Discriminating between viable and membrane-damaged cells of the plant pathogen Xylella fastidiosa.

Xylella fastidiosa is a plant pathogenic bacterium with devastating consequences to several crops of economic importance across the world. While this pathogen has been studied for over a century in the United States, several aspects of its biology remain to be investigated. Determining the physiological state of bacteria is essential to understand the effects of its interactions with different biotic and abiotic factors on cell viability. Although X. fastidiosa is culturable, its slow growing nature makes this technique cumbersome to assess the physiological state of cells present in a given environment. PMA-qPCR, i.e. the use of quantitative PCR combined with the pre-treatment of cells with the dye propidium monoazide, has been successfully used in a number of studies on human pathogens to calculate the proportion of viable cells, but has less frequently been tested on plant pathogens. We found that the use of a version of PMA, PMAxx, facilitated distinguishing between viable and non-viable cells based on cell membrane integrity in vitro and in planta. Additional experiments comparing the number of culturable, viable, and total cells in planta would help further confirm our initial results. Enhancers, intended to improve the efficacy of PMAxx, were not effective and appeared to be slightly toxic to X. fastidiosa

The Mqsra Toxin-antitoxin System From Xylella Fastidiosa Plays A Key Role In Bacterial Fitness, Pathogenicity, And Persister Cell Formation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Through the formation of persister cells, bacteria exhibit tolerance to multidrug and other environmental stresses without undergoing genetic changes. The toxin-antitoxin (TA) systems are involved in the formation of persister cells because they are able to induce cell dormancy. Among the TA systems, the MqsRA system has been observed to be highly induced in persister cells of Xylella fastidiosa (causal agent of citrus variegated chlorosis-CVC) activated by copper stress, and has been described in Escherichia coil as related to the formation of persister cells and biofilms. Thus, we evaluated the role of this TA system in X. fastidiosa by overexpressing the MqsR toxin, and verified that the toxin positively regulated biofilm formation and negatively cell movement, resulting in reduced pathogenicity in citrus plants. The overexpression of MqsR also increased the formation of persister cells under copper stress. Analysis of the gene and protein expression showed that this system likely has an autoregulation mechanism to express the toxin and antitoxin in the most beneficial ratio for the cell to oppose stress. Our results suggest that this TA system plays a key role in the adaptation and survival of X fastidiosa and reveal new insights into the physiology of phytopathogen host interactions.7Fundacao de Amparo a Pesquisa do Estado de Sao Paulo [2010/50712-9, 2013/17485-7]CNPqFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

The MqsRA toxin-antitoxin system from xylella fastidiosa plays a key role in bacterial fitness, pathogenicity, and persister cell formation

Through the formation of persister cells, bacteria exhibit tolerance to multidrug and other environmental stresses without undergoing genetic changes. The toxin-antitoxin (TA) systems are involved in the formation of persister cells because they are able to induce cell dormancy. Among the TA systems, the MqsRA system has been observed to be highly induced in persister cells of Xylella fastidiosa (causal agent of citrus variegated chlorosis-CVC) activated by copper stress, and has been described in Escherichia coil as related to the formation of persister cells and biofilms. Thus, we evaluated the role of this TA system in X. fastidiosa by overexpressing the MqsR toxin, and verified that the toxin positively regulated biofilm formation and negatively cell movement, resulting in reduced pathogenicity in citrus plants. The overexpression of MqsR also increased the formation of persister cells under copper stress. Analysis of the gene and protein expression showed that this system likely has an autoregulation mechanism to express the toxin and antitoxin in the most beneficial ratio for the cell to oppose stress. Our results suggest that this TA system plays a key role in the adaptation and survival of X fastidiosa and reveal new insights into the physiology of phytopathogen host interactions7CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPnão tem2010/50712-9; 2013/17485-7; 2013/02014-

Draft Genome Sequence Of 11399, A Transformable Citrus-pathogenic Strain Of Xylella Fastidiosa

The draft genome of Xylella fastidiosa subsp. pauca strain 11399, a transformable citrus-pathogenic strain, is reported here. The 11399 genome size is 2,690,704 bp and has a G+C content of 52.7%. The draft genome of 11399 reveals the absence of four type I restriction-modification system genes. © 2016 Niza et al.4

Determinação de metais pesados no caranguejo comestível Ucides cordatus (Crustacea: Decapoda)

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Determinação de metais pesados no caranguejo comestível Ucides cordatus (Crustacea: Decapoda)

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Determinação de metais pesados no caranguejo comestível Ucides cordatus (Crustacea: Decapoda)

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Study of the functional role of the toxin-antitoxin system XF2490/XF2491 of Xylella fastidiosa under copper stress

