RESUMO
Virulent lactococcal phages of the Siphoviridae family are responsible for the industrial milk fermentation failures worldwide. Lactococcus lactis, a Gram-positive bacterium widely used for the manufacture of fermented dairy products, is subjected to infections by virulent phages, predominantly those of the 936 group, including phage p2. Among the proteins coded by lactococcal phage genomes, of special interest are those expressed early, which are crucial to efficiently carry out the phage lytic cycle. We previously identified and solved the 3D structure of lactococcal phage p2 ORF34, a single stranded DNA binding protein (SSBp2). Here we investigated the molecular basis of ORF34 binding mechanism to DNA. DNA docking on SSBp2 and Molecular Dynamics simulations of the resulting complex identified R15 as a crucial residue for ssDNA binding. Electrophoretic Mobility Shift Assays (EMSA) and Atomic Force Microscopy (AFM) imaging revealed the inability of the Arg15Ala mutant to bind ssDNA, as compared to the native protein. Since R15 is highly conserved among lactococcal SSBs, we propose that its role in the SSBp2/DNA complex stabilization might be extended to all the members of this protein family.
Assuntos
Bacteriófago P2/metabolismo , DNA de Cadeia Simples/metabolismo , DNA Viral/metabolismo , Proteínas de Ligação a DNA/metabolismo , Lactococcus lactis/virologia , Proteínas Virais/metabolismo , Bacteriófago P2/genética , DNA de Cadeia Simples/genética , DNA Viral/genética , Proteínas de Ligação a DNA/genética , Ensaio de Desvio de Mobilidade Eletroforética/métodos , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Microscopia de Força Atômica/métodos , Simulação de Acoplamento Molecular/métodos , Simulação de Dinâmica Molecular , Mutação , Dobramento de Proteína , Proteínas Virais/genéticaRESUMO
At weaning, piglets are exposed to many stressors, such as separation from the sow, mixing with other litters, end of lactational immunity, and a change in their environment and gut microbiota. The sudden change of feeding regime after weaning causes morphological and histological changes in the small intestine which are critical for the immature digestive system. Sixteen female piglets were studied to assess the effect of sorbic acid supplementation on the small intestine tissue transcriptome. At weaning day (T0, piglet age 28 days), four piglets were sacrificed and ileal tissue samples collected. The remaining 12 piglets were weighed and randomly assigned to different postweaning (T5, piglet age 33 days) diets. Diet A (n = 6) contained 5 g/kg of sorbic acid. In diet B (n = 6), the organic acids were replaced by barley flour. Total RNA was isolated and then hybridized to CombiMatrix CustomArray™ 90-K platform microarrays, screening about 30 K genes. Even though diet had no detectable effect on the transcriptome during the first 5 days after weaning, results highlighted some of the response mechanisms to the stress of weaning occurring in the piglet gut. A total of 205 differentially expressed genes were used for functional analysis using the bioinformatics tools BLAST2GO, Ingenuity Pathway Analysis 8.0, and Dynamic Impact Approach (DIA). Bioinformatic analysis revealed that apoptosis, RIG-I-like, and NOD-like receptor signaling were altered as a result of weaning. Interferons and caspases gene families were the most activated after weaning in response to piglets to multiple stressors. Results suggest that immune and inflammatory responses were activated and likely are a cause of small intestine atrophy as revealed by a decrease in villus height and villus/crypt ratio.
Assuntos
Imunidade , Inflamação/imunologia , Inflamação/patologia , Intestino Delgado/imunologia , Intestino Delgado/patologia , Sus scrofa/imunologia , Desmame , Animais , Animais Recém-Nascidos , Dieta , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Ontologia Genética , Redes Reguladoras de Genes/genética , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Transdução de Sinais/genética , Sus scrofa/sangue , Sus scrofa/genéticaRESUMO
The recent evolution of cattle is marked by fluctuations in body size. Height in the Bos taurus lineage was reduced by a factor of ~1.5 from the Neolithic to the Middle Ages, and increased again only during the Early Modern Ages. Using haplotype analysis, we found evidence that the bovine PLAG1 mutation (Q) with major effects on body size, weight and reproduction is a >1,000 years old derived allele that increased rapidly in frequency in Northwestern European B. taurus between the 16th and 18th centuries. Towards the 19th and 20th centuries, Q was introgressed into non-European B. taurus and Bos indicus breeds. These data implicate a major role of Q in recent changes in body size in modern cattle, and represent one of the first examples of a genomic sweep in livestock that was driven by selection on a complex trait.