A soluble diacylglycerol acyltransferase is involved in triacylglycerol biosynthesis in the oleaginous yeast Rhodotorula glutinis

The biosynthesis of triacylglycerol (TAG) occurs in the microsomal membranes of eukaryotes. Here, we report the identification and functional characterization of diacylglycerol acyltransferase (DGAT), a member of the 10 S cytosolic TAG biosynthetic complex (TBC) in Rhodotorula glutinis. Both a full-length and an N-terminally truncated cDNA clone of a single gene were isolated from R. glutinis. The DGAT activity of the protein encoded by RgDGAT was confirmed in vivo by the heterologous expression of cDNA in a Saccharomyces cerevisiae quadruple mutant (H1246) that is defective in TAG synthesis. RgDGAT overexpression in yeast was found to be capable of acylating diacylglycerol (DAG) in an acyl-CoA-dependent manner. Quadruple mutant yeast cells exhibit growth defects in the presence of oleic acid, but wild-type yeast cells do not. In an in vivo fatty acid supplementation experiment, RgDGAT expression rescued quadruple mutant growth in an oleate-containing medium. We describe a soluble acyl-CoA-dependent DAG acyltransferase from R. glutinis that belongs to the DGAT3 class of enzymes. The study highlights the importance of an alternative TAG biosynthetic pathway in oleaginous yeasts.

Salmonella methylglyoxal detoxification by STM3117-encoded lactoylglutathione lyase affects virulence in coordination with Salmonella pathogenicity island 2 and phagosomal acidification

Intracellular pathogens such as Salmonella enterica serovar Typhimurium (S. Typhimurium) manipulate their host cells through the interplay of various virulence factors. A multitude of such virulence factors are encoded on the genome of S. Typhimurium and are usually organized in pathogenicity islands. The virulence-associated genomic stretch of STM3117-3120 has structural features of pathogenicity islands and is present exclusively in non-typhoidal serovars of Salmonella. It encodes metabolic enzymes predicted to be involved in methylglyoxal metabolism. STM3117-encoded lactoylglutathione lyase significantly impacts the proliferation of intracellular Salmonella. The deletion mutant of STM3117 (Delta lgl) fails to grow in epithelial cells but hyper-replicates in macrophages. This difference in proliferation outcome was the consequence of failure to detoxify methylglyoxal by Delta lgl, which was also reflected in the form of oxidative DNA damage and upregulation of kefB in the mutant. Within macrophages, the toxicity of methylglyoxal adducts elicits the potassium efflux channel (KefB) in the mutant which subsequently modulates the acidification of mutant-containing vacuoles (MCVs). The perturbation in the pH of the MCV milieu and bacterial cytosol enhances the Salmonella pathogenicity island 2 translocation in Delta lgl, increasing its net growth within macrophages. In epithelial cells, however, the maturation of Delta lgl-containing vacuoles were affected as these non-phagocytic cells maintain less acidic vacuoles compared to those in macrophages. Remarkably, ectopic expression of Toll-like receptors 2 and 4 on epithelial cells partially restored the survival of Delta lgl. This study identified a novel metabolic enzyme in S. Typhimurium whose activity during intracellular infection within a given host cell type differentially affected the virulence of the bacteria.

Dual role of MsRbpA: transcription activation and rescue of transcription from the inhibitory effect of rifampicin

MsRbpA is an RNA polymerase (RNAP) binding protein from Mycobacterium smegmatis. According to previous studies, MsRbpA rescues rifampicin-induced transcription inhibition upon binding to the RNAP. Others have shown that RbpA from Mycobacterium tuberculosis (MtbRbpA) is a transcription activator. In this study, we report that both MsRbpA and MtbRbpA activate transcription as well as rescue rifampicin-induced transcription inhibition. Transcription activation is achieved through the increased formation of closed RNAP-promoter complex as well as enhanced rate of conversion of this complex to a stable transcriptionally competent RNAP promoter complex. When a 16 aa peptide fragment (Asp 58 to Lys 73) was deleted from MsRbpA, the resulting protein showed 1000-fold reduced binding with core RNAP. The deletion results in abolition of transcription activation and rescue of transcription from the inhibitory effect of rifampicin. Through alanine scanning of this essential region of MsRbpA, Gly 67, Val 69, Pro 70 and Pro 72 residues are identified to be important for MsRbpA function. Furthermore, we report here that the protein is indispensable for M. smegmatis, and it appears to help the organism grow in the presence of the antibiotic rifampicin.