Orientador: Alessandra Alves de SouzaDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Através da formação de células persistentes, as bactérias são capazes de apresentar tolerância à multidrogas e à outros estresses ambientais sem que ocorram mudanças genéticas. Os sistemas toxina-antitoxina (TA) estão envolvidos com o processo de formação de células persistentes, uma vez que, eles são capazes de induzir a dormência celular. Entre os sistemas TA, o sistema mqsRA tem sido descrito em Escherichia coli como relacionado tanto com a formação de células persistentes, como com a formação de biofilme. Em X. fastidiosa, agente causal da Clorose Variegada dos Citros (CVC), o sistema TA mqsRA também foi previamente observado como altamente induzido em células persistentes desse organismo ativadas por estresse ao cobre. Desse modo, nesse trabalho foi avaliado o papel funcional desse sistema TA em X. fastidiosa. Através da superexpressão da toxina mqsR em X. fastidiosa, foi visto que esse sistema também está relacionado com o estilo de vida desta bactéria, pois induziu a formação de biofilme e reduziu a motilidade twitching. Esses resultados corroboraram com a expressão gênica, pois a superexpressão de mqsR induziu genes relacionados com o crescimento em biofilme e reprimiu genes relacionados com o movimento celular, evidenciando a capacidade regulatória desse sistema TA. Assim, essas características observadas resultaram na diminuição da patogenicidade in planta dessa bactéria. A formação de células persistentes sob estresse por cobre também aumentou com a superexpressão de mqsR, o que foi evidenciado pela maior taxa de sobrevivência celular e pela maior formação de células alongadas. Através da análise da expressão de mqsR e mqsA por PCR quantitativo em tempo real e das proteínas por Western blot, foi possível observar que esse sistema provavelmente possui um mecanismo de autorregulação que permite expressar a toxina e a antitoxina na proporção mais benéfica para a célula combater o estresse e retomar o crescimento rapidamente quando as condições se tornarem favoráveis. Desse modo, os resultados sugerem que esse sistema TA possui um papel fundamental na adaptação e sobrevivência da X. fastidiosa nos seus diferentes hospedeirosAbstract: Through the formation of persister cells, bacteria are capable of exhibiting tolerance to multidrug and other environmental stresses without undergoing genetic changes. The toxin-antitoxin (TA) systems are involved in the process of persister cells formation, since they are able to induce cell dormancy. Among the TA systems, the mqsRA system has been described in Escherichia coli as related both to the formation of persister cells, as with biofilm formation. In X. fastidiosa, the causal agent of Citrus Variegated Chlorosis (CVC), the mqsRA TA system has also been previously observed to be highly induced in persister cells of this organism activated by copper stress. Thus, in this study we evaluated the functional role of this TA system in X. fastidiosa. By overexpressing the mqsR toxin in X. fastidiosa, it was seen that this system is also related to the lifestyle of this bacterium, as it induced biofilm formation and reduced twitching motility. These results corroborated with the gene expression profile, since the mqsR overexpression induced genes related to growth in biofilm and repressed genes related to cell movement, indicating the regulatory ability of this TA system. Thus, these observed characteristics resulted in a reduced pathogenicity of this bacterium in planta. The formation of persister cells under copper stress also increased with the overexpression of mqsR, which was seen by increased cell survival and increased formation of elongated cells. Through the analysis of the expression of mqsR and mqsA by real-time quantitative PCR and of the proteins by Western blot, we observed that this system probably possess a self-regulatory mechanism that allows expressing the toxin and antitoxin in the most beneficial ratio for the cell to oppose the stress and resume growth quickly when conditions become favorable. Thus, the results suggest that this TA system has a key role in the adaptation and survival of X. fastidiosa in its different hostsMestradoGenetica de MicroorganismosMestre em Genética e Biologia Molecular2013/02014-9FAPES

Fitness strategies of fastidious prokaryotes

Fastidious prokaryotes are slow-growing bacteria that either require specific media to grow or are not able to grow axenically. ‘Candidatus Liberibacter asiaticus’ (CLas) and Xylella fastidiosa are important fastidious plant pathogenic bacteria that threaten agriculture worldwide. CLas is associated with Huanglongbing, the most devastating disease of citrus globally, whereas X. fastidiosa harms many economically important crops worldwide, including grapevine, citrus, and olive. In this study, we sought to understand the fitness of CLas in its natural environments based on published studies and aimed to replicate these conditions in vitro to increase its culturability, since CLas is hitherto unculturable axenically. Commercial grapefruit juice (GJ), which was used as base culture medium, was amended with a wide range of compounds, and incubated under different conditions to evaluate the optimization of CLas growth. A reproducible growth behavior in which CLas growth ratios were inversely proportional to the initial inoculum concentration was observed. Additionally, strategies to reduce the cell density allowed CLas to reach maximum growth as fast as 3 days post inoculation, but with no sustained exponential growth over time. Conversely, we performed a complete functional analysis of the molecular components of the type IV pili (TFP) machinery of X. fastidiosa that mediates both its twitching motility and natural competence, an important mechanism to generate genetic diversity and likely modulate its fitness within natural hosts. We determined the core components of TFP involved in these two traits, as well as screened the role for virulence in planta for some of these components. Remarkably, we identified a novel minor pilin, FimT3, which is the DNA receptor of the X. fastidiosa TFP, being thus essential for its natural competence by binding DNA. Moreover, we identified recently recombined genes within X. fastidiosa strains by performing whole genome analyses. Identified genes are involved in host colonization, regulation and signaling, host evasion, and nutrient acquisition, which are all important for the ecology of X. fastidiosa. We hope our results may help to better understand the fitness of these two pathogens, which may aid the development of strategies for controlling them