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1.
Nucleic Acids Res ; 2024 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-38884209

RESUMO

Generalized transduction is pivotal in bacterial evolution but lacks comprehensive understanding regarding the facilitating features and variations among phages. We addressed this gap by sequencing and comparing the transducing particle content of three different Salmonella Typhimurium phages (i.e. Det7, ES18 and P22) that share a headful packaging mechanism that is typically initiated from a cognate pac site within the phage chromosome. This revealed substantial disparities in both the extent and content of transducing particles among these phages. While Det7 outperformed ES18 in terms of relative number of transducing particles, both phages contrasted with P22 in terms of content. In fact, we found evidence for the presence of conserved P22 pac-like sequences in the host chromosome that direct tremendously increased packaging and transduction frequencies of downstream regions by P22. More specifically, a ca. 561 kb host region between oppositely oriented pac-like sequences in the purF and minE loci was identified as highly packaged and transduced during both P22 prophage induction and lytic infection. Our findings underscore the evolution of phage transducing capacity towards attenuation, promiscuity or directionality, and suggest that pac-like sequences in the host chromosome could become selected as sites directing high frequency of transduction.

2.
PLoS Biol ; 20(4): e3001608, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35389980

RESUMO

Virulence gene expression can represent a substantial fitness cost to pathogenic bacteria. In the model entero-pathogen Salmonella Typhimurium (S.Tm), such cost favors emergence of attenuated variants during infections that harbor mutations in transcriptional activators of virulence genes (e.g., hilD and hilC). Therefore, understanding the cost of virulence and how it relates to virulence regulation could allow the identification and modulation of ecological factors to drive the evolution of S.Tm toward attenuation. In this study, investigations of membrane status and stress resistance demonstrate that the wild-type (WT) expression level of virulence factors embedded in the envelope increases membrane permeability and sensitizes S.Tm to membrane stress. This is independent from a previously described growth defect associated with virulence gene expression in S.Tm. Pretreating the bacteria with sublethal stress inhibited virulence expression and increased stress resistance. This trade-off between virulence and stress resistance could explain the repression of virulence expression in response to harsh environments in S.Tm. Moreover, we show that virulence-associated stress sensitivity is a burden during infection in mice, contributing to the inherent instability of S.Tm virulence. As most bacterial pathogens critically rely on deploying virulence factors in their membrane, our findings could have a broad impact toward the development of antivirulence strategies.


Assuntos
Regulação Bacteriana da Expressão Gênica , Salmonella typhimurium , Animais , Proteínas de Bactérias/metabolismo , Camundongos , Permeabilidade , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Virulência/genética , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
3.
Cell Mol Life Sci ; 80(12): 360, 2023 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-37971522

RESUMO

Mechanisms underlying deviant cell size fluctuations among clonal bacterial siblings are generally considered to be cryptic and stochastic in nature. However, by scrutinizing heat-stressed populations of the model bacterium Escherichia coli, we uncovered the existence of a deterministic asymmetry in cell division that is caused by the presence of intracellular protein aggregates (PAs). While these structures typically locate at the cell pole and segregate asymmetrically among daughter cells, we now show that the presence of a polar PA consistently causes a more distal off-center positioning of the FtsZ division septum. The resulting increased length of PA-inheriting siblings persists over multiple generations and could be observed in both E. coli and Bacillus subtilis populations. Closer investigation suggests that a PA can physically perturb the nucleoid structure, which subsequently leads to asymmetric septation.


Assuntos
Proteínas de Bactérias , Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Bactérias/metabolismo , Agregados Proteicos , Divisão Celular , Bactérias/metabolismo , Bacillus subtilis/metabolismo
4.
Biochem Biophys Res Commun ; 681: 291-297, 2023 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-37801778

RESUMO

Mycophage endolysins are highly diverse and modular enzymes composed of domains involved in peptidoglycan binding and degradation. Mostly, they are characterized by a three-module design: an N-terminal peptidase domain, a central catalytic domain and a C-terminal peptidoglycan binding domain. Previously, the affinity of cell wall binding domains (CBDs) to the mycobacterial peptidoglycan layer was shown for some of these endolysins. In this study, an in depth screening was performed on twelve mycophage endolysins. The discovered CBDs were characterized for their binding affinity to Mycobacterium (M.) bovis bacille Calmette-Guérin (BCG), a largely unexplored target and an attenuated strain of M. bovis, responsible for bovine tuberculosis. Using homology-based annotation, only four endolysins showed the presence of a known peptidoglycan binding domain, the previously characterized pfam 01471 domain. However, analysis of the secondary structure aided by AlphaFold predictions revealed the presence of a C-terminal domain in the other endolysins. These were hypothesized as new, uncharacterized CBDs. Fusion proteins composed of these domains linked to GFP were constructed and positively assayed for their affinity to M. bovis BCG in a peptidoglycan binding assay. Moreover, two CBDs were able to fluorescently label M. bovis BCG in milk samples, highlighting the potential to further explore their possibility to function as CBD-based diagnostics.


