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1.
Gene ; 765: 145091, 2021 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-32898604

RESUMO

Sequencing transposon mutant libraries have been pivotal in annotating essential and non-essential genes in bacteria. This is particularly very helpful in the case of Mycobacterium tuberculosis with a large part of its genome without known function. It is not known whether there are any variations in the essentiality states as a function of optimal growth in the absence of any selection pressure. We here grow a high-density mutant library of M. tuberculosis through serial cultures and monitor the temporal fluctuations in insertion frequencies across all TA dinucleotides in the genome. Genes that cause morphological and physiological heterogeneity or enable metabolic bypass were found to gradually lose insertions, while genes comprising the toxin-antitoxin systems were found to get enriched with insertions during growth in nutrient replete conditions. High levels of fluctuations were observed in genes involved in cell wall and cell processes, intermediary metabolism, and genes involved in virulence, suggesting new modes of adaptation undertaken by the mutants. We also report the essentiality status of several newly annotated genetic features.


Assuntos
Elementos de DNA Transponíveis/genética , Genes Essenciais/genética , Mycobacterium tuberculosis/genética , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Mutagênese Insercional/genética , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidade , Sistemas Toxina-Antitoxina/genética , Virulência/genética
2.
Nucleic Acids Res ; 48(19): 11054-11067, 2020 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-33045733

RESUMO

The two-gene module HEPN/MNT is predicted to be the most abundant toxin/antitoxin (TA) system in prokaryotes. However, its physiological function and neutralization mechanism remains obscure. Here, we discovered that the MntA antitoxin (MNT-domain protein) acts as an adenylyltransferase and chemically modifies the HepT toxin (HEPN-domain protein) to block its toxicity as an RNase. Biochemical and structural studies revealed that MntA mediates the transfer of three AMPs to a tyrosine residue next to the RNase domain of HepT in Shewanella oneidensis. Furthermore, in vitro enzymatic assays showed that the three AMPs are transferred to HepT by MntA consecutively with ATP serving as the substrate, and this polyadenylylation is crucial for reducing HepT toxicity. Additionally, the GSX10DXD motif, which is conserved among MntA proteins, is the key active motif for polyadenylylating and neutralizing HepT. Thus, HepT/MntA represents a new type of TA system, and the polyadenylylation-dependent TA neutralization mechanism is prevalent in bacteria and archaea.


Assuntos
Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Shewanella/metabolismo , Sistemas Toxina-Antitoxina
3.
Nucleic Acids Res ; 48(15): 8545-8561, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32735661

RESUMO

A crucial bacterial strategy to avoid killing by antibiotics is to enter a growth arrested state, yet the molecular mechanisms behind this process remain elusive. The conditional overexpression of mazF, the endoribonuclease toxin of the MazEF toxin-antitoxin system in Staphylococcus aureus, is one approach to induce bacterial growth arrest, but its targets remain largely unknown. We used overexpression of mazF and high-throughput sequence analysis following the exact mapping of non-phosphorylated transcriptome ends (nEMOTE) technique to reveal in vivo toxin cleavage sites on a global scale. We obtained a catalogue of MazF cleavage sites and unearthed an extended MazF cleavage specificity that goes beyond the previously reported one. We correlated transcript cleavage and abundance in a global transcriptomic profiling during mazF overexpression. We observed that MazF affects RNA molecules involved in ribosome biogenesis, cell wall synthesis, cell division and RNA turnover and thus deliver a plausible explanation for how mazF overexpression induces stasis. We hypothesize that autoregulation of MazF occurs by directly modulating the MazEF operon, such as the rsbUVW genes that regulate the sigma factor SigB, including an observed cleavage site on the MazF mRNA that would ultimately play a role in entry and exit from bacterial stasis.


