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1.
Nat Commun ; 11(1): 4915, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33004811

RESUMO

A phenotype of Escherichia coli and Klebsiella pneumoniae, resistant to piperacillin/tazobactam (TZP) but susceptible to carbapenems and 3rd generation cephalosporins, has emerged. The resistance mechanism associated with this phenotype has been identified as hyperproduction of the ß-lactamase TEM. However, the mechanism of hyperproduction due to gene amplification is not well understood. Here, we report a mechanism of gene amplification due to a translocatable unit (TU) excising from an IS26-flanked pseudo-compound transposon, PTn6762, which harbours blaTEM-1B. The TU re-inserts into the chromosome adjacent to IS26 and forms a tandem array of TUs, which increases the copy number of blaTEM-1B, leading to TEM-1B hyperproduction and TZP resistance. Despite a significant increase in blaTEM-1B copy number, the TZP-resistant isolate does not incur a fitness cost compared to the TZP-susceptible ancestor. This mechanism of amplification of blaTEM-1B is an important consideration when using genomic data to predict susceptibility to TZP.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Infecções por Escherichia coli/tratamento farmacológico , Proteínas de Escherichia coli/genética , Escherichia coli/genética , beta-Lactamases/genética , Antibacterianos/uso terapêutico , Cromossomos Bacterianos/genética , Elementos de DNA Transponíveis/genética , DNA Bacteriano/genética , Quimioterapia Combinada/métodos , Escherichia coli/efeitos dos fármacos , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/microbiologia , Amplificação de Genes , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano/genética , Humanos , Testes de Sensibilidade Microbiana , Piperacilina/farmacologia , Piperacilina/uso terapêutico , Polimorfismo de Fragmento de Restrição , RNA Ribossômico 16S/genética , Tazobactam/farmacologia , Tazobactam/uso terapêutico , Sequenciamento Completo do Genoma
2.
Mol Cell ; 80(2): 175-177, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-33065017

RESUMO

Eisenbart et al. (2020) find an SSR-associated sRNA, NikS, that is subject to variable repeat-controlled expression. NikS regulates H. pylori virulence by post-transcriptionally repressing pathogenicity factors, including CagA and VacA, via base-pairing to their mRNAs.


Assuntos
Helicobacter pylori , Fatores de Virulência , DNA , Regulação Bacteriana da Expressão Gênica , Helicobacter pylori/genética , RNA Bacteriano/genética , Virulência/genética
3.
Nat Commun ; 11(1): 4365, 2020 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-32868761

RESUMO

Current approaches explore bacterial genes that change transcriptionally upon stress exposure as diagnostics to predict antibiotic sensitivity. However, transcriptional changes are often specific to a species or antibiotic, limiting implementation to known settings only. While a generalizable approach, predicting bacterial fitness independent of strain, species or type of stress, would eliminate such limitations, it is unclear whether a stress-response can be universally captured. By generating a multi-stress and species RNA-Seq and experimental evolution dataset, we highlight the strengths and limitations of existing gene-panel based methods. Subsequently, we build a generalizable method around the observation that global transcriptional disorder seems to be a common, low-fitness, stress response. We quantify this disorder using entropy, which is a specific measure of randomness, and find that in low fitness cases increasing entropy and transcriptional disorder results from a loss of regulatory gene-dependencies. Using entropy as a single feature, we show that fitness and quantitative antibiotic sensitivity predictions can be made that generalize well beyond training data. Furthermore, we validate entropy-based predictions in 7 species under antibiotic and non-antibiotic conditions. By demonstrating the feasibility of universal predictions of bacterial fitness, this work establishes the fundamentals for potentially new approaches in infectious disease diagnostics.