Assuntos
Mycobacterium bovis , Peptidoglicano , Peptidoglicano/metabolismo , Mycobacterium bovis/metabolismo , Endopeptidases/metabolismo , Parede Celular/metabolismo
5.
Int J Mol Sci ; 23(3)2022 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-35163175

RESUMO

Many phage genes lack sequence similarity to any other open reading frame (ORF) in current databases. These enigmatic ORFan genes can have a tremendous impact on phage propagation and host interactions but often remain experimentally unexplored. We previously revealed a novel interaction between phage P22 and its Salmonella Typhimurium host, instigated by the ORFan gene pid (for phage P22 encoded instigator of dgo expression) and resulting in derepression of the host dgoRKAT operon. The pid gene is highly expressed in phage carrier cells that harbor a polarly located P22 episome that segregates asymmetrically among daughter cells. Here, we discovered that the pid locus is fitted with a weak promoter, has an exceptionally long 5' untranslated region that is instructive for a secondary pid mRNA species, and has a 3' Rho-independent termination loop that is responsible for stability of the pid transcript.


Assuntos
Bacteriófago P22/genética , Regulação Viral da Expressão Gênica/genética , Bacteriófagos/genética , Expressão Gênica/genética , Fases de Leitura Aberta/genética , Óperon , Regiões Promotoras Genéticas/genética , Fagos de Salmonella/genética , Salmonella typhimurium/genética , Salmonella typhimurium/virologia
6.
PLoS Biol ; 16(8): e2003853, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30153247

RESUMO

Protein misfolding and aggregation are typically perceived as inevitable and detrimental processes tied to a stress- or age-associated decline in cellular proteostasis. A careful reassessment of this paradigm in the E. coli model bacterium revealed that the emergence of intracellular protein aggregates (PAs) was not related to cellular aging but closely linked to sublethal proteotoxic stresses such as exposure to heat, peroxide, and the antibiotic streptomycin. After removal of the proteotoxic stress and resumption of cellular proliferation, the polarly deposited PA was subjected to limited disaggregation and therefore became asymmetrically inherited for a large number of generations. Many generations after the original PA-inducing stress, the cells inheriting this ancestral PA displayed a significantly increased heat resistance compared to their isogenic, PA-free siblings. This PA-mediated inheritance of heat resistance could be reproduced with a conditionally expressed, intracellular PA consisting of an inert, aggregation-prone mutant protein, validating the role of PAs in increasing resistance and indicating that the resistance-conferring mechanism does not depend on the origin of the PA. Moreover, PAs were found to confer robustness to other proteotoxic stresses, as imposed by reactive oxygen species or streptomycin exposure, suggesting a broad protective effect. Our findings therefore reveal the potential of intracellular PAs to serve as long-term epigenetically inheritable and functional memory elements, physically referring to a previous cellular insult that occurred many generations ago and meanwhile improving robustness to a subsequent proteotoxic stress. The latter is presumably accomplished through the PA-mediated asymmetric inheritance of protein quality control components leading to their specific enrichment in PA-bearing cells.


Assuntos
Adaptação Fisiológica/genética , Epigênese Genética , Proteínas de Escherichia coli/química , Escherichia coli/genética , Proteínas de Choque Térmico/química , Estresse Fisiológico/genética , Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Temperatura Alta , Peróxido de Hidrogênio/farmacologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Agregados Proteicos/efeitos dos fármacos , Dobramento de Proteína/efeitos dos fármacos , Proteostase/efeitos dos fármacos , Proteostase/genética , Análise de Célula Única , Estreptomicina/farmacologia , Proteína Vermelha Fluorescente
7.
Metab Eng ; 62: 287-297, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32979485

