Cytosolic protein degradation is crucial for cellular homeostasis as it orchestrates protein turnover by destruction of misfolded, unstable and abnormal proteins. This process has two main stages: (i) an ATP-dependent stage mediated by unfoldases and proteases, and (ii) an ATP-independent stage mediated by various peptidases. The ATP dependent proteases recognise target proteins and cleave them into smaller peptides. These enzymes comprise the ATPase-family-associated-with-various-cellular-activities domain that is important for unfolding target proteins. Subsequently, unfolded proteins enter a barrel-shaped proteolytic chamber, an architecture conserved throughout prokaryotes, archea and eukaryotes, where the peptide bond is hydrolysed in an ATP-independent manner. The smaller peptides released are broken down by ATP-independent peptidases into free amino acids recycled into the cellular pool. In prokaryotes, major cellular protein degradation functions are performed by Clp and Lon proteases. Earlier studies in our laboratory have shown the role of an ATP-independent peptidase, AminopeptidaseN, in sodium salicylate (NaSal) induced growth inhibition. NaSal belongs to the family of Non-Steroidal Anti-Inflammatory Drugs and its acetyl ester, Aspirin, is a very widely used analgesic. It is produced by plants as a defence response and is known to cause different effects, including xenobiotic stress i. e. stress mediated by compounds which are not naturally produced or expected to be present in the organism in bacteria. In bacteria, salicylate modulates outer membrane proteins, virulence factors, and reduces motility. In addition, NaSal is able induce “phenotypic antibiotic resistance” by binding to MarR and de-repressing the mar operon. NaSal promotes the dissociation of MarR from the promoter site leading to transcription of MarA, a transcription factor that induces several genes that encode the AcrAB-TolC pump which effluxes multiple antibiotics from the cell. The present study investigates the possible roles of ATP-dependent proteases, Lon and Clp, during growth reduction of E. coli induced with high (2-3 mM) amounts of NaSal. The growth of the Lon mutant (lon), but not clpP, was found to be greatly reduced with high doses of NaSal in the media. Our hypothesis was that the lack of Lon led to the accumulation of high amounts of substrate proteins, which led to its greater sensitivity with high doses of NaSal. To identify the substrate protein/s and to better understand the mechanism of action, single and double mutants (with lon) of E. coli lacking several prominent Lon substrates, i.e. MarA, RcsA, StpA, SulA and UmuD, were generated and screened for rescue of growth with 2-3 mM NaSal. MarA, a transcription factor, was identified to be important during NaSal-induced growth reduction. It modulates outer membrane proteins and induces the AcrAB-TolC pump that increases the efflux of antibiotics from the cell. Also, RT-PCR analysis revealed that the levels of marA and its targets, acrA and acrB, were higher in the lon strain suggesting that the MarA protein levels were stabilised the cell in the absence of Lon. Further studies using approriate strains demonstrated that one of the effectors of MarA, i.e. the AcrAB-TolC efflux pump, was not involved in the NaSal-induced growth inhibition of lon. Therefore, in presence of higher doses of NaSal, MarA is upregulated due to de-repression of the operon. The levels of MarA are regulated by Lon via degradation but in the absence of Lon, MarA levels are stabilised and lead to upregulation of MarA and its target genes like AcrAB-TolC. This study identifies higher amounts of MarA to be responsible for NaSal-induced growth inhibition of lon. Subsequently, experiments were conducted to demonstrate the role of MarA and its targets in antibiotic resistance with low dose (0.5 mM) NaSal that does not affect growth. This low dose of NaSal was able to upregulate marA and its target genes, acrA, acrB and tolC. Quantification of antibiotic resistance further revealed an induction in resistance by 0.5 mM NaSal in a MarA- and AcrB-dependent fashion. Studies using atomic force microscopy demonstrated that ciprofloxacin-induced cell elongation was lower in lon due to higher levels of MarA. Therefore, low dose of NaSal is capable of upregulating MarA and inducing antibiotic resistance but does not affect cell growth. This part of the study addresses the roles of Lon protease, its substrate MarA and MarA-induced targets, e.g. AcrB, during NaSal-mediated growth reduction and antibiotic resistance. The factors contributing to antibiotic resistance in bacteria are an important area of study for the global public health care system. Antibiotic resistance can be acquired by transmittance of genetic material, accumulation of antibiotic resistant mutations in the target molecule or can be induced by certain compounds present in the environment like NaSal. For rapid identification of compounds that may behave in a similar fashion as NaSal, a 96-well plate based screen was developed utilising the growth inhibition feature of the lon strain. The compounds were selected on the basis of their structural (phenolic compounds) and functional (Non-Steroidal Anti Inflammatory Drugs or NSAIDs) similarity to NaSal. Through this screen, four compounds were identified that lowered the growth of lon more than that of wild type strain and may be important in inducing phenotypic antibiotic resistance: Acetaminophen (anti-pyretic), Ibuprofen (NSAID), and two phenolic uncouplers, Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and 2,4-dinitrophenol (2,4-DNP). Notably, another compound Phenylbutazone (NSAID), which is used to lower inflammation in animals, did not reduce the growth of E. coli. RNA expression analysis revealed that these four compounds, but not phenylbutazone, induced the expression of marA and its target gene involved in antibiotic efflux, acrB. Furthermore, dose dependent and comparative studies with Nasal demonstrated differential effects of these four compounds in inducing antibiotic resistance with respect to ciprofloxacin, tetracycline and nalidixic acid. The two uncouplers were much more effective in inducing antibiotic resistance at lower doses than the NSAIDs. As NSAIDs are clinically important compounds, the study suggests that it would be desirable to screen them for induction of antibiotic resistance. The approach elucidated in this study has the potential to identify additional compounds present in the environment that may contribute to antibiotic resistance in bacteria. Overall, this study delineates the roles of Lon protease and its substrate, MarA, during NaSal-mediated responses, involving antibiotic resistance and/or growth reduction in E. coli. In addition, four other compounds were identified that could induce phenotypic antibiotic resistance in E. coli in a MarA-dependent manner. These observations may have implications in the adaptation of bacteria under different environmental conditions.