Assuntos
Proteínas de Ligação a DNA/genética , Endorribonucleases/genética , Proteínas de Escherichia coli/genética , Staphylococcus aureus/genética , Sistemas Toxina-Antitoxina/genética , Antibacterianos/farmacologia , Proliferação de Células/efeitos dos fármacos , Proteínas de Ligação a DNA/química , Escherichia coli/genética , Humanos , Óperon/genética , RNA Mensageiro/genética , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/genética , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/patogenicidade , Especificidade por Substrato , Transcriptoma/genética
4.
Nucleic Acids Res ; 48(18): 10527-10541, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-32845304

RESUMO

YoeB-YefM, the widespread type II toxin-antitoxin (TA) module, binds to its own promoter to autoregulate its transcription: repress or induce transcription under normal or stress conditions, respectively. It remains unclear how YoeB-YefM regulates its transcription depending on the YoeB to YefM TA ratio. We find that YoeB-YefM complex from S.aureus exists as two distinct oligomeric assemblies: heterotetramer (YoeB-YefM2-YoeB) and heterohexamer (YoeB-YefM2-YefM2-YoeB) with low and high DNA-binding affinities, respectively. Structures of the heterotetramer alone and heterohexamer bound to promoter DNA reveals that YefM C-terminal domain undergoes disorder to order transition upon YoeB binding, which allosterically affects the conformation of N-terminal DNA-binding domain. At TA ratio of 1:2, unsaturated binding of YoeB to the C-terminal regions of YefM dimer forms an optimal heterohexamer for DNA binding, and two YefM dimers with N-terminal domains dock into the adjacent major grooves of DNA to specifically recognize the 5'-TTGTACAN6AGTACAA-3' palindromic sequence, resulting in transcriptional repression. In contrast, at TA ratio of 1:1, binding of two additional YoeB molecules onto the heterohexamer induces the completely ordered conformation of YefM and disassembles the heterohexamer into two heterotetramers, which are unable to bind the promoter DNA optimally due to steric clashes, hence derepresses TA operon transcription.


Assuntos
Proteínas de Bactérias/ultraestrutura , Endorribonucleases/ultraestrutura , Proteínas de Escherichia coli/genética , Staphylococcus aureus/ultraestrutura , Sistemas Toxina-Antitoxina/genética , Antitoxinas/genética , Antitoxinas/ultraestrutura , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Proteínas de Ligação a DNA/genética , Endorribonucleases/química , Endorribonucleases/genética , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/ultraestrutura , Óperon/genética , Regiões Promotoras Genéticas , Ligação Proteica/genética , Multimerização Proteica/genética , Staphylococcus aureus/química , Staphylococcus aureus/genética
5.
Int J Food Microbiol ; 334: 108819, 2020 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-32818765

RESUMO

In a viable but nonculturable (VBNC) state, bacteria are no longer culturable on standard laboratory media, but still, remain a pathogenic potential and present possible health risks. In this study, we investigated ampicillin's ability, which is commonly used in dairy cattle disease treatment, to induce Cronobacter sakazakii into the VBNC state. After treatment with ampicillin, the counts of culturable cells decreased from 108 CFU/mL to an undetected level 7-30 days post-treatment. Meanwhile, viable cells were still approximately 104-105 cells/mL, and could be resuscitated under appropriate conditions. Fluorescence microscopy showed that VBNC cell maintained apparent cellular integrity, but that the morphology of VBNC cells differed visibly from that of normal cells. Moreover, the respiratory chain activity of VBNC cells were confirmed by flow cytometry (FCM) analysis, suggesting that cells in a VBNC state were physiologically active. Finally, transcriptomics analysis and real-time PCR (qPCR) validation were used to explore the underlying mechanisms of VBNC cell formation. Over-expression of relA, lon, ppx, and ppk in the toxin-antitoxin (TA) trigger system contributed to VBNC cell formation. In the TA trigger system, RelA and exopolyphosphatases/guanosine pentaphosphate phosphohydrolases (PPX/GPPA) synthesize ppGpp, which activates polyphosphate kinase (PPK), the cellular enzyme that accumulates plyphosphate (PolyP). PolyP combines with and stimulates Lon to degrade the antitoxins, thereby activating the toxins that induce a VBNC state. The results of our research will facilitate a better understanding of the survival strategies that bacteria develop to deal with ampicillin pressure and the health risks associated with VBNC Cronobacter sakazakii induced by antibiotics.