RESUMO

Although high hydrostatic pressure (HHP) is an interesting parameter to be applied in bioprocessing, its potential is currently limited by the lack of bacterial chassis capable of surviving and maintaining homeostasis under pressure. While several efforts have been made to genetically engineer microorganisms able to grow at sublethal pressures, there is little information for designing backgrounds that survive more extreme pressures. In this investigation, we analyzed the genome of an extreme HHP-resistant mutant of E. coli MG1655 (designated as DVL1), from which we identified four mutations (in the cra, cyaA, aceA and rpoD loci) causally linked to increased HHP resistance. Analysing the functional effect of these mutations we found that the coupled effect of downregulation of cAMP/CRP, Cra and the glyoxylate shunt activity, together with the upregulation of RpoH and RpoS activity, could mechanistically explain the increased HHP resistance of the mutant. Using combinations of three mutations, we could synthetically engineer E. coli strains able to comfortably survive pressures of 600-800 MPa, which could serve as genetic backgrounds for HHP-based biotechnological applications.


Assuntos
Bactérias , Escherichia coli , Escherichia coli/genética , Pressão Hidrostática , Mutação
8.
Food Microbiol ; 87: 103388, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31948629

RESUMO

The growing demand for minimally processed foods with clean labels has stimulated research into mild processing methods and natural antimicrobials to replace intensive heating and conventional preservatives, respectively. However, we have previously demonstrated that repetitive exposure of some bacteria to mild heat or subinhibitory concentrations of essential oil constituents (EOCs) may induce the emergence of mutants with increased resistance to these treatments. Since the combination of mild heat with some EOCs has a synergistic effect on microbial inactivation, we evaluated the potential of such combinations against our resistant E. coli mutants. While citral, carvacrol and t-cinnamaldehyde synergistically increased heat inactivation (53.0 °C, 10 min) of the wild-type MG1655 suspended in buffer, only the combination with carvacrol (200 µl/l) was able to mitigate the increased resistance of all the mutants. Moreover, the combination of heat and carvacrol acted synergistically inactivating heat-resistant variants of E. coli O157:H7 (ATCC 43888). This combined treatment could synergistically achieve more than 5 log10 reductions of the most resistant mutants in coconut water, although the temperature had to be raised to 57.0 °C. Therefore, the combination of mild heat with carvacrol appears to hold promise for mild processing, and it is expected to counteract the development of heat resistance.


Assuntos
Antibacterianos/farmacologia , Cocos/química , Escherichia coli O157/efeitos dos fármacos , Óleos Voláteis/farmacologia , Extratos Vegetais/farmacologia , Óleos de Plantas/farmacologia , Acroleína/análogos & derivados , Acroleína/farmacologia , Monoterpenos Acíclicos/farmacologia , Cimenos/farmacologia , Farmacorresistência Bacteriana , Escherichia coli O157/crescimento & desenvolvimento , Temperatura Alta
9.
Curr Genet ; 65(4): 865-869, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30820637

RESUMO

The concept of phenotypic heterogeneity preparing a subpopulation of isogenic cells to better cope with anticipated stresses has been well established. However, less is known about how stress itself can drive subsequent cellular individualization in clonal populations. In this perspective, we focus on the impact of stress-induced cellular protein aggregates, and how their segregation and disaggregation can act as a deterministic incentive for heterogeneity in the population emerging from a stressed ancestor.


Assuntos
Heterogeneidade Genética , Agregados Proteicos/genética , Estresse Fisiológico/genética , Escherichia coli/genética
10.
Nucleic Acids Res ; 45(9): 5323-5332, 2017 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-28369499

RESUMO

A sub-lethal hydrostatic pressure (HP) shock of ∼100 MPa elicits a RecA-dependent DNA damage (SOS) response in Escherichia coli K-12, despite the fact that pressure cannot compromise the covalent integrity of DNA. Prior screens for HP resistance identified Mrr (Methylated adenine Recognition and Restriction), a Type IV restriction endonuclease (REase), as instigator for this enigmatic HP-induced SOS response. Type IV REases tend to target modified DNA sites, and E. coli Mrr activity was previously shown to be elicited by expression of the foreign M.HhaII Type II methytransferase (MTase), as well. Here we measured the concentration and stoichiometry of a functional GFP-Mrr fusion protein using in vivo fluorescence fluctuation microscopy. Our results demonstrate that Mrr is a tetramer in unstressed cells, but shifts to a dimer after HP shock or co-expression with M.HhaII. Based on the differences in reversibility of tetramer dissociation observed for wild-type GFP-Mrr and a catalytic mutant upon HP shock compared to M.HhaII expression, we propose a model by which (i) HP triggers Mrr activity by directly pushing inactive Mrr tetramers to dissociate into active Mrr dimers, while (ii) M.HhaII triggers Mrr activity by creating high affinity target sites on the chromosome, which pull the equilibrium from inactive tetrameric Mrr toward active dimer.