Assuntos
Bactérias/genética , Evolução Molecular Direcionada , Farmacorresistência Bacteriana/genética , Estresse Fisiológico , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/metabolismo , Fenômenos Fisiológicos Bacterianos , Doenças Transmissíveis/diagnóstico , Entropia , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Genoma Bacteriano , Análise de Sequência de RNA , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/metabolismo , Transcriptoma
4.
Nat Commun ; 11(1): 4817, 2020 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-32968056

RESUMO

Lysozymes are among the best-characterized enzymes, acting upon the cell wall substrate peptidoglycan. Here, examining the invasive bacterial periplasmic predator Bdellovibrio bacteriovorus, we report a diversified lysozyme, DslA, which acts, unusually, upon (GlcNAc-) deacetylated peptidoglycan. B. bacteriovorus are known to deacetylate the peptidoglycan of the prey bacterium, generating an important chemical difference between prey and self walls and implying usage of a putative deacetyl-specific "exit enzyme". DslA performs this role, and ΔDslA strains exhibit a delay in leaving from prey. The structure of DslA reveals a modified lysozyme superfamily fold, with several adaptations. Biochemical assays confirm DslA specificity for deacetylated cell wall, and usage of two glutamate residues for catalysis. Exogenous DslA, added ex vivo, is able to prematurely liberate B. bacteriovorus from prey, part-way through the predatory lifecycle. We define a mechanism for specificity that invokes steric selection, and use the resultant motif to identify wider DslA homologues.


Assuntos
Bdellovibrio bacteriovorus/enzimologia , Bdellovibrio bacteriovorus/metabolismo , Muramidase/química , Muramidase/metabolismo , Periplasma/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bdellovibrio bacteriovorus/genética , Parede Celular , Escherichia coli , Regulação Bacteriana da Expressão Gênica , Modelos Moleculares , Muramidase/genética , Mutação , Peptidoglicano/metabolismo , Fenótipo , Conformação Proteica , Especificidade por Substrato
5.
Nat Commun ; 11(1): 4827, 2020 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-32973167

RESUMO

In bacteria, translation re-initiation is crucial for synthesizing proteins encoded by genes that are organized into operons. The mechanisms regulating translation re-initiation remain, however, poorly understood. We now describe the ribosome termination structure (RTS), a conserved and stable mRNA secondary structure localized immediately downstream of stop codons, and provide experimental evidence for its role in governing re-initiation efficiency in a synthetic Escherichia coli operon. We further report that RTSs are abundant, being associated with 18%-65% of genes in 128 analyzed bacterial genomes representing all phyla, and are selectively depleted when translation re-initiation is advantageous yet selectively enriched so as to insulate translation when re-initiation is deleterious. Our results support a potentially universal role for the RTS in controlling translation termination-insulation and re-initiation across bacteria.


Assuntos
Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Óperon/genética , RNA Mensageiro/química , RNA Mensageiro/fisiologia , Bactérias/classificação , Bactérias/genética , Códon de Terminação/metabolismo , Escherichia coli/metabolismo , Genes Bacterianos/genética , Iniciação Traducional da Cadeia Peptídica , Estrutura Secundária de Proteína , RNA Mensageiro/genética , Ribossomos/metabolismo
6.
PLoS Pathog ; 16(9): e1008552, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32966346

RESUMO

Type VI secretion systems (T6SSs) are complex macromolecular injection machines which are widespread in Gram-negative bacteria. They are involved in host-cell interactions and pathogenesis, required to eliminate competing bacteria, or are important for the adaptation to environmental stress conditions. Here we identified regulatory elements controlling the T6SS4 of Yersinia pseudotuberculosis and found a novel type of hexameric transcription factor, RovC. RovC directly interacts with the T6SS4 promoter region and activates T6SS4 transcription alone or in cooperation with the LysR-type regulator RovM. A higher complexity of regulation was achieved by the nutrient-responsive global regulator CsrA, which controls rovC expression on the transcriptional and post-transcriptional level. In summary, our work unveils a central mechanism in which RovC, a novel key activator, orchestrates the expression of the T6SS weapons together with a global regulator to deploy the system in response to the availability of nutrients in the species' native environment.


Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Regiões Promotoras Genéticas , Sistemas de Secreção Tipo VI/metabolismo , Yersinia pseudotuberculosis/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Conformação Proteica , Estresse Fisiológico , Sistemas de Secreção Tipo VI/química , Sistemas de Secreção Tipo VI/genética , Yersinia pseudotuberculosis/genética
7.
Nat Commun ; 11(1): 4531, 2020 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-32913225

RESUMO

RNAs begin to fold and function during transcription. Riboswitches undergo cotranscriptional switching in the context of transcription elongation, RNA folding, and ligand binding. To investigate how these processes jointly modulate the function of the folate stress-sensing Fusobacterium ulcerans ZTP riboswitch, we apply a single-molecule vectorial folding (VF) assay in which an engineered superhelicase Rep-X sequentially releases fluorescently labeled riboswitch RNA from a heteroduplex in a 5'-to-3' direction, at ~60 nt s-1 [comparable to the speed of bacterial RNA polymerase (RNAP)]. We demonstrate that the ZTP riboswitch is kinetically controlled and that its activation is favored by slower unwinding, strategic pausing between but not before key folding elements, or a weakened transcription terminator. Real-time single-molecule monitoring captures folding riboswitches in multiple states, including an intermediate responsible for delayed terminator formation. These results show how individual nascent RNAs occupy distinct channels within the folding landscape that controls the fate of the riboswitch.


Assuntos
Fusobacterium/genética , Regulação Bacteriana da Expressão Gênica , Dobramento de RNA/genética , RNA Bacteriano/genética , Riboswitch/genética , Aminoimidazol Carboxamida/metabolismo , Fusobacterium/metabolismo , Conformação de Ácido Nucleico , RNA Bacteriano/metabolismo , Ribonucleotídeos/metabolismo , Imagem Individual de Molécula , Transcrição Genética
8.
Nat Commun ; 11(1): 4126, 2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32807804

RESUMO

Neisseria gonorrhoeae is an urgent public health threat due to rapidly increasing incidence and antibiotic resistance. In contrast with the trend of increasing resistance, clinical isolates that have reverted to susceptibility regularly appear, prompting questions about which pressures compete with antibiotics to shape gonococcal evolution. Here, we used genome-wide association to identify loss-of-function (LOF) mutations in the efflux pump mtrCDE operon as a mechanism of increased antibiotic susceptibility and demonstrate that these mutations are overrepresented in cervical relative to urethral isolates. This enrichment holds true for LOF mutations in another efflux pump, farAB, and in urogenitally-adapted versus typical N. meningitidis, providing evidence for a model in which expression of these pumps in the female urogenital tract incurs a fitness cost for pathogenic Neisseria. Overall, our findings highlight the impact of integrating microbial population genomics with host metadata and demonstrate how host environmental pressures can lead to increased antibiotic susceptibility.


Assuntos
Proteínas de Bactérias/metabolismo , Colo do Útero/microbiologia , Neisseria gonorrhoeae/efeitos dos fármacos , Neisseria gonorrhoeae/genética , Animais , Proteínas de Bactérias/genética , Resistência Microbiana a Medicamentos/genética , Feminino , Regulação Bacteriana da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Testes de Sensibilidade Microbiana , Mutação/genética , Neisseria gonorrhoeae/metabolismo , Óperon/genética , Regiões Promotoras Genéticas/genética
9.
Nat Commun ; 11(1): 4202, 2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32826900

RESUMO

Antibiotic biosynthetic gene clusters (BGCs) produce bioactive metabolites that impart a fitness advantage to their producer, providing a mechanism for natural selection. This selection drives antibiotic evolution and adapts BGCs for expression in different organisms, potentially providing clues to improve heterologous expression of antibiotics. Here, we use phage-assisted continuous evolution (PACE) to achieve bioactivity-dependent adaptation of the BGC for the antibiotic bicyclomycin (BCM), facilitating improved production in a heterologous host. This proof-of-principle study demonstrates that features of natural bioactivity-dependent evolution can be engineered to access unforeseen routes of improving metabolic pathways and product yields.