O objetivo do trabalho foi o desenvolvimento de tecnologias para melhoria da qualidade e vida de vaso em rosas (Rosa hybrida.) Vega. Os tratamentos précolheita foram realizados com aplicação de silício (0, 200, 400 e 800 mg L-1) por meio de fertirrigação via solo e via foliar. Foram realizadas seis aplicações semanais de silicato de potássio (12% Si) em rosas Vega, distribuídas em blocos casualisados com quatro repetições, e seis avaliações pós-colheita (dias 0, 2, 4, 6, 8 e 10). Após a colheita, foram avaliados: comprimento de haste, diâmetro de haste e de pedúnculo, comprimento de botão e a massa das hastes. As flores foram armazenadas em vasos com água deionizada a 20±1°C e 65±5% UR, sendo avaliadas: variação da massa fresca, conteúdo relativo de água (CRA) de pétalas e folhas, atividade de peroxidase, abertura floral, curvatura do pedúnculo, turgescência e escurecimento de pétalas, e coloração de pétalas. A aplicação de 400 mg L-1 de silício proporcionou maior comprimento de haste, maior CRA e cromaticidade das pétalas e menor atividade de peroxidase. A aplicação foliar também apresentou maior comprimento de botão, CRA de folhas, e luminosidade de pétalas, e a aplicação via solo também apresentou maior diâmetro de haste, maior massa inicial, menor variação de massa fresca e maior abertura floral, indicando que a aplicação de silício pode contribuir para melhorar a qualidade de rosas Vega. Os tratamentos pós-colheita foram realizados com aplicações de 0,1 mM de salicilato de metila (MeSa), 1 L L-1 de 1-metilciclopropeno (1-MCP), e 1 mM de ácido salicílico (AS). Após a aplicação dos tratamentos por 12 horas, as flores foram mantidas em vasos com água deionizada, armazenadas a 25±1°C e 65±5% U R. O delineamento experimental foi inteiramente casualisado, com quatro repetições por tratamento e seis avaliações (dias 0, 2, 4, 6, 8 e 10). Os tratamentos com AS e 1-MCP reduziram a atividade respiratória, produção de etileno, perda de massa e a curvatura do pedúnculo, tendo permitido maior manutenção do conteúdo relativo de água das pétalas, maior abertura floral, maior luminosidade das pétalas, e maior atividade de peroxidase. O AS também proporcionou melhor manutenção da turgescência, do conteúdo relativo de água das folhas, e menor escurecimento das pétalas. / The objective of this study was the development of technologies to increase the quality and vase life of cut roses (Rosa hybrida.) Vega. The pre-harvest treatments were the application of silicon (0, 200, 400 and 800 mg L-1) via foliar spray and root feeding, in quality of cut roses. Six weekly applications of potassium silicate (12% Si) were performed on roses Vega, distributed in a randomized block design with four replicates per treatment, and six evaluations (days 0, 2, 4, 6, 8 and 10). After harvest, the stem length, stem diameter, peduncle diameter, bud length and mass of the stems were evaluated. The flowers were kept in vases with deionized water, stored at 20±1°C and 65±5% RH, with evaluati ons of loss of fresh weight, leaf and petal relative water content (RWC), peroxidase activity, flower opening, bent neck, turgidity, petal darkening, and color of petals. The application of 400 mg L-1 of silicon provided increase in stem length, in petal RWC, petal chromaticity and reduced peroxidase activity. The application via foliar spray also increased bud length, the leaf RWC and the petal luminosity. The root feeding also increased the stem diameter, the floral opening, the initial mass, and reduced the fresh weight loss, indicating that the application of silicon can improve the quality of roses Vega. The postharvest treatments were the application of 0,1 mM of methyl salicylate (MeSa), 1 L L-1 of 1-methylcyclopropene (1-MCP), and 1 mM of salicylic acid (SA), in postharvest quality conservation of Vega roses. After the application of treatments for 12 hours, the flowers were kept in vases with deionized water, stored at 25±1°C and 65±5% RH. The experimental design was completely randomized, with four replications and six evaluations (days 0, 2, 4, 6, 8 and 10). The treatments with SA and 1-MCP reduced respiratory activity, ethylene production, loss of fresh weight and bent neck; and enable better maintenance of petal relative water content, largest flower opening and higher petal luminosity. SA also provided better maintenance of turgidity and leaf relative water content and less petal darkening.