Assuntos
Enzimas de Restrição do DNA/metabolismo , Escherichia coli K12/metabolismo , Pressão , Multimerização Proteica , Biocatálise , Cromatografia em Gel , Ativação Enzimática , Fluorescência , Proteínas de Fluorescência Verde/metabolismo , Modelos Biológicos , Proteínas Mutantes/metabolismo , Mutação/genética , Estresse Fisiológico
11.
Food Microbiol ; 78: 171-178, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30497599

RESUMO

High hydrostatic pressure (HHP) is an interesting hurdle in minimal food processing that aims to synergistically combine different stresses to improve food microbiological safety and stability without compromising quality. For a proper understanding and design of hurdle technology, the cellular impact of the applied stresses on foodborne pathogens should be well-established. To study the mechanism of HHP-mediated cell injury and death, we screened for loss-of-function mutations in E. coli MG1655 that affected HHP sensitivity. More specifically, ca. 6000 random transposon insertion mutants were individually exposed to HHP, after which the phenotype of the most resistant or sensitive mutations was confirmed by de novo gene deletions in the parental strain. We found that disruption of rbsK, rbsR, hdfR and crl decreased HHP resistance, while disruption of sucC and sucD (encoding subunits of the succinyl-CoA synthetase) increased HHP resistance. More detailed study of the tricarboxylic acid cycle enzymes encoded by the sdhCDAB-sucABCD operon surprisingly showed that disruption of the sucA or sucB gene (encoding subunits of the 2-oxoglutarate dehydrogenase complex) notably decreased HHP survival. We also found that the increased HHP resistance of a ΔsucC and ΔsucD mutant was mediated by increased basal RpoS activity levels, although it did not correlate with their heat resistance. Our results reveal that compromising TCA cycle enzymes can profoundly affect HHP resistance in E. coli.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Genes Bacterianos , Ciclo do Ácido Cítrico , Manipulação de Alimentos/métodos , Regulação Bacteriana da Expressão Gênica , Temperatura Alta , Pressão Hidrostática , Mutação , Óperon , Proteínas Repressoras/deficiência , Proteínas Repressoras/genética
12.
PLoS Genet ; 11(12): e1005770, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26720743

RESUMO

Monitoring the complex transmission dynamics of a bacterial virus (temperate phage P22) throughout a population of its host (Salmonella Typhimurium) at single cell resolution revealed the unexpected existence of a transiently immune subpopulation of host cells that emerged from peculiarities preceding the process of lysogenization. More specifically, an infection event ultimately leading to a lysogen first yielded a phage carrier cell harboring a polarly tethered P22 episome. Upon subsequent division, the daughter cell inheriting this episome became lysogenized by an integration event yielding a prophage, while the other daughter cell became P22-free. However, since the phage carrier cell was shown to overproduce immunity factors that are cytoplasmically inherited by the P22-free daughter cell and further passed down to its siblings, a transiently resistant subpopulation was generated that upon dilution of these immunity factors again became susceptible to P22 infection. The iterative emergence and infection of transiently resistant subpopulations suggests a new bet-hedging strategy by which viruses could manage to sustain both vertical and horizontal transmission routes throughout an infected population without compromising a stable co-existence with their host.


Assuntos
Bacteriófago P22/imunologia , Bacteriófago P22/patogenicidade , Salmonella typhimurium/imunologia , Salmonella typhimurium/virologia , Bacteriófago P22/genética , Cromossomos/metabolismo , Citoplasma/genética , Citoplasma/imunologia , Interações Hospedeiro-Patógeno/imunologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Mutação , Salmonella typhimurium/genética , Análise de Célula Única , Proteínas Virais/genética , Proteínas Virais/imunologia , Proteínas Virais/metabolismo
13.
Environ Microbiol ; 19(2): 511-523, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27449737