Assuntos
Antibacterianos/biossíntese , Vias Biossintéticas/genética , Família Multigênica , Produtos Biológicos/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Clonagem Molecular , Escherichia coli , Regulação Bacteriana da Expressão Gênica , Engenharia Metabólica , Pseudomonas fluorescens/genética , Pseudomonas fluorescens/metabolismo
10.
Nat Commun ; 11(1): 3970, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32769975

RESUMO

The rise of antibiotic resistance in many bacterial pathogens has been driven by the spread of a few successful strains, suggesting that some bacteria are genetically pre-disposed to evolving resistance. Here, we test this hypothesis by challenging a diverse set of 222 isolates of Staphylococcus aureus with the antibiotic ciprofloxacin in a large-scale evolution experiment. We find that a single efflux pump, norA, causes widespread variation in evolvability across isolates. Elevated norA expression potentiates evolution by increasing the fitness benefit provided by DNA topoisomerase mutations under ciprofloxacin treatment. Amplification of norA provides a further mechanism of rapid evolution in isolates from the CC398 lineage. Crucially, chemical inhibition of NorA effectively prevents the evolution of resistance in all isolates. Our study shows that pre-existing genetic diversity plays a key role in shaping resistance evolution, and it may be possible to predict which strains are likely to evolve resistance and to optimize inhibitor use to prevent this outcome.


Assuntos
Proteínas de Bactérias/metabolismo , Resistência Microbiana a Medicamentos , Evolução Molecular , Staphylococcus aureus/genética , Staphylococcus aureus/isolamento & purificação , Ciprofloxacino/farmacologia , Resistência Microbiana a Medicamentos/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genoma Bacteriano , Mutação/genética , Filogenia , Staphylococcus aureus/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos , Transcriptoma/genética
11.
PLoS Pathog ; 16(8): e1008856, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32845936

RESUMO

Copper and superoxide are used by the phagocytes to kill bacteria. Copper is a host effector encountered by uropathogenic Escherichia coli (UPEC) during urinary tract infection in a non-human primate model, and in humans. UPEC is exposed to higher levels of copper in the gut prior to entering the urinary tract. Effects of pre-exposure to copper on bacterial killing by superoxide has not been reported. We hypothesized that copper-replete E. coli is more sensitive to killing by superoxide in vitro, and in activated macrophages. We utilized wild-type UPEC strain CFT073, and its isogenic mutants lacking copper efflux systems, superoxide dismutases (SODs), regulators of a superoxide dismutase, and complemented mutants to address this question. Surprisingly, our results reveal that copper protects UPEC against killing by superoxide in vitro. This copper-dependent protection was amplified in the mutants lacking copper efflux systems. Increased levels of copper and manganese were detected in UPEC exposed to sublethal concentration of copper. Copper activated the transcription of sodA in a SoxR- and SoxS-dependent manner resulting in enhanced levels of SodA activity. Importantly, pre-exposure to copper increased the survival of UPEC within RAW264.7 and bone marrow-derived murine macrophages. Loss of SodA, but not SodB or SodC, in UPEC obliterated copper-dependent protection from superoxide in vitro, and from killing within macrophages. Collectively, our results suggest a model in which sublethal levels of copper trigger the activation of SodA and SodC through independent mechanisms that converge to promote the survival of UPEC from killing by superoxide. A major implication of our findings is that bacteria colonizing copper-rich milieus are primed for efficient detoxification of superoxide.


Assuntos
Cobre/farmacologia , Infecções por Escherichia coli/tratamento farmacológico , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Superóxido Dismutase/metabolismo , Superóxidos/toxicidade , Infecções Urinárias/tratamento farmacológico , Escherichia coli Uropatogênica/efeitos dos fármacos , Animais , Infecções por Escherichia coli/induzido quimicamente , Infecções por Escherichia coli/microbiologia , Feminino , Regulação Bacteriana da Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Superóxido Dismutase/genética , Infecções Urinárias/induzido quimicamente , Infecções Urinárias/microbiologia
12.
PLoS One ; 15(8): e0237135, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32822422

