Para tanto, foram definidos os seguintes objetivos específicos:
- Selecionar clones de C. violaceum que expressem genes relacionados à tolerância e/ou resistência a luz ultravioleta (UVC);
- Seqüenciar clones selecionados e analisar as seqüências in silico para identificação de genes e predição de possíveis funções;
- Analisar os clones de interesse por ensaios de complementação funcional e mutagenicidade;
- Identificar por RT-PCR (Transcriptase Reversa) as possíveis ORFs em operon, bem como, analisar sua expressão a partir de variações e análise relativa.
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6 Conclusões
- Neste trabalho um operon em C. Violaceum indutível em resposta ao tratamento foi identificado envolvido com resposta ao estresse. Apesar deste operon ser encontrado em outros organismos, sua ativação sob estresse não havia sido relatada anteriormente;
- O sinergismo observado em relação ao aumento da sobrevivência de DH10B na presença das ORFs CV_3722 e CV_3724 após tratamento com UV sugere que as proteínas codificadas por estes genes atuam em resposta ao estresse em vias independentes;
- A ORF CV_3724 é homologa a uma proteína hipotética conservada e nossos dados abrem perspectivas para a caracterização de sua função em relação à resposta ao estresse;
- Podemos destacar uma possível contribuição das ORFs CV_3721 (PilZ) e CV_3723 (TMK), uma vez que estas podem estar envolvidas em processos que podem favorecer o crescimento sob estresse, como a produção de biofilme e síntese de desoxinucleotídeos, respectivamente;
- A ORF CV_3722 (HolB) devido a sua contribuição a mutagênese pode complementar a deficiência de RecA por sua atividade sobre a ativação do clamp
ter um efeito estimulante na atividade da Pol IV;
- Os clones PLE1G, PLE7B, PLE10B e PLE12H complementam a cepa DH10B por desempenharem funções à resposta celular frente ao estresse;
- A ORF CV_1667 (PLE1G) que apresenta domínio GGDEF com função relacionada a biossíntese do nucleotídeo c-di-GMP (2º mensageiro) deve favorecer a sobrevivência do organismo devido a função regulatória deste mensageiro, o que permite uma resposta inter-relacionada com outras proteínas, como a PilZ (ORF CV_3721) na formação de biofilme;
- A ORF CV_0391 que codifica uma proteína hipotética com domínios ICL/PEMP) (PLE7B) pode estar atuando no aumento de mutagenicidade no clone devido à atividade desta proteína no fornecimento de energia, evento primordial aos processos celulares de defesa e sobrevivência;
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- A capacidade de complementação e mutagenicidade do clone PLE10B na da cepa DH10B não pode ser indicado devido a não identificação das ORFs hipotéticas (CV_1311 e 1312 sem domínios conservados), bem como, da não associação de função específica da Isonitrila Hidratase (ORF CV_1313) relacionada a estas atividades;
- Mesmo de forma redundante, a ORF CV_4160 (subunidade a da RNA Pol de C. violaceum) deve ter contribuído na complementação (viabilidade) da cepa DH10B, já que atua na transcrição, processo essencial na resposta celular a agentes estressores;
- Os dados obtidos neste trabalho são relevantes uma vez que a resposta ao estresse em bactérias de vida livre é mal compreendido.
86
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