Plasmid pUM505 encodes a Toxin-Antitoxin system conferring plasmid stability and increased Pseudomonas aeruginosa virulence.

Pseudomonas aeruginosa plasmid pUM505 possesses a pathogenicity island that contains the pumAB genes that encode products with sequence similarity to Toxin-Antitoxin (TA) modules. RT-PCR assays on the overlapping regions of the pumAB genes generated a bicistronic messenger RNA, suggesting that they form an operon. When the pumAB genes were cloned into the pJET vector, recombinant plasmid pJET-pumAB was maintained under nonselective conditions in Escherichia coli cells after six daily subcultures, whereas pJET without pumAB genes was lost. These data indicate that pumAB genes confer post-segregational plasmid stability. In addition, overexpression of the PumA protein in the E. coli BL21 strain resulted in a significant growth inhibition, while BL21 co-expressing the PumA and PumB proteins did not show growth inhibition. These results indicate that pumAB genes encode a TA system where the PumB protein counters the toxic effects of the PumA toxin. Furthermore, P. aeruginosa PAO1 transformants with the pumA gene increased Caenorhabditis elegans and mouse mortality rate and improved mouse organ invasion, effects neutralized by the PumB protein. Moreover, purified recombinant His-PumA protein decreased the viability of C. elegans, indicating that the PumA protein could acts as a toxin. These results indicate that PumA has the potential to promoter the PAO1 virulence against C. elegans and mice when is expressed in absence of PumB. This is the first description, to our knowledge, of a plasmid-encoded TA system that confers plasmid stability and encoded a toxin with the possible ability to increase the P. aeruginosa virulence.

Genes from pUM505 plasmid contribute to Pseudomonas aeruginosa virulence.

The pUM505 plasmid was isolated from a clinical strain of Pseudomonas aeruginosa. This plasmid contains a genomic island with sequence similar to islands found in chromosomes of virulent P. aeruginosa clinical isolates. The objective of this work was to determine whether pUM505 increases the virulence of P. aeruginosa and to identify the genes responsible for this property. First, using the lettuce-leaf model, we found that pUM505 significantly increases the virulence of P. aeruginosa reference strain PAO1. pUM505 also increased the PAO1 virulence in a murine model and increased cytotoxicity of this strain toward HeLa cells. Thus, we generated a pUM505 gene library of 103 clones in the pUCP20 binary vector. The library was transferred to Escherichia coli TOP10 and P. aeruginosa PAO1 to identify genes. The lettuce-leaf model allowed us to identify three recombinant plasmids that increased the virulence of both E. coli and P. aeruginosa strains. These recombinant plasmids also increased the virulence of the PAO1 strain in mice and induced a cytotoxic effect in HeLa cells. Eleven genes were identified in the virulent transformants. Of these genes, only the pUM505 ORF 2 has homology with a gene previously implicated in virulence. These results indicate that pUM505 contains several genes that encode virulence factors, suggesting that the plasmid may contribute directly to bacterial virulence.

[Effect of dietary fiber in the quantitative expression of butyrate receptor GPR43 in rats colon]. / Efecto del consumo de la fibra dietética en la expresión cuantitativa del receptor de butirato GPR43 en colon de ratas.

INTRODUCTION: Short chain fatty acids (SCFA) acetate, propionate and butyrate are the major anions produced by the bacterial fermentation of dietary fiber (DF) in colon. Recently, butyrate has been recently studied because is important to maintain colonic functions and because it has been related with a protective effect in colorectal cancer, which is mainly, explained by its potential to regulate gene expression by inhibiting enzyme histonedeacetylase (HDAC). Several investigationsshown that SCFAreceptor GPR43 is involved insignal transduction mechanisms once they bind to ligands such as butyrate to generate different physiological effects in colonocytes. OBJECTIVE: Determine if dietary fiber consumption from nopal (Opuntia ficus I.) containing a ratio of soluble-insoluble fiber 40/60, has a direct influence on the quantitative expression of butyrate-specific receptor GPR43. METHODS: Wistar rats were fed with four different diets formulated at different concentrations of dietary fiber of 0, 5, 15 and 25% of dietary fiber from opuntia, respectively. RESULTS AND DISCUSSION: The results shown an increase in the expression of GPR43 (93.1%) when rats was fed with a 5% fiber diet, using ß-actin as a reference gene. The results of this investigation will contribute to determinate the relation of diet with intestinal health for the purpose of expanding the knowledge of butyric acid on colonic functions.