RESUMO

DNA-binding Transcription Factors (TFs) play a central role in regulation of gene expression in prokaryotic organisms, and similarities at the sequence level have been reported. These proteins are predicted with different abundances as a consequence of genome size, where small organisms contain a low proportion of TFs and large genomes contain a high proportion of TFs. In this work, we analyzed a collection of 668 experimentally validated TFs across 30 different species from diverse taxonomical classes, including Escherichia coli K-12, Bacillus subtilis 168, Corynebacterium glutamicum, and Streptomyces coelicolor, among others. This collection of TFs, together with 111 hidden Markov model profiles associated with DNA-binding TFs collected from diverse databases such as PFAM and DBD, was used to identify the repertoire of proteins putatively devoted to gene regulation in 1321 representative genomes of Archaea and Bacteria. The predicted regulatory proteins were posteriorly analyzed in terms of their genomic context, allowing the prediction of functions for TFs and their neighbor genes, such as genes involved in virulence, enzymatic functions, phosphorylation mechanisms, and antibiotic resistance. The functional analysis associated with PFAM groups showed diverse functional categories were significantly enriched in the collection of TFs and the proteins encoded by the neighbor genes, in particular, small-molecule binding and amino acid transmembrane transporter activities associated with the LysR family and proteins devoted to cellular aromatic compound metabolic processes or responses to drugs, stress, or abiotic stimuli in the MarR family. We consider that with the increasing data derived from new technologies, novel TFs can be identified and help improve the predictions for this class of proteins in complete genomes. The complete collection of experimentally characterized and predicted TFs is available at http://web.pcyt.unam.mx/EntrafDB/.


Assuntos
Archaea/genética , Proteínas Arqueais/genética , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Escherichia coli K12/genética , Fatores de Transcrição/genética , Archaea/patogenicidade , Proteínas Arqueais/metabolismo , Proteínas de Bactérias/metabolismo , Sítios de Ligação , DNA Arqueal/metabolismo , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Escherichia coli K12/patogenicidade , Regulação da Expressão Gênica em Archaea , Regulação Bacteriana da Expressão Gênica , Genoma Arqueal , Genoma Bacteriano , Ligação Proteica , Fatores de Transcrição/metabolismo , Virulência/genética
13.
Proc Natl Acad Sci U S A ; 117(33): 20202-20210, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32747578

RESUMO

In biology, it is often critical to determine the identity of an organism and phenotypic traits of interest. Whole-genome sequencing can be useful for this but has limited power for trait prediction. However, we can take advantage of the inherent information content of phenotypes to bypass these limitations. We demonstrate, in clinical and environmental bacterial isolates, that growth dynamics in standardized conditions can differentiate between genotypes, even among strains from the same species. We find that for pairs of isolates, there is little correlation between genetic distance, according to phylogenetic analysis, and phenotypic distance, as determined by growth dynamics. This absence of correlation underscores the challenge in using genomics to infer phenotypes and vice versa. Bypassing this complexity, we show that growth dynamics alone can robustly predict antibiotic responses. These findings are a foundation for a method to identify traits not easily traced to a genetic mechanism.


Assuntos
Enterobacteriaceae/crescimento & desenvolvimento , Enterobacteriaceae/genética , Antibacterianos/farmacologia , DNA Bacteriano/genética , Farmacorresistência Bacteriana Múltipla , Enterobacteriaceae/efeitos dos fármacos , Microbiologia Ambiental , Regulação Bacteriana da Expressão Gênica , Polimorfismo de Nucleotídeo Único , Especificidade da Espécie , Fatores de Tempo
14.
PLoS Pathog ; 16(8): e1008708, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32785266