RESUMO

Inactivation of bacterial pathogens is of critical importance in fields ranging from antimicrobial therapy to food preservation. The efficacy of an antimicrobial treatment is often experimentally determined through viable plate counts that inherently provide a poor focus on the mechanisms and distribution of (sub)lethal injury and subsequent inactivation or resuscitation behavior of the stressed cells, which are increasingly important features for the proper understanding and design of inactivation strategies. In this report, we employ a live cell biology approach focusing on the energy-dependent motion of intracellular protein aggregates to investigate the heterogeneity within heat stressed Escherichia coli populations. As such, we were able to identify differential dynamics of cellular resuscitation and inactivation that are impossible to distinguish using more traditional approaches. Moreover, our data indicate the existence of late-resuscitating cells that remain physiologically active and are able to persist in the presence of antibiotics before resuscitation.


Assuntos
Escherichia coli/metabolismo , Agregados Proteicos , Estresse Fisiológico , Escherichia coli/genética , Temperatura Alta , Transporte Proteico
14.
Crit Rev Microbiol ; 43(6): 709-730, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28407717

RESUMO

Transposable elements (TE), small mobile genetic elements unable to exist independently of the host genome, were initially believed to be exclusively deleterious genomic parasites. However, it is now clear that they play an important role as bacterial mutagenic agents, enabling the host to adapt to new environmental challenges and to colonize new niches. This review focuses on the impact of insertion sequences (IS), arguably the smallest TE, on bacterial genome plasticity and concomitant adaptability of phenotypic traits, including resistance to antibacterial agents, virulence, pathogenicity and catabolism. The direct consequence of IS transposition is the insertion of one DNA sequence into another. This event can result in gene inactivation as well as in modulation of neighbouring gene expression. The latter is usually mediated by de-repression or by the introduction of a complete or partial promoter located within the element. Furthermore, transcription and transposition of IS are affected by host factors and in some cases by environmental signals offering the host an adaptive strategy and promoting genetic variability to withstand the environmental challenges.


Assuntos
Bactérias/genética , Plasticidade Celular/genética , Elementos de DNA Transponíveis/genética , Regulação Bacteriana da Expressão Gênica/genética , Genoma Bacteriano/genética , Bactérias/metabolismo , Bactérias/patogenicidade , Farmacorresistência Bacteriana/genética , Virulência/genética
15.
RNA Biol ; 14(1): 6-10, 2017 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-27834591

RESUMO

In a recent publication, we reported a unique interaction between a protein encoded by the giant myovirus phiKZ and the Pseudomonas aeruginosa RNA degradosome. Crystallography, site-directed mutagenesis and interactomics approaches revealed this 'degradosome interacting protein' or Dip, to adopt an 'open-claw' dimeric structure that presents acidic patches on its outer surface which hijack 2 conserved RNA binding sites on the scaffold domain of the RNase E component of the RNA degradosome. This interaction prevents substrate RNAs from being bound and degraded by the RNA degradosome during the virus infection cycle. In this commentary, we provide a perspective into the biological role of Dip, its structural analysis and its mysterious evolutionary origin, and we suggest some therapeutic and biotechnological applications of this distinctive viral protein.


Assuntos
Bactérias/genética , Bactérias/virologia , Bacteriófagos/fisiologia , Interações Hospedeiro-Patógeno/genética , RNA Bacteriano/genética , Bactérias/efeitos dos fármacos , Bactérias/metabolismo , Endorribonucleases/genética , Endorribonucleases/metabolismo , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Polirribonucleotídeo Nucleotidiltransferase/genética , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , Ligação Proteica , Pseudomonas aeruginosa/fisiologia , Pseudomonas aeruginosa/virologia , RNA Helicases/genética , RNA Helicases/metabolismo , Estabilidade de RNA , RNA Bacteriano/metabolismo
16.
Antimicrob Agents Chemother ; 60(6): 3480-8, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27021321