Assuntos
Ampicilina/farmacologia , Antibacterianos/farmacologia , Cronobacter sakazakii/efeitos dos fármacos , Cronobacter sakazakii/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Contagem de Colônia Microbiana , Cronobacter sakazakii/genética , Cronobacter sakazakii/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Viabilidade Microbiana/efeitos dos fármacos , Sistemas Toxina-Antitoxina/genética
6.
Mol Plant Microbe Interact ; 33(9): 1142-1149, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32720865

RESUMO

High-value pharmaceutical products are already successfully produced in contained facilities using Agrobacterium-mediated transient transformation of plants. However, transfection methods suitable for open field applications are still desirable as a cheaper alternative. Biosafety concerns related to the use of recombinant agrobacteria in an industrial transfection process include possible transformation or transfection of unintended hosts or spread of the genetically modified agrobacteria in the environment. In this paper, we explored a novel biocontrol approach resulting in greater biosafety of the transient expression process in plants. Our proposed solution involves inducible expression of Agrobacterium tumefaciens toxin PemK and antitoxin PemI that provides for strictly regulated T-DNA transfer from agrobacteria to plants. We also identified several other toxins from putative Agrobacterium toxin-antitoxin modules and demonstrate their potential usefulness in the control of Agrobacterium tumefaciens as a DNA vector.


Assuntos
Agrobacterium tumefaciens , DNA Bacteriano/genética , Plantas Geneticamente Modificadas , Sistemas Toxina-Antitoxina , Transformação Genética , Agrobacterium tumefaciens/genética , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Plantas/genética
7.
PLoS One ; 15(6): e0230652, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32603331

RESUMO

Toxin-antitoxin systems (TAS) are commonly found on bacterial plasmids and are generally involved in plasmid maintenance. In addition to plasmid maintenance, several plasmid-mediated TAS are also involved in bacterial stress response and virulence. Even though the same TAS are present in a variety of plasmid types and bacterial species, differences in their sequences, expression and functions are not well defined. Here, we aimed to identify commonly occurring plasmid TAS in Escherichia coli and Klebsiella pneumoniae and compare the sequence, expression and plasmid stability function of their variants. 27 putative type II TAS were identified from 1063 plasmids of Klebsiella pneumoniae in GenBank. Among these, ccdAB and pemIK were found to be most common, also occurring in plasmids of E. coli. Comparisons of ccdAB variants, taken from E. coli and K. pneumoniae, revealed sequence differences, while pemIK variants from IncF and IncL/M plasmids were almost identical. Similarly, the expression and plasmid stability functions of ccdAB variants varied according to the host strain and species, whereas the expression and functions of pemIK variants were consistent among host strains. The specialised functions of some TAS may determine the host specificity and epidemiology of major antibiotic resistance plasmids.


Assuntos
Enterobacteriaceae/genética , Plasmídeos/genética , Sistemas Toxina-Antitoxina/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência Conservada , Farmacorresistência Bacteriana/genética , Enterobacteriaceae/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica
8.
Nucleic Acids Res ; 48(15): 8617-8625, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32597957

RESUMO

Type II toxin-antitoxins systems are widespread in prokaryotic genomes. Typically, they comprise two proteins, a toxin, and an antitoxin, encoded by adjacent genes and forming a complex in which the enzymatic activity of the toxin is inhibited. Under stress conditions, the antitoxin is degraded liberating the active toxin. Though thousands of various toxin-antitoxins pairs have been predicted bioinformatically, only a handful has been thoroughly characterized. Here, we describe the AtaT2 toxin from a toxin-antitoxin system from Escherichia coli O157:H7. We show that AtaT2 is the first GNAT (Gcn5-related N-acetyltransferase) toxin that specifically targets charged glycyl tRNA. In vivo, the AtaT2 activity induces ribosome stalling at all four glycyl codons but does not evoke a stringent response. In vitro, AtaT2 acetylates the aminoacyl moiety of isoaccepting glycyl tRNAs, thus precluding their participation in translation. Our study broadens the known target specificity of GNAT toxins beyond the earlier described isoleucine and formyl methionine tRNAs, and suggest that various GNAT toxins may have evolved to specificaly target other if not all individual aminoacyl tRNAs.