O objetivo do trabalho foi o desenvolvimento de tecnologias para melhoria da qualidade e vida de vaso em rosas (Rosa hybrida.) Vega. Os tratamentos précolheita foram realizados com aplicação de silício (0, 200, 400 e 800 mg L-1) por meio de fertirrigação via solo e via foliar. Foram realizadas seis aplicações semanais de silicato de potássio (12% Si) em rosas Vega, distribuídas em blocos casualisados com quatro repetições, e seis avaliações pós-colheita (dias 0, 2, 4, 6, 8 e 10). Após a colheita, foram avaliados: comprimento de haste, diâmetro de haste e de pedúnculo, comprimento de botão e a massa das hastes. As flores foram armazenadas em vasos com água deionizada a 20±1°C e 65±5% UR, sendo avaliadas: variação da massa fresca, conteúdo relativo de água (CRA) de pétalas e folhas, atividade de peroxidase, abertura floral, curvatura do pedúnculo, turgescência e escurecimento de pétalas, e coloração de pétalas. A aplicação de 400 mg L-1 de silício proporcionou maior comprimento de haste, maior CRA e cromaticidade das pétalas e menor atividade de peroxidase. A aplicação foliar também apresentou maior comprimento de botão, CRA de folhas, e luminosidade de pétalas, e a aplicação via solo também apresentou maior diâmetro de haste, maior massa inicial, menor variação de massa fresca e maior abertura floral, indicando que a aplicação de silício pode contribuir para melhorar a qualidade de rosas Vega. Os tratamentos pós-colheita foram realizados com aplicações de 0,1 mM de salicilato de metila (MeSa), 1 L L-1 de 1-metilciclopropeno (1-MCP), e 1 mM de ácido salicílico (AS). Após a aplicação dos tratamentos por 12 horas, as flores foram mantidas em vasos com água deionizada, armazenadas a 25±1°C e 65±5% U R. O delineamento experimental foi inteiramente casualisado, com quatro repetições por tratamento e seis avaliações (dias 0, 2, 4, 6, 8 e 10). Os tratamentos com AS e 1-MCP reduziram a atividade respiratória, produção de etileno, perda de massa e a curvatura do pedúnculo, tendo permitido maior manutenção do conteúdo relativo de água das pétalas, maior abertura floral, maior luminosidade das pétalas, e maior atividade de peroxidase. O AS também proporcionou melhor manutenção da turgescência, do conteúdo relativo de água das folhas, e menor escurecimento das pétalas. / The objective of this study was the development of technologies to increase the quality and vase life of cut roses (Rosa hybrida.) Vega. The pre-harvest treatments were the application of silicon (0, 200, 400 and 800 mg L-1) via foliar spray and root feeding, in quality of cut roses. Six weekly applications of potassium silicate (12% Si) were performed on roses Vega, distributed in a randomized block design with four replicates per treatment, and six evaluations (days 0, 2, 4, 6, 8 and 10). After harvest, the stem length, stem diameter, peduncle diameter, bud length and mass of the stems were evaluated. The flowers were kept in vases with deionized water, stored at 20±1°C and 65±5% RH, with evaluati ons of loss of fresh weight, leaf and petal relative water content (RWC), peroxidase activity, flower opening, bent neck, turgidity, petal darkening, and color of petals. The application of 400 mg L-1 of silicon provided increase in stem length, in petal RWC, petal chromaticity and reduced peroxidase activity. The application via foliar spray also increased bud length, the leaf RWC and the petal luminosity. The root feeding also increased the stem diameter, the floral opening, the initial mass, and reduced the fresh weight loss, indicating that the application of silicon can improve the quality of roses Vega. The postharvest treatments were the application of 0,1 mM of methyl salicylate (MeSa), 1 L L-1 of 1-methylcyclopropene (1-MCP), and 1 mM of salicylic acid (SA), in postharvest quality conservation of Vega roses. After the application of treatments for 12 hours, the flowers were kept in vases with deionized water, stored at 25±1°C and 65±5% RH. The experimental design was completely randomized, with four replications and six evaluations (days 0, 2, 4, 6, 8 and 10). The treatments with SA and 1-MCP reduced respiratory activity, ethylene production, loss of fresh weight and bent neck; and enable better maintenance of petal relative water content, largest flower opening and higher petal luminosity. SA also provided better maintenance of turgidity and leaf relative water content and less petal darkening.