RESUMO

Bacteriophage-encoded endolysins have shown promise as a novel class of antibacterials with a unique mode of action, i.e., peptidoglycan degradation. However, Gram-negative pathogens are generally not susceptible due to their protective outer membrane. Artilysins overcome this barrier. Artilysins are optimized, engineered fusions of selected endolysins with specific outer membrane-destabilizing peptides. Artilysin Art-175 comprises a modified variant of endolysin KZ144 with an N-terminal fusion to SMAP-29. Previously, we have shown the high susceptibility of Pseudomonas aeruginosa to Art-175. Here, we report that Art-175 is highly bactericidal against stationary-phase cells of multidrug-resistant Acinetobacter baumannii, even resulting in a complete elimination of large inocula (≥10(8) CFU/ml). Besides actively dividing cells, Art-175 also kills persisters. Instantaneous killing of A. baumannii upon contact with Art-175 could be visualized after immobilization of the bacteria in a microfluidic flow cell. Effective killing of a cell takes place through osmotic lysis after peptidoglycan degradation. The killing rate is enhanced by the addition of 0.5 mM EDTA. No development of resistance to Art-175 under selection pressure and no cross-resistance with existing resistance mechanisms could be observed. In conclusion, Art-175 represents a highly active Artilysin against both A. baumannii and P. aeruginosa, two of the most life-threatening pathogens of the order Pseudomonadales.


Assuntos
Infecções por Acinetobacter/tratamento farmacológico , Acinetobacter baumannii/efeitos dos fármacos , Antibacterianos/farmacologia , Catelicidinas/farmacologia , Endopeptidases/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Infecções por Acinetobacter/microbiologia , Farmacorresistência Bacteriana Múltipla , Ácido Edético/farmacologia , Endopeptidases/química , Humanos , Testes de Sensibilidade Microbiana , Técnicas Analíticas Microfluídicas , Pseudomonas aeruginosa/efeitos dos fármacos
17.
Appl Environ Microbiol ; 82(22): 6656-6663, 2016 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-27590820

RESUMO

The development of resistance in foodborne pathogens to food preservation techniques is an issue of increasing concern, especially in minimally processed foods where safety relies on hurdle technology. In this context, mild heat can be used in combination with so-called nonthermal processes, such as high hydrostatic pressure (HHP), at lower individual intensities to better retain the quality of the food. However, mild stresses may increase the risk of (cross-)resistance development in the surviving population, which in turn might compromise food safety. In this investigation, we examined the evolution of Escherichia coli O157:H7 strain ATCC 43888 after recurrent exposure to progressively intensifying mild heat shocks (from 54.0°C to 60.0°C in 0.5°C increments) with intermittent resuscitation and growth of survivors. As such, mutant strains were obtained after 10 cycles of selection with ca. 106-fold higher heat resistance than that for the parental strain at 58.0°C, although this resistance did not extend to temperatures exceeding 60.0°C. Moreover, these mutant strains typically displayed cross-resistance against HHP shock and displayed signs of enhanced RpoS and RpoH activity. Interestingly, additional cycles of selection maintaining the intensity of the heat shock constant (58.5°C) selected for mutant strains in which resuscitation speed, rather than resistance, appeared to be increased. Therefore, it seems that resistance and resuscitation speed are rapidly evolvable traits in E. coli ATCC 43888 that can compromise food safety. IMPORTANCE: In this investigation, we demonstrated that Escherichia coli O157:H7 ATCC 43888 rapidly acquires resistance to mild heat exposure, with this resistance yielding cross-protection to high hydrostatic pressure treatment. In addition, mutants of E. coli ATCC 43888 in which resuscitation speed, rather than resistance, appeared to be improved were selected. As such, both resistance and resuscitation speed seem to be rapidly evolvable traits that can compromise the control of foodborne pathogens in minimal processing strategies, which rely on the efficacy of combined mild preservation stresses for food safety.


Assuntos
Escherichia coli O157/crescimento & desenvolvimento , Resposta ao Choque Térmico , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Contagem de Colônia Microbiana , Evolução Molecular Direcionada , Escherichia coli O157/genética , Escherichia coli O157/patogenicidade , Fast Foods/microbiologia , Manipulação de Alimentos , Microbiologia de Alimentos , Conservação de Alimentos , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Temperatura Alta , Humanos , Pressão Hidrostática , Mutação , Fator sigma/genética , Fator sigma/metabolismo
18.
Nucleic Acids Res ; 42(6): 3908-18, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24423871

RESUMO

In this study, we examined the intracellular whereabouts of Mrr, a cryptic type IV restriction endonuclease of Escherichia coli K12, in response to different conditions. In absence of stimuli triggering its activity, Mrr was found to be strongly associated with the nucleoid as a number of discrete foci, suggesting the presence of Mrr hotspots on the chromosome. Previously established elicitors of Mrr activity, such as exposure to high (hydrostatic) pressure (HP) or expression of the HhaII methyltransferase, both caused nucleoid condensation and an unexpected coalescence of Mrr foci. However, although the resulting Mrr/nucleoid complex was stable when triggered with HhaII, it tended to be only short-lived when elicited with HP. Moreover, HP-mediated activation of Mrr typically led to cellular blebbing, suggesting a link between chromosome and cellular integrity. Interestingly, Mrr variants could be isolated that were specifically compromised in either HhaII- or HP-dependent activation, underscoring a mechanistic difference in the way both triggers activate Mrr. In general, our results reveal that Mrr can take part in complex spatial distributions on the nucleoid and can be engaged in distinct modes of activity.