Characterization of a dual-active enzyme, DcpA, involved in cyclic diguanosine monophosphate turnover in Mycobacterium smegmatis

We have reported previously that the long-term survival of Mycobacterium smegmatis is facilitated by a dual-active enzyme MSDGC-1 (renamed DcpA), which controls the cellular turnover of cyclic diguanosine monophosphate (c-di-GMP). Most mycobacterial species possess at least a single copy of a DcpA orthologue that is highly conserved in terms of sequence similarity and domain architecture. Here, we show that DcpA exists in monomeric and dimeric forms. The dimerization of DcpA is due to non-covalent interactions between two protomers that are arranged in a parallel orientation. The dimer shows both synthesis and hydrolysis activities, whereas the monomer shows only hydrolysis activity. In addition, we have shown that DcpA is associated with the cytoplasmic membrane and exhibits heterogeneous cellular localization with a predominance at the cell poles. Finally, we have also shown that DcpA is involved in the change in cell length and colony morphology of M. smegmatis. Taken together, our study provides additional evidence about the role of the bifunctional protein involved in c-di-GMP signalling in M. smegmatis.

Reduction in DNA topoisomerase I level affects growth, phenotype and nucleoid architecture of Mycobacterium smegmatis

The steady-state negative supercoiling of eubacterial genomes is maintained by the action of DNA topoisomerases. Topoisomerase distribution varies in different species of mycobacteria. While Mycobacterium tuberculosis (Mtb) contains a single type I (Topol) and a single type II (Gyrase) enzyme, Mycobacterium smegmatis (Msm) and other members harbour additional relaxases. Topol is essential for Mtb survival. However, the necessity of Topol or other relaxases in Msm has not been investigated. To recognize the importance of Topol for growth, physiology and gene expression of Msm, we have developed a conditional knock-down strain of Topol in Msm. The Topol-depleted strain exhibited extremely slow growth and drastic changes in phenotypic characteristics. The cessation of growth indicates the essential requirement of the enzyme for the organism in spite of having additional DNA relaxation enzymes in the cell. Notably, the imbalance in Topol level led to the altered expression of topology modulatory proteins, resulting in a diffused nucleoid architecture. Proteomic and transcript analysis of the mutant indicated reduced expression of the genes involved in central metabolic pathways and core DNA transaction processes. RNA polymerase (RNAP) distribution on the transcription units was affected in the Topol-depleted cells, suggesting global alteration in transcription. The study thus highlights the essential requirement of Topol in the maintenance of cellular phenotype, growth characteristics and gene expression in mycobacteria. A decrease in Topol level led to altered RNAP occupancy and impaired transcription elongation, causing severe downstream effects.

Peptide-utilizing carbon starvation gene yjiY is required for flagella-mediated infection caused by Salmonella

Peptide metabolism forms an important part of the metabolic network of Salmonella and to acquire these peptides the pathogen possesses a number of peptide transporters. Whilst various peptide transporters known in Salmonella are well studied, very little is known about the carbon starvation (cst) genes cstA and yjiY, which are also predicted to be involved in peptide metabolism. We investigated the role of these genes in the metabolism and pathogenesis of Salmonella, and demonstrated for the first time, to the best of our knowledge, that cst genes actually participate in transport of specific peptides in Salmonella. Furthermore, we established that the carbon starvation gene yjiY affects the expression of flagella, leading to poor adhesion of the bacterium to host cells. In contrast to the previously reported role of cstA in virulence of Salmonella in Caenorhabditis elegans, we showed that yjiY is required for successful colonization of Salmonella in the mouse gut. Thus, cst genes not only contribute to the metabolism of Salmonella, but also influence its virulence.

Physiological role of FolD (methylenetetrahydrofolate dehydrogenase), FchA (methenyltetrahydrofolate cyclohydrolase) and Fhs (formyltetrahydrofolate synthetase) from Clostridium perfringens in a heterologous model of Escherichia coli

Most organisms possess bifunctional FolD 5,10-methylenetetrahydrofolate (5,10-CH2-THF) dehydrogenase-cyclohydrolase] to generate NADPH and 10-formyltetrandrofolate (10-CHO-THF) required in various metabolic steps. In addition, some organisms including Clostridium perfringens possess another protein, Fhs (formyltetrahydrofolate synthetase), to synthesize 10-CHO-THF. Here, we show that unlike the bifunctional FolD of Escherichia coli (Eco FolD), and contrary to its annotated bifunctional nature, C. perfringens FolD (Cpe FoID) is a monofunctional 5,10-CH2-THF dehydrogenase. The dehydrogenase activity of Cpe FoID is about five times more efficient than that of Eco FolD. The 5,10-methenyltetrahydrofolate (5,10-CH+-THF) cyclohydrolase activity in C. perfringens is provided by another protein, FchA (5,10-CH+-THF cyclohydrolase), whose cyclohydrolase activity is similar to 10 times more efficient than that of Eco FolD. Kinetic parameters for Cpe Fhs were also determined for utilization of all of its substrates. Both Cpe FoID and Cpe FchA are required to substitute for the single bifunctional FolD in E. coli. The simultaneous presence of Cpe FoID and Cpe FchA is also necessary to rescue an E coli folD deletion strain (harbouring Cpe Fhs support) for its formate and glycine auxotrophies, and to alleviate its susceptibility to trimethoprim (an antifolate drug) or UV light. The presence of the three clostridial proteins (FolD, FchA and Fhs) is required to maintain folate homeostasis in the cell.