Assuntos
Acetiltransferases/genética , Escherichia coli O157/genética , Glicina-tRNA Ligase/genética , Biossíntese de Proteínas/genética , Antitoxinas/genética , Toxinas Bacterianas/genética , Escherichia coli O157/patogenicidade , Sistemas Toxina-Antitoxina/genética
9.
Mol Cell ; 79(2): 280-292.e8, 2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32533919

RESUMO

Toxin-antitoxin (TA) systems are ubiquitous genetic elements in bacterial genomes, but their functions are controversial. Although they are frequently postulated to regulate cell growth following stress, few null phenotypes for TA systems have been reported. Here, we show that TA transcript levels can increase substantially in response to stress, but toxin is not liberated. We find that the growth of an Escherichia coli strain lacking ten TA systems encoding endoribonuclease toxins is not affected following exposure to six stresses that each trigger TA transcription. Additionally, using RNA sequencing, we find no evidence of mRNA cleavage following stress. Stress-induced transcription arises from antitoxin degradation and relief of transcriptional autoregulation. Importantly, although free antitoxin is readily degraded in vivo, antitoxin bound to toxin is protected from proteolysis, preventing release of active toxin. Thus, transcription is not a reliable marker of TA activity, and TA systems do not strongly promote survival following individual stresses.


Assuntos
Toxinas Bacterianas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Estresse Fisiológico , Sistemas Toxina-Antitoxina , Transcrição Genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/crescimento & desenvolvimento , Plasmídeos/genética , Proteólise , RNA Bacteriano/metabolismo , RNA-Seq , Sistemas Toxina-Antitoxina/genética
10.
Proc Natl Acad Sci U S A ; 117(19): 10500-10510, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32345719

RESUMO

Under stressful conditions, bacterial RelA-SpoT Homolog (RSH) enzymes synthesize the alarmone (p)ppGpp, a nucleotide second messenger. (p)ppGpp rewires bacterial transcription and metabolism to cope with stress, and, at high concentrations, inhibits the process of protein synthesis and bacterial growth to save and redirect resources until conditions improve. Single-domain small alarmone synthetases (SASs) are RSH family members that contain the (p)ppGpp synthesis (SYNTH) domain, but lack the hydrolysis (HD) domain and regulatory C-terminal domains of the long RSHs such as Rel, RelA, and SpoT. We asked whether analysis of the genomic context of SASs can indicate possible functional roles. Indeed, multiple SAS subfamilies are encoded in widespread conserved bicistronic operon architectures that are reminiscent of those typically seen in toxin-antitoxin (TA) operons. We have validated five of these SASs as being toxic (toxSASs), with neutralization by the protein products of six neighboring antitoxin genes. The toxicity of Cellulomonas marina toxSAS FaRel is mediated by the accumulation of alarmones ppGpp and ppApp, and an associated depletion of cellular guanosine triphosphate and adenosine triphosphate pools, and is counteracted by its HD domain-containing antitoxin. Thus, the ToxSAS-antiToxSAS system with its multiple different antitoxins exemplifies how ancient nucleotide-based signaling mechanisms can be repurposed as TA modules during evolution, potentially multiple times independently.


Assuntos
Bactérias/crescimento & desenvolvimento , Guanosina Pentafosfato/metabolismo , Sistemas Toxina-Antitoxina/fisiologia , Nucleotídeos de Adenina/metabolismo , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Bases de Dados Genéticas , Regulação Bacteriana da Expressão Gênica/genética , Guanosina Tetrafosfato/metabolismo , Guanosina Trifosfato/metabolismo , Ligases/metabolismo , Pirofosfatases/metabolismo , Transdução de Sinais , Estresse Fisiológico/fisiologia
11.
Mol Biotechnol ; 62(6-7): 335-343, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32236842