Assuntos
Enzimas de Restrição do DNA/análise , Enzimas de Restrição do DNA/metabolismo , Proteínas de Escherichia coli/análise , Proteínas de Escherichia coli/metabolismo , Enzimas de Restrição do DNA/genética , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo III , Proteínas de Escherichia coli/genética , Pressão Hidrostática , Mutação
19.
PLoS Genet ; 9(2): e1003269, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23483857

RESUMO

We discovered a novel interaction between phage P22 and its host Salmonella Typhimurium LT2 that is characterized by a phage mediated and targeted derepression of the host dgo operon. Upon further investigation, this interaction was found to be instigated by an ORFan gene (designated pid for phage P22 encoded instigator of dgo expression) located on a previously unannotated moron locus in the late region of the P22 genome, and encoding an 86 amino acid protein of 9.3 kDa. Surprisingly, the Pid/dgo interaction was not observed during strict lytic or lysogenic proliferation of P22, and expression of pid was instead found to arise in cells that upon infection stably maintained an unintegrated phage chromosome that segregated asymmetrically upon subsequent cell divisions. Interestingly, among the emerging siblings, the feature of pid expression remained tightly linked to the cell inheriting this phage carrier state and became quenched in the other. As such, this study is the first to reveal molecular and genetic markers authenticating pseudolysogenic development, thereby exposing a novel mechanism, timing, and populational distribution in the realm of phage-host interactions.


Assuntos
Bacteriófago P22 , Interações Hospedeiro-Patógeno/genética , Salmonella typhimurium , Bacteriófago P22/genética , Bacteriófago P22/crescimento & desenvolvimento , Portador Sadio , Regulação Bacteriana da Expressão Gênica , Genoma , Lisogenia/genética , Lisogenia/fisiologia , Salmonella typhimurium/genética , Salmonella typhimurium/fisiologia
20.
Environ Microbiol ; 17(5): 1586-99, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25142185

RESUMO

Many bacteria are able to assume a transient cell wall-deficient (or L-form) state under favourable osmotic conditions. Cell wall stress such as exposure to ß-lactam antibiotics can enforce the transition to and maintenance of this state. L-forms actively proliferate and can return to the walled state upon removal of the inducing agent. We have adopted Escherichia coli as a model system for the controlled transition to and reversion from the L-form state, and have studied these dynamics with genetics, cell biology and 'omics' technologies. As such, a transposon mutagenesis screen underscored the requirement for the Rcs phosphorelay and colanic acid synthesis, while proteomics show only little differences between rods and L-forms. In contrast, metabolome comparison reveals the high abundance of lysophospholipids and phospholipids with unsaturated or cyclopropanized fatty acids in E. coli L-forms. This increase of membrane lipids associated with increased membrane fluidity may facilitate proliferation through bud formation. Visualization of the residual peptidoglycan with a fluorescently labelled peptidoglycan binding protein indicates de novo cell wall synthesis and a role for septal peptidoglycan synthesis during bud constriction. The DD-carboxypeptidases PBP5 and PBP6 are threefold and fourfold upregulated in L-forms, indicating a specific role for regulation of crosslinking during L-form proliferation.


Assuntos
Parede Celular/metabolismo , Escherichia coli/metabolismo , Lipídeos de Membrana/metabolismo , Peptidoglicano/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/genética , Biblioteca Gênica , Modelos Biológicos , Proteínas de Ligação às Penicilinas/biossíntese , Proteínas de Ligação às Penicilinas/genética , D-Ala-D-Ala Carboxipeptidase Tipo Serina/biossíntese , D-Ala-D-Ala Carboxipeptidase Tipo Serina/genética , Resistência beta-Lactâmica/genética , beta-Lactamas/farmacologia
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