Characterization of a heat-resistant extracellular protease from Pseudomonas fluorescens 07A shows that low temperature treatments are more effective in deactivating its proteolytic activity

This work discusses the biological and biochemical characterization of an extracellular protease produced by Pseudomonas fluorescens. The enzyme has a molecular weight of 49.486 kDa and hydrolyzes gelatin, casein, and azocasein, but not BSA. Its maximum activity is found at 37°C and pH 7.5, but it retained almost 70% activity at pH 10.0. It was shown to be a metalloprotease inhibited by Cu2+, Ni2+, Zn2+, Hg2+, Fe2+, and Mg2+, but induced by Mn2+. After incubation at 100°C for 5 min, the enzyme presented over 40% activity, but only 14 to 30% when submitted to milder heat treatments. This behavior may cause significant problems under conditions commonly used for the processing and storage of milk and dairy products, particularly UHT milk. A specific peptide sequenced by mass spectrometer analysis allowed the identification of gene that encodes this extracellular protease in the genome of Pseudomonas fluorescens 07A strain. The enzyme has 477 AA and highly conserved Ca2+- and Zn2+-binding domains, indicating that Ca2+, the main ion in milk, is also a cofactor. This work contributes to the understanding of the biochemical aspects of enzyme activity and associates them with its sequence and structure. These findings are essential for the full understanding and control of these enzymes and the technological problems they cause in the dairy industry

Comparative genomics of Staphylococcus aureus associated with subclinical and clinical bovine mastitis.

Many efforts have been made to understand the pathogenesis of bovine mastitis to reduce losses and promote animal welfare. Staphylococcus aureus may cause bovine clinical mastitis, but it is mainly associated with subclinical infection, which is usually persistent and can easily reoccur. Here, we conducted a comparative genomic analysis between strains of S. aureus causing subclinical infection (Sau170, 302, 1269, 1364), previously sequenced by our group, and two well-characterized strains causing clinical mastitis (N305 and RF122) to find differences that could be linked to mastitis outcome. A total of 146 virulence-associated genes were compared and no appreciable differences were found between the bacteria. However, several nonsynonymous single nucleotide polymorphisms (SNPs) were identified in genes present in the subclinical strains when compared to RF122 and N305, especially in genes encoding host immune evasion and surface proteins. The secreted and surface proteins predicted by in silico tools were compared through multidimensional scaling analysis (MDS), revealing a high degree of similarity among the strains. The comparison of orthologous genes by OrthoMCL identified a membrane transporter and a lipoprotein as exclusive of bacteria belonging to the subclinical and clinical groups, respectively. No hit was found in RF122 and N305 for the membrane transporter using BLAST algorithm. For the lipoprotein, sequences of Sau170, 302, 1269, and 1364 with identities between 68-73% were found in the MDS dataset. A conserved region found only in the lipoprotein genes of RF122 and N305 was used for primer design. Although the polymerase chain reaction (PCR) on field isolates of S. aureus did not validate the findings for the transporter, the lipoprotein was able to separate the clinical from the subclinical isolates. These results show that sequence variation among bovine S. aureus, and not only the presence/absence of virulence factors, is an important aspect to consider when comparing isolates causing different mastitis outcomes

Evaluation of the genetic variability found in Brazilian commercial vaccines for infectious bronchitis virus

Infectious bronchitis virus (IBV) is currently one of the most important pathogens in the poultry industry. The H120 and Ma5 are the only viral strains approved by the Brazilian government as the constituent of vaccines. Despite the systematic vaccination in Brazil, IBV has not yet been controlled and diseases associated with this virus have been reported in vaccinated chickens. Here, we investigated the genetic variability of H120 and Ma5 strains present in the IBV vaccines from different Brazilian manufacturers. We performed DNA sequencing analyses of the S1 spike glycoprotein gene to investigate its genetic variability and the presence of viral subpopulations among vaccines, between batches, and also in each vaccine after a single passage was performed in chicken embryonated eggs. Our results revealed up to 13 amino acid substitutions among vaccines and some of them were localized in regions of the S1 glycoprotein that play a role in virus–host interaction. Secondary nucleotide peaks identified in the chromatogram for the S1 gene sequence revealed that all original vaccines (H120 and Ma5) were composed by different subpopulations of IBV. Moreover, new viral subpopulations were also found in vaccines after a single passage in chicken embryonated eggs. These findings indicate that H120 and Ma5 viral strains used in vaccines market in Brazil can still mutate very rapidly during replication, leading to amino acid substitutions in proteins involved in the stimulation of the immune response, such as the S1 glycoprotein. Therefore, our data suggest that the genetic variability of these viral strains should be taken into consideration to ensure an effective immune response against IBV

Dry matter production and distribution in three cassava (Manihot esculenta Crantz) cultivars during the second vegetative plant cycle