Efecto del consumo de la fibra dietética en la expresión cuantitativa del receptor de butirato GPR43 en colon de ratas / Effect of dietary fiber in the quantitative expression of butyrate receptor GPR43 in rats colon

Introducción: Los ácidos grasos de cadena corta (AGCC) acetato, propionato y butirato, son productos de fermentación de la fibra dietética (FD) en el intestino grueso. Recientemente, el butirato ha sido estudiado ya que es considerado indispensable para el mantenimiento de las funciones del colon y por su relación con la protección del cáncer colorrectal. Esto se atribuye a la capacidad de butirato de regular la expresión génica por mecanismos como la inhibición de la enzima histona deacetilasa. Se ha reportado que el receptor de AGCC, GPR43 está involucrado en el proceso de transducción de señales intracelulares una vez que se unen a ligandos como butirato para generar los efectos fisiológicos del butirato en los colonocitos. Objetivo: Determinar si el consumo de FD de nopal (Opuntia ficus I) tiene influencia directa sobre la expresión cuantitativa del receptor específico de butirato GPR43. Métodos: Ratas adultas Wistar se sometieron a cuatro diferentes dietas variando el contenido de FD en 0, 5, 15 y 25% de FD denopal, respectivamente. Resultados y discusión: Los resultados mostraron un aumento significativo de la expresión relativa de GPR43 (93,1%) cuando se suministró a las ratas una dieta conteniendo 5% de FD de nopal, usando como gen de referencia β-actina. Los resultados de esta investigación aportarán nuevos datos a los estudios que determinan la relación de la dieta con la salud intestinal, con el fin de ampliar el conocimiento sobre los efectos del ácido butírico en las funciones colónicas (AU)

Introduction: Short chain fatty acids (SCFA) acetate, propionate and butyrate are the major anions produced by the bacterial fermentation of dietary fiber (DF) in colon. Recently, butyrate has been recently studied because is important to maintain colonic functions and because it has been related with a protective effect in colorectal cancer, which is mainly, explained by its potential to regulate gene expression by inhibiting enzyme histonedeacetylase (HDAC). Several investigationsshown that SCFAreceptor GPR43 is involved insignal transduction mechanisms once they bind to ligands such as butyrate to generate different physiological effects in colonocytes. Objective: Determine if dietary fiber consumption from nopal (Opuntia ficus I.) containing a ratio of soluble-insoluble fiber 40/60, has a direct influence on the quantitative expression of butyrate-specific receptor GPR43. Methods: Wistar rats were fed with four different diets formulated at different concentrations of dietary fiber of 0, 5, 15 and 25% of dietary fiber from opuntia, respectively. Results and discussion: The results shown an increase in the expression of GPR43 (93.1%) when rats was fed with a 5% fiber diet, using β-actin as a reference gene. The results of this investigation will contribute to determinate the relation of diet with intestinal health for the purpose of expanding the knowledge of butyric acid on colonic functions (AU)

Transformation of Mucor circinelloides with autoreplicative vectors containing homologous and heterologous ARS elements and the dominant Cbx(r) carboxine-resistance gene.

Mucor circinelloides transformants prototrophic to leucine and resistant to carboxine (Leu(+) Cbx(r)) have been obtained by treatment of protoplasts with plasmid constructs containing homologous leuA gene and adjacent autonomously replicating sequences (ARS) element combined with the Cbx(r)(carboxine-resistance) gene of Ustilago maydis and ARS sequences from this basidiomycete (plasmid pGG37) or from the 2 mu plasmid of Saccharomyces cerevisiae (plasmid pGG43). The presence in the same plasmid molecule of the M. circinelloides leuA gene and adjacent ARS element together with heterologous ARS elements produced an increase in the transformation frequency of about 65-120%. The presence of autoreplicating plasmid molecules in the transformants was demonstrated by mitotic stability experiments, by Southern analysis, and by the rescue of plasmids from transformed bacterial cells.