RESUMO

Emergence of multidrug resistant strains and extremely drug resistant strains of Mycobacterium tuberculosis is due to its ability to form persister cells. The formation of persister cells is assumed to be triggered due to the presence of large number of toxin-antitoxin (TA) systems in its genome. Mtb genome encodes 47 VapBC TA systems. In this work, we aim to biochemically characterize VapC46 toxin of the VapBC46 TA operon from Mycobacterium tuberculosis. Heterologous expression of VapC46 in E. coli is shown to exhibit bacteriostasis and toxicity alters the surface morphology of the E. coli cells. VapC46 is shown to possess ribonuclease activity in a magnesium-dependent manner. Using FRET and pull down assay, VapC46 is shown to interact with VapB46 antitoxin. A model of VapC46 is shown to resemble PIN domain family of proteins and reveals the putative active site required for its ribonuclease activity.


Assuntos
Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Mycobacterium tuberculosis/metabolismo , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Genoma Bacteriano/genética , Mycobacterium tuberculosis/genética , Ribonucleases/genética , Ribonucleases/metabolismo , Sistemas Toxina-Antitoxina/genética , Sistemas Toxina-Antitoxina/fisiologia
12.
Nucleic Acids Res ; 48(8): 4357-4370, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32232417

RESUMO

The Klebsiella pneumoniae species complex includes important opportunistic pathogens which have become public health priorities linked to major hospital outbreaks and the recent emergence of multidrug-resistant hypervirulent strains. Bacterial virulence and the spread of multidrug resistance have previously been linked to toxin-antitoxin (TA) systems. TA systems encode a toxin that disrupts essential cellular processes, and a cognate antitoxin which counteracts this activity. Whilst associated with the maintenance of plasmids, they also act in bacterial immunity and antibiotic tolerance. However, the evolutionary dynamics and distribution of TA systems in clinical pathogens are not well understood. Here, we present a comprehensive survey and description of the diversity of TA systems in 259 clinically relevant genomes of K. pneumoniae. We show that TA systems are highly prevalent with a median of 20 loci per strain. Importantly, these toxins differ substantially in their distribution patterns and in their range of cognate antitoxins. Classification along these properties suggests different roles of TA systems and highlights the association and co-evolution of toxins and antitoxins.


Assuntos
Evolução Molecular , Klebsiella pneumoniae/genética , Sistemas Toxina-Antitoxina/genética , Simulação por Computador , Farmacorresistência Bacteriana/genética , Genoma Bacteriano , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/patogenicidade , Fenótipo , Fatores de Virulência/genética
13.
Mol Genet Genomics ; 295(4): 891-909, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32189066

RESUMO

Wolbachia is an obligate intracellular Gram-negative alpha-proteobacterium that has diverse effects on reproduction of arthropod hosts, including cytoplasmic incompatibility, male killing, feminization, and parthenogenesis. Some of these effects have important potential for control of insect pests, including mosquitoes that vector pathogens of humans. In mosquitoes, and in most other arthropods, elimination of Wolbachia by antibiotic treatment has no effect on host survival and reverses the Wolbachia-associated phenotype. Elimination of Wolbachia strain wFol, which enables parthenogenetic reproduction of the Collembolan, Folsomia candida, would result in population extinction. However, F. candida adults remain viable and resume reproduction when antibiotics are removed, suggesting that wFol survives antibiotic treatment in a quiescent persister state similar to that induced by chromosomally encoded toxin-antitoxin (TA) modules in free-living bacteria. Computational approaches were used to document the presence of antitoxin genes upstream of Wolbachia RelE/ParE, Fic, and AbiEii toxin genes. Moreover, this analysis revealed that Wolbachia RatA toxin is encoded by a single copy gene associated with an ssrS noncoding RNA gene. Documentation of potentially functional TA modules expands our understanding of the metabolic capabilities of Wolbachia, and provides an explanation for variable and sometimes contradictory results of antibiotic treatments. The presence of chromosomal TA modules in Wolbachia genomes suggests that wFol, and potentially other strains of Wolbachia, can enter a quiescent persister state.