RESUMO

The intestinal pathogen Clostridioides difficile exhibits heterogeneity in motility and toxin production. This phenotypic heterogeneity is achieved through phase variation by site-specific recombination via the DNA recombinase RecV, which reversibly inverts the "flagellar switch" upstream of the flgB operon. A recV mutation prevents flagellar switch inversion and results in phenotypically locked strains. The orientation of the flagellar switch influences expression of the flgB operon post-transcription initiation, but the specific molecular mechanism is unknown. Here, we report the isolation and characterization of spontaneous suppressor mutants in the non-motile, non-toxigenic recV flg OFF background that regained motility and toxin production. The restored phenotypes corresponded with increased expression of flagellum and toxin genes. The motile suppressor mutants contained single-nucleotide polymorphisms (SNPs) in rho, which encodes the bacterial transcription terminator Rho factor. Analyses using transcriptional reporters indicate that Rho contributes to heterogeneity in flagellar gene expression by preferentially terminating transcription of flg OFF mRNA within the 5' leader sequence. Additionally, Rho is important for initial colonization of the intestine in a mouse model of infection, which may in part be due to the sporulation and growth defects observed in the rho mutants. Together these data implicate Rho factor as a regulator of gene expression affecting phase variation of important virulence factors of C. difficile.


Assuntos
Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Infecções por Clostridium/microbiologia , Clostridium difficile/metabolismo , Flagelos/metabolismo , Fator Rho/metabolismo , Animais , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Clostridium difficile/genética , Clostridium difficile/patogenicidade , Feminino , Flagelos/genética , Regulação Bacteriana da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Óperon , Fator Rho/genética , Virulência
15.
PLoS One ; 15(8): e0238151, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32833990

RESUMO

Bacteria often possess relatively flexible genome structures and adaptive genetic variants that allow survival in unfavorable growth conditions. Bacterial survival tactics in disadvantageous microenvironments include mutations that are beneficial against threats in their niche. Here, we report that the aerobic rice bacterial pathogen Burkholderia glumae BGR1 changes a specific gene for improved survival in static culture conditions. Static culture triggered formation of colony variants with deletions or point mutations in the gene bspP (BGLU_RS28885), which putatively encodes a protein that contains PDC2, PAS-9, SpoIIE, and HATPase domains. The null mutant of bspP survived longer in static culture conditions and produced a higher level of bis-(3'-5')-cyclic dimeric guanosine monophosphate than the wild type. Expression of the bacterial cellulose synthase regulator (bcsB) gene was upregulated in the mutant, consistent with the observation that the mutant formed pellicles faster than the wild type. Mature pellicle formation was observed in the bspP mutant before pellicle formation in wild-type BGR1. However, the population density of the bspP null mutant decreased substantially when grown in Luria-Bertani medium with vigorous agitation due to failure of oxalate-mediated detoxification of the alkaline environment. The bspP null mutant was less virulent and exhibited less effective colonization of rice plants than the wild type. All phenotypes caused by mutations in bspP were recovered to those of the wild type by genetic complementation. Thus, although wild-type B. glumae BGR1 prolonged viability by spontaneous mutation under static culture conditions, such genetic changes negatively affected colonization in rice plants. These results suggest that adaptive gene sacrifice of B. glumae to survive unfavorable growth conditions is not always desirable as it can adversely affect adaptability in the host.


Assuntos
Adaptação Biológica/genética , Burkholderia/genética , Burkholderia/metabolismo , Burkholderia/patogenicidade , Regulação Bacteriana da Expressão Gênica/genética , Genoma Bacteriano/genética , Genômica/métodos , Mutação , Oryza/microbiologia , Doenças das Plantas/microbiologia , Percepção de Quorum/genética , Virulência/genética
16.
PLoS One ; 15(8): e0237367, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32810148

