (Table 5). The hipB anti-toxin neutralizes the HipA toxin, a serine/threonine kinase inhibiting cell growth where hipBA modules are found in divergent bacterial genomes, and many are related to the persistence of antibiotic resistance [63]. ...
Background
Emerging pathogenic bacteria are an increasing threat to public health. Two recently described species of the genus Aliarcobacter, A. faecis and A. lanthieri, isolated from human or livestock feces, are closely related to Aliarcobacter zoonotic pathogens (A. cryaerophilus, A. skirrowii, and A. butzleri). In this study, comparative genomics analysis was carried out to examine the virulence-related, including virulence, antibiotic, and toxin (VAT) factors in the reference strains of A. faecis and A. lanthieri that may enable them to become potentially opportunistic zoonotic pathogens.
Results
Our results showed that the genomes of the reference strains of both species have flagella genes (flaA, flaB, flgG, flhA, flhB, fliI, fliP, motA and cheY1) as motility and export apparatus, as well as genes encoding the Twin-arginine translocation (Tat) (tatA, tatB and tatC), type II (pulE and pulF) and III (fliF, fliN and ylqH) secretory pathways, allowing them to secrete proteins into the periplasm and host cells. Invasion and immune evasion genes (ciaB, iamA, mviN, pldA, irgA and fur2) are found in both species, while adherence genes (cadF and cj1349) are only found in A. lanthieri. Acid (clpB), heat (clpA and clpB), osmotic (mviN), and low-iron (irgA and fur2) stress resistance genes were observed in both species, although urease genes were not found in them. In addition, arcB, gyrA and gyrB were found in both species, mutations of which may mediate the resistance to quaternary ammonium compounds (QACs). Furthermore, 11 VAT genes including six virulence (cadF, ciaB, irgA, mviN, pldA, and tlyA), two antibiotic resistance [tet(O) and tet(W)] and three cytolethal distending toxin (cdtA, cdtB, and cdtC) genes were validated with the PCR assays. A. lanthieri tested positive for all 11 VAT genes. By contrast, A. faecis showed positive for ten genes except for cdtB because no PCR assay for this gene was available for this species.
Conclusions
The identification of the virulence, antibiotic-resistance, and toxin genes in the genomes of A. faecis and A. lanthieri reference strains through comparative genomics analysis and PCR assays highlighted the potential zoonotic pathogenicity of these two species. However, it is necessary to extend this study to include more clinical and environmental strains to explore inter-species and strain-level genetic variations in virulence-related genes and assess their potential to be opportunistic pathogens for animals and humans.
... A complication is, however, that TAs are often present in several paralogous copies per genome. Although some cases of cross-neutralization have been reported [7][8][9] , insulation of numerous paralogous TAs, where there is high specificity with no cross-pairing, suggests that a neutralizing cross-interaction is detrimental [10][11][12][13] . In the case of TA systems, evolution of insulation without disruption of a cognate interaction is critical because of the unforgiving nature of neutralization loss. ...
Toxin–antitoxin (TA) systems are a large family of genes implicated in the regulation of bacterial growth and its arrest in response to attacks. These systems encode nonsecreted toxins and antitoxins that specifically pair, even when present in several paralogous copies per genome. Salmonella enterica serovar Typhimurium contains three paralogous TacAT systems that block bacterial translation. We determined the crystal structures of the three TacAT complexes to understand the structural basis of specific TA neutralization and the evolution of such specific pairing. In the present study, we show that alteration of a discrete structural add-on element on the toxin drives specific recognition by their cognate antitoxin underpinning insulation of the three pairs. Similar to other TA families, the region supporting TA-specific pairing is key to neutralization. Our work reveals that additional TA interfaces beside the main neutralization interface increase the safe space for evolution of pairing specificity. Structural characterization of three paralogous toxin–antitoxin complexes illuminates how each antitoxin specifically targets its cognate toxin, including auxiliary neutralization interfaces that confer evolvability while preventing loss of activity.