Assuntos
Partenogênese/genética , Reprodução/genética , Sistemas Toxina-Antitoxina/genética , Wolbachia/genética , Animais , Cromossomos Bacterianos/genética , Culicidae/microbiologia , DNA Topoisomerase IV/genética , Genoma Bacteriano/genética , Humanos , Masculino , Controle de Pragas , Simbiose/genética , Wolbachia/patogenicidade
14.
Appl Microbiol Biotechnol ; 104(7): 3081-3095, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32043192

RESUMO

Here, for the first time, we have investigated the hipBAXn toxin-antitoxin (TA) module from entomopathogenic bacterium Xenorhabdus nematophila. It is a type II TA module that consists of HipAXn toxin and HipBXn antitoxin protein and located in the complementary strand of chromosome under XNC1_operon 0810 locus tag. For functional analysis, hipAXn toxin, hipBXn antitoxin, and an operon having both genes were cloned in pBAD/His C vector and transformed in Escherichia coli cells. The expression profiles and endogenous toxicity assay were performed in these cells. To determine the active amino acid residues responsible for the toxicity of HipAXn toxin, site-directed mutagenesis (SDM) was performed. SDM results showed that amino acid residues S149, D306, and D329 in HipAXn toxin protein were significantly essential for its toxicity. For transcriptional analysis, the 157 bp upstream region of the hipBAXn TA module was identified as a promoter with bioinformatics tools. Further, the LacZ reporter construct with promoter region was prepared and LacZ assays as well as reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was performed under different stress conditions. Electrophoretic mobility shift assay (EMSA) was also performed with recombinant HipAXn toxin, HipBXn antitoxin protein, and 157 bp promoter region. Results showed that the hipBAXn TA module is a well-regulated system in which the upregulation of gene expression was also found compulsive in different SOS conditions. KEY POINTS: •Functional characterization of hipBA Xn TA module from Xenorhabdus nematophila. •hipBA Xn TA module is a functional type II TA module. •Transcriptional characterization of hipBA Xn TA module. •hipBA Xn TA module is a well regulated TA module. Graphical abstract.


Assuntos
Proteínas de Bactérias/fisiologia , Proteínas de Ligação a DNA/fisiologia , Sistemas Toxina-Antitoxina/fisiologia , Xenorhabdus/fisiologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Óperon , Regiões Promotoras Genéticas , Estresse Fisiológico , Sistemas Toxina-Antitoxina/genética , Xenorhabdus/genética
15.
Int J Med Microbiol ; 310(2): 151400, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32001143

RESUMO

A plethora of toxin-antitoxin systems exist in bacteria and has multilateral roles in bacterial pathogenesis and virulence. Toxin-antitoxin systems have been involved in persister cell formation in Escherichia coli and Mycobacterium but have not been reported to be associated with Staphylococcus aureus persistence. Persistence is the ability of bacterial cells to tolerate unfavorable conditions and multiple stresses. There are less known and more unknown factors that either alleviate or aggravate bacterial persistence phenomenon. For the first time, we reported a new chromosomally encoded tripartite toxin-antitoxin system and its role in S. aureus persister cell formation. The toxin gene is bacteriostatic in action and counterbalanced by antitoxin RNA that could basepair with the toxin mRNA and formed a duplex. The transcriptional regulator positively regulates the toxin expression under certain stress conditions. The toxin ectopic induction increased S. aureus susceptibility to norfloxacin, ciprofloxacin, and ofloxacin. Whole-genome RNA sequencing revealed that MDR efflux pump norA is significantly down-regulated by toxin ectopic induction. The deletion of norA from S. aureus genome reduced resistance toward ciprofloxacin, norfloxacin, and ofloxacin, as well as resulted in a decrease in minimal inhibitory concentration while complementation of norA successfully restored the phenotypes. The persistence assay of the norA mutant revealed that deletion of norA increased persister cell survival in S. aureus. Altogether, we have provided insight into the first tripartite type-I TA system and revealed the role of MDR NorA in the persister cell formation of S. aureus.