RESUMO

Bacterial group II introns mostly behave as versatile retromobile genetic elements going through distinct cycles of gain and loss. These large RNA molecules are also ribozymes splicing autocatalytically from their interrupted pre-mRNA transcripts by two different concurrent pathways, branching and circularization. These two splicing pathways were shown to release in bacterial cells significant amounts of branched intron lariats and perfect end-to-end intron circles respectively. On one hand, released intron lariats can invade new sites in RNA and/or DNA by reverse branching while released intron circles are dead end spliced products since they cannot reverse splice through circularization. The presence of two parallel and competing group II intron splicing pathways in bacteria led us to investigate the conditions that influence the overall circle to lariat ratio in vivo. Here we unveil that removing a prominent processing site within the Ll.LtrB group II intron, raising growth temperature of Lactococcus lactis host cells and increasing the expression level of the intron-interrupted gene all increased the relative amount of released intron circles compared to lariats. Strengthening and weakening the base pairing interaction between the intron and its upstream exon respectively increased and decreased the overall levels of released intron circles in comparison to lariats. Host environment was also found to impact the circle to lariat ratio of the Ll.LtrB and Ll.RlxA group II introns from L. lactis and the Ef.PcfG intron from Enterococcus faecalis. Overall, our data show that multiple factors significantly influence the balance between released intron circles and lariats in bacterial cells.


Assuntos
Íntrons/genética , Lactococcus lactis/genética , Regulação Bacteriana da Expressão Gênica , Temperatura
17.
Nature ; 585(7823): 124-128, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32848247

RESUMO

Tight coupling of transcription and translation is considered a defining feature of bacterial gene expression1,2. The pioneering ribosome can both physically associate and kinetically coordinate with RNA polymerase (RNAP)3-11, forming a signal-integration hub for co-transcriptional regulation that includes translation-based attenuation12,13 and RNA quality control2. However, it remains unclear whether transcription-translation coupling-together with its broad functional consequences-is indeed a fundamental characteristic of bacteria other than Escherichia coli. Here we show that RNAPs outpace pioneering ribosomes in the Gram-positive model bacterium Bacillus subtilis, and that this 'runaway transcription' creates alternative rules for both global RNA surveillance and translational control of nascent RNA. In particular, uncoupled RNAPs in B. subtilis explain the diminished role of Rho-dependent transcription termination, as well as the prevalence of mRNA leaders that use riboswitches and RNA-binding proteins. More broadly, we identified widespread genomic signatures of runaway transcription in distinct phyla across the bacterial domain. Our results show that coupled RNAP-ribosome movement is not a general hallmark of bacteria. Instead, translation-coupled transcription and runaway transcription constitute two principal modes of gene expression that determine genome-specific regulatory mechanisms in prokaryotes.


Assuntos
Bacillus subtilis/genética , Regulação Bacteriana da Expressão Gênica , Biossíntese de Proteínas , Transcrição Genética , Regiões 5' não Traduzidas/genética , Bacillus subtilis/enzimologia , Bacillus subtilis/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Filogenia , RNA Bacteriano/biossíntese , RNA Bacteriano/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fator Rho/metabolismo , Ribossomos/metabolismo , Riboswitch/genética
18.
PLoS Pathog ; 16(8): e1008776, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32845938

RESUMO

Enteroaggregative Escherichia coli (EAEC) is a diarrheagenic pathotype associated with traveler's diarrhea, foodborne outbreaks and sporadic diarrhea in industrialized and developing countries. Regulation of virulence in EAEC is mediated by AggR and its negative regulator Aar. Together, they control the expression of at least 210 genes. On the other hand, we observed that about one third of Aar-regulated genes are related to metabolism and transport. In this study we show the AggR/Aar duo controls the metabolism of lipids. Accordingly, we show that AatD, encoded in the AggR-regulated aat operon (aatPABCD) is an N-acyltransferase structurally similar to the essential Apolipoprotein N-acyltransferase Lnt and is required for the acylation of Aap (anti-aggregation protein). Deletion of aatD impairs post-translational modification of Aap and causes its accumulation in the bacterial periplasm. trans-complementation of 042aatD mutant with the AatD homolog of ETEC or with the N-acyltransferase Lnt reestablished translocation of Aap. Site-directed mutagenesis of the E207 residue in the putative acyltransferase catalytic triad disrupted the activity of AatD and caused accumulation of Aap in the periplasm due to reduced translocation of Aap at the bacterial surface. Furthermore, Mass spectroscopy revealed that Aap is acylated in a putative lipobox at the N-terminal of the mature protein, implying that Aap is a lipoprotein. Lastly, deletion of aatD impairs bacterial colonization of the streptomycin-treated mouse model. Our findings unveiled a novel N-acyltransferase family associated with bacterial virulence, and that is tightly regulated by AraC/XylS regulators in the order Enterobacterales.