A study was carried out in Araruna County, State of Paraná, to understand the relationship between the total dry matter yield and its proportion allocated to the storage roots of cassava (Manihot esculenta Crantz) plants in the second vegetative cycle. The experimental design was a randomized complete block in split-plot scheme with four replications. The plots consisted of the Mico, IAC 13 and IAC 14 cultivars and the monthly harvesting dates were assessed in the sub-plots. The results showed that the Mico and IAC 13 cultivars were more efficient in allocating dry matter to the storage roots. The IAC 14 cultivar allocated a higher proportion of assimilates to stems compared with the other two cultivars. With regard to the influence of harvesting time, the lowest harvest indexes were observed in the periods of more intense vegetative growth. However, the highest carbohydrate proportions were allocated to the storage roots during periods of low vegetative growth.<br>Com o objetivo de uma melhor compreensão da relação entre a produtividade total de massa seca e a proporção de alocação desta nas raízes tuberosas em plantas de mandioca, foi conduzido no município de Araruna-PR, um experimento em delineamento de blocos casualizados, em esquema de parcelas subdivididas com quatro repetições. Nas parcelas foram dispostas três cultivares (Mico, IAC 13 e IAC 14), e nas subparcelas dez épocas de colheita mensais, a partir do início do segundo ciclo vegetativo das plantas. As cultivares Mico e IAC 13 foram mais eficientes do que a IAC 14 em alocar massa seca nas raízes tuberosas, ao passo que esta última alocou maior proporção de massa seca em suas hastes. Em relação às épocas, os menores índices de colheita ocorreram em períodos de mais intenso crescimento vegetativo das plantas, ao passo que a maior proporção de carboidratos foi alocada nas raízes tuberosas em períodos de baixo crescimento vegetativo

Genetic divergence in sweet cassava cultivars using morphological agronomic traits and RAPD molecular markers

In this work the genetic divergence among 14 sweet cassava cultivars was estimated by their morphological agronomic traits and RAPD molecular markers. The Tocher cluster analysis and the Nearest Neighbor Method were applied. The most dissimilar cultivars were Pão and Guaíra, Fécula Branca and Pão, and Pão and Caipira, while the most similar cultivar were the Fécula Branca and Branca 1, Branca 3 and Branca 1, and Guaíra and Branca 1. The Jaccard's coefficient showed that the most similar cultivars were Guaíra and Quarenta Quilos, while the most dissimilar were Branca 3 and Amarela da Rama Cinza. The divergence analysis indicated that promising crosses could be made between the Branca 3 cultivar and the Pão, Amarela 1, Fécula Branca and Amarela 2 cultivars for the high genetic divergence, favorable agronomic and culinary traits, and disease resistance on the part of at least one of the parents involved in the cross.<br>A divergência genética entre 14 cultivares de mandioca-de-mesa foi estimada mediante o uso de caracteres morfoagronômicos e de marcadores moleculares RAPD. As análises de agrupamento de Tocher e do Vizinho Mais Próximo foram efetuadas. As cultivares mais divergentes foram Pão e Guaíra, Fécula Branca e Pão, e Pão e Caipira, enquanto as mais similares foram Fécula Branca e Branca 1, Branca 3 e Branca 1, e Guaíra e Branca 1. O coeficiente de Jaccard indicou que as cultivares mais similares foram Guaíra e Quarenta Quilos, enquanto as mais dissimilares foram Branca 3 e Amarela da Rama Cinza. A análise de divergência indicou que cruzamentos promissores deveriam ser efetuados entre as cultivares Branca 3, Pão, Amarela 1, Fécula Branca e Amarela 2 pela alta divergência genética, características agronômicas e culinárias favoráveis, e resistência à doenças de parte de pelo menos um dos parentais envolvidos no cruzamento

Comparison of methods for phenotypic stability analysis of cassava (Manihot esculenta Crantz) genotypes for yield and storage root dry matter content

The objective of this work was to compare different phenotypic stability methods by using yield and storage root dry matter content data of eight cassava genotypes, assessed in eight environments in northwest of Paraná State, Brazil. All the methodologies applied showed to be able to study the stability of cassava genotypes, but each with its peculiarities. The methodologies of Eskridge, Annicchiarico and Lin and Binns were the most appropriated on situation with smaller effect of G x E interaction. The AMMI analysis and the Toler and Burrows methodology were the most specific on detailing specific adaptations of cassava genotypes to favorable and unfavorable environments. It could be suggested to use simultaneous AMMI analysis and Toler and Burrows methodology. The clone IAC 190-89 was the most promising.<br>O objetivo deste trabalho foi comparar diferentes metodologias de análise de estabilidade fenotípica considerando produção e teor de matéria seca nas raízes tuberosas de oito genótipos de mandioca, avaliados em oito ambientes na região Noroeste do Paraná. Todas as metodologias aplicadas se mostraram aptas no estudo da estabilidade dos genótipos avaliados, cada uma delas com suas particularidades. As metodologias de Eskridge, Annicchiarico e Lin e Binns se mostraram mais adequadas para situações de menor efeito da interação G x A. A análise AMMI e a metodologia de Toler e Burrows propiciaram um melhor detalhamento das adaptações específicas dos genótipos a ambientes favoráveis e desaforáveis. É sugerido o uso simultâneo da análise AMMI e da metodologia de Toler e Burrows. O clone IAC 190-89 mostrou-se mais promissor