Assuntos
Proteínas de Bactérias/genética , Farmacorresistência Bacteriana Múltipla/genética , Regulação Bacteriana da Expressão Gênica , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Staphylococcus aureus/genética , Staphylococcus aureus/fisiologia , Sistemas Toxina-Antitoxina/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Testes de Sensibilidade Microbiana , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Sequenciamento Completo do Genoma
16.
Sci Rep ; 10(1): 2865, 2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-32071324

RESUMO

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 neutralizes the toxin. E. coli HipA inactivates the glutamyl-tRNA synthetase GltX, which inhibits translation and triggers the highly conserved stringent response. Although hipBA operons are widespread in bacterial genomes, it is unknown if this mechanism is conserved in other species. Here we describe the functions of three hipBA modules in the alpha-proteobacterium Caulobacter crescentus. The HipA toxins have different effects on growth and macromolecular syntheses, and they phosphorylate distinct substrates. HipA1 and HipA2 contribute to antibiotic persistence during stationary phase by phosphorylating the aminoacyl-tRNA synthetases GltX and TrpS. The stringent response regulator SpoT is required for HipA-mediated antibiotic persistence, but persister cells can form in the absence of all hipBA operons or spoT, indicating that multiple pathways lead to persister cell formation in C. crescentus.


Assuntos
Caulobacter crescentus/genética , Proteínas de Ligação a DNA/genética , Proteínas de Escherichia coli/genética , Sistemas Toxina-Antitoxina/genética , Aminoacil-tRNA Sintetases/genética , Antibacterianos/farmacologia , Caulobacter crescentus/enzimologia , Escherichia coli/genética , Genoma Bacteriano/genética , Glutamato-tRNA Ligase/genética , Óperon/genética , Proteínas Quinases/genética
17.
PLoS Genet ; 16(2): e1008607, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32053596

RESUMO

RHS elements are components of conserved toxin-delivery systems, wide-spread within the bacterial kingdom and some of the most positively selected genes known. However, very little is known about how Rhs toxins affect bacterial biology. Salmonella Typhimurium contains a full-length rhs gene and an adjacent orphan rhs gene, which lacks the conserved delivery part of the Rhs protein. Here we show that, in addition to the conventional delivery, Rhs toxin-antitoxin pairs encode for functional type-II toxin-antitoxin (TA) loci that regulate S. Typhimurium proliferation within macrophages. Mutant S. Typhimurium cells lacking both Rhs toxins proliferate 2-times better within macrophages, mainly because of an increased growth rate. Thus, in addition to providing strong positive selection for the rhs loci under conditions when there is little or no toxin delivery, internal expression of the toxin-antitoxin system regulates growth in the stressful environment found inside macrophages.


Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Macrófagos/microbiologia , Salmonella typhimurium/genética , Sistemas Toxina-Antitoxina/genética , Animais , Proteínas de Bactérias/metabolismo , Loci Gênicos/genética , Camundongos , Mutação , Fases de Leitura Aberta/genética , Regiões Promotoras Genéticas/genética , Células RAW 264.7 , Salmonella typhimurium/crescimento & desenvolvimento , Salmonella typhimurium/patogenicidade , Seleção Genética
18.
PLoS One ; 15(1): e0217255, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31931516

RESUMO

Natural competence allows bacteria to respond to environmental and nutritional cues by taking up free DNA from their surroundings, thus gaining both nutrients and genetic information. In the Gram-negative bacterium Haemophilus influenzae, the genes needed for DNA uptake are induced by the CRP and Sxy transcription factors in response to lack of preferred carbon sources and nucleotide precursors. Here we show that one of these genes, HI0659, encodes the antitoxin of a competence-regulated toxin-antitoxin operon ('toxTA'), likely acquired by horizontal gene transfer from a Streptococcus species. Deletion of the putative toxin (HI0660) restores uptake to the antitoxin mutant. The full toxTA operon was present in only 17 of the 181 strains we examined; complete deletion was seen in 22 strains and deletions removing parts of the toxin gene in 142 others. In addition to the expected Sxy- and CRP-dependent-competence promoter, HI0659/660 transcript analysis using RNA-seq identified an internal antitoxin-repressed promoter whose transcription starts within toxT and will yield nonfunctional protein. We propose that the most likely effect of unopposed toxin expression is non-specific cleavage of mRNAs and arrest or death of competent cells in the culture. Although the high frequency of toxT and toxTA deletions suggests that this competence-regulated toxin-antitoxin system may be mildly deleterious, it could also facilitate downregulation of protein synthesis and recycling of nucleotides under starvation conditions. Although our analyses were focused on the effects of toxTA, the RNA-seq dataset will be a useful resource for further investigations into competence regulation.