Assuntos
Acetiltransferases/metabolismo , Fator de Transcrição AraC/metabolismo , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Escherichia coli/patogenicidade , Regulação Bacteriana da Expressão Gênica , Acetiltransferases/genética , Acilação , Animais , Fator de Transcrição AraC/química , Fator de Transcrição AraC/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Óperon , Filogenia , Conformação Proteica , Virulência
19.
PLoS Pathog ; 16(8): e1008512, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32776984

RESUMO

Bordetella bronchiseptica and Bordetella pertussis are closely related respiratory pathogens that evolved from a common bacterial ancestor. While B. bronchiseptica has an environmental reservoir and mostly establishes chronic infections in a broad range of mammals, B. pertussis is a human-specific pathogen causing acute pulmonary pertussis in infants and whooping cough illness in older humans. Both species employ a type III secretion system (T3SS) to inject a cytotoxic BteA effector protein into host cells. However, compared to the high BteA-mediated cytotoxicity of B. bronchiseptica, the cytotoxicity induced by B. pertussis BteA (Bp BteA) appears to be quite low and this has been attributed to the reduced T3SS gene expression in B. pertussis. We show that the presence of an alanine residue inserted at position 503 (A503) of Bp BteA accounts for its strongly attenuated cytotoxic potency. The deletion of A503 from Bp BteA greatly enhanced the cytotoxic activity of B. pertussis B1917 on mammalian HeLa cells and expression of Bp BteAΔA503 was highly toxic to Saccharomyces cerevisiae cells. Vice versa, insertion of A503 into B. bronchiseptica BteA (Bb BteA) strongly decreased its cytotoxicity to yeast and HeLa cells. Moreover, the production of Bp BteAΔA503 increased virulence of B. pertussis B1917 in the mouse model of intranasal infection (reduced LD50) but yielded less inflammatory pathology in infected mouse lungs at sublethal infectious doses. This suggests that A503 insertion in the T3SS effector Bp BteA may represent an evolutionary adaptation that fine-tunes B. pertussis virulence and host immune response.


Assuntos
Alanina/metabolismo , Proteínas de Bactérias/metabolismo , Bordetella pertussis/fisiologia , Regulação Bacteriana da Expressão Gênica , Coqueluche/patologia , Alanina/genética , Animais , Proteínas de Bactérias/genética , Feminino , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Sistemas de Secreção Tipo III/genética , Sistemas de Secreção Tipo III/metabolismo , Virulência , Coqueluche/genética , Coqueluche/microbiologia
20.
Mol Cell ; 79(5): 797-811.e8, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32750314

RESUMO

Pausing by RNA polymerase (RNAP) during transcription elongation, in which a translocating RNAP uses a "stepping" mechanism, has been studied extensively, but pausing by RNAP during initial transcription, in which a promoter-anchored RNAP uses a "scrunching" mechanism, has not. We report a method that directly defines the RNAP-active-center position relative to DNA with single-nucleotide resolution (XACT-seq; "crosslink-between-active-center-and-template sequencing"). We apply this method to detect and quantify pausing in initial transcription at 411 (∼4,000,000) promoter sequences in vivo in Escherichia coli. The results show initial-transcription pausing can occur in each nucleotide addition during initial transcription, particularly the first 4 to 5 nucleotide additions. The results further show initial-transcription pausing occurs at sequences that resemble the consensus sequence element for transcription-elongation pausing. Our findings define the positional and sequence determinants for initial-transcription pausing and establish initial-transcription pausing is hard coded by sequence elements similar to those for transcription-elongation pausing.


Assuntos
DNA Bacteriano/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Regiões Promotoras Genéticas , Análise de Sequência de DNA/métodos , Domínio Catalítico , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Transcrição Genética
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