Peptide-utilizing carbon starvation gene yjiY is required for flagella-mediated infection caused by Salmonella

Peptide metabolism forms an important part of the metabolic network of Salmonella and to acquire these peptides the pathogen possesses a number of peptide transporters. Whilst various peptide transporters known in Salmonella are well studied, very little is known about the carbon starvation (cst) genes cstA and yjiY, which are also predicted to be involved in peptide metabolism. We investigated the role of these genes in the metabolism and pathogenesis of Salmonella, and demonstrated for the first time, to the best of our knowledge, that cst genes actually participate in transport of specific peptides in Salmonella. Furthermore, we established that the carbon starvation gene yjiY affects the expression of flagella, leading to poor adhesion of the bacterium to host cells. In contrast to the previously reported role of cstA in virulence of Salmonella in Caenorhabditis elegans, we showed that yjiY is required for successful colonization of Salmonella in the mouse gut. Thus, cst genes not only contribute to the metabolism of Salmonella, but also influence its virulence.

El aprendizaje activo de la microbiología en el Grado de Podología incrementa el rendimiento académico

Se ha planteado como objetivo la mejora de la calidad de la docencia de la Microbiología mediante la actualización de la metodología docente , introduciendo como actividad docente el aprendizaje activo basado en preguntas (inquirybased learning:IBL) para conseguir mejorar las competencias que deberán adquirir los estudiantes como parte de su formación integral. En este estudio han participado 55 alumnos de Segundo Curso del Grado de Podología (Curso 2014-2015),y se ha calculado el porcentaje de alumnos que participaron en las 3 pruebas (3 IBL), en 2 (2 IBL), en 1 (1 IBL), y los que no participaron en ninguna y posteriormente se relacionó con las calificaciones obtenidas en la asignatura de Microbiología. Se incluyeron las preguntas IBL que se realizaron en clase en el campus virtual de la asignatura, pero sin incluir la corrección de las mismas. En los alumnos que realizaron alguna prueba IBL se obtuvieron calificaciones mejores en las preguntas diseñadas para analizar la síntesis de conocimientos y el análisis de datos que en aquellos que no habían participado en ninguna. Al finalizar la actividad se realizó un estudio transversal a través de un cuestionario autocumplimentado en el que se valoraba la opinión de los alumnos sobre el aprendizaje activo mediante IBL valorando positivamente esta actividad para medir el aprendizaje y mejorar la preparación del examen. Consideramos que el uso del campus virtual unido a la actualización en la metodología docente puede mejorar el rendimiento académico de los estudiantes de Microbiología.

Flanking and internal regions of chromosomal genes mediating aerobactin iron uptake systems in enteroinvasive Escherichia coli and Shigella flexneri

We have investigated the presence of the aerobactin system and the location of the aerobactin genes in enteroinvasive strains of Escherichia coli. Also, we cloned the aerobactin region and its flanking sequences from the chromosome of a strain of Shigella flexneri and compared the molecular organization of the aerobactin genes in the two genera. Of the 11 enteroinvasive E. coli strains studied, 5 possessed the aerobactin genes, which were located on the chromosome in each case. These strains produced and utilized aerobactin and also were susceptible to the bacteriocin cloacin-DF13. Restriction endonuclease mapping and hybridization experiments showed that the regions corresponding to the aerobactin-specific sequences were very similar in both enteroinvasive E. coli and S. flexneri. However, differences were found in the region corresponding to the aerobactin receptor gene. The regions flanking the aerobactin system in enteroinvasive E. coli and S. flexneri exhibited some similarities but were different from those in pColV-K30. Under iron-limiting conditions, aerobactin-producing enteroinvasive E. coli and S. flexneri synthesized outer-membrane proteins of 76 and 77 kDa, respectively, which cross-reacted immunologically with rabbit antiserum raised against the 74 kDa pColV-K30-encoded ferric aerobactin receptor.

Immunochemical characterization of a polysaccharide antigen of Bacteroides fragilis with an IgM monoclonal antibody

An IgM mouse monoclonal antibody (McAb) Bf4 was produced to a surface polysaccharide of Bacteroides fragilis NCTC 9343. Immunoblotting showed that McAb Bf4 reacted strongly with a high molecular mass structure which was sensitive to oxidation with periodate but resisted