Assuntos
DNA/genética , Haemophilus influenzae/genética , Streptococcus/genética , Sistemas Toxina-Antitoxina/genética , Fatores de Transcrição/genética , Antitoxinas/genética , DNA/metabolismo , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , Transferência Genética Horizontal/genética , Óperon/genética , Regiões Promotoras Genéticas , Biossíntese de Proteínas/genética , RNA-Seq , Transativadores/genética , Transformação Bacteriana/genética
19.
J Bacteriol ; 202(7)2020 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-31932311

RESUMO

Type II toxin-antitoxin (TA) systems are small genetic elements composed of a toxic protein and its cognate antitoxin protein, the latter counteracting the toxicity of the former. While TA systems were initially discovered on plasmids, functioning as addiction modules through a phenomenon called postsegregational killing, they were later shown to be massively present in bacterial chromosomes, often in association with mobile genetic elements. Extensive research has been conducted in recent decades to better understand the physiological roles of these chromosomally encoded modules and to characterize the conditions leading to their activation. The diversity of their proposed roles, ranging from genomic stabilization and abortive phage infection to stress modulation and antibiotic persistence, in conjunction with the poor understanding of TA system regulation, resulted in the generation of simplistic models, often refuted by contradictory results. This review provides an epistemological and critical retrospective on TA modules and highlights fundamental questions concerning their roles and regulations that still remain unanswered.


Assuntos
Antitoxinas/genética , Antitoxinas/imunologia , Toxinas Bacterianas/genética , Toxinas Bacterianas/imunologia , Evolução Biológica , Sistemas Toxina-Antitoxina , Estudos de Associação Genética , Genoma Bacteriano , Fenótipo , Sistemas Toxina-Antitoxina/genética , Sistemas Toxina-Antitoxina/imunologia
20.
Appl Microbiol Biotechnol ; 104(4): 1423-1435, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31900550

RESUMO

Since the last 20 years, bacteria of the genus Acinetobacter have been the leading cause of hospital-acquired infections. In addition to the ability of Acinetobacter species to acquire rapid antibiotic resistance, limited knowledge on the mechanisms of multidrug resistance to antibiotics limits the treatment options for such infections. Here, we present a review of cellular processes, including oxidative stress defense, energy metabolism, ppGpp signaling, toxin-antitoxin system, and quorum sensing network in Acinetobacter species and their roles in antimicrobial resistance. Although inhibition of stress responses is an attractive approach to the development of effective antimicrobial therapeutic agents, it is crucial to understand the mechanisms that cause antibiotic resistance in Acinetobacter species, as they are not as well studied as those in other pathogenic bacteria. RelA/SpoT has been shown to be involved in ppGpp synthesis in all 50 genomes of 35 Acinetobacter species. However, toxin-antitoxin (TA) systems are present in less than 30% of the 50 genomes (28/30% of SplT/A; 14/14% of HigB/A; 4/6% of HicA/B), except the RelE/B system (30/78%). These data suggested that ppGpp signaling is conserved in Acinetobacter species, but TA systems are not. This review describes our current knowledge on stress responses with respect to antibiotic resistance or tolerance in pathogenic and non-pathogenic Acinetobacter species.


Assuntos
Acinetobacter/efeitos dos fármacos , Acinetobacter/genética , Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Estresse Fisiológico/efeitos dos fármacos , Acinetobacter/metabolismo , Metabolismo Energético , Genoma Bacteriano , Estresse Oxidativo , Percepção de Quorum , Sistemas Toxina-Antitoxina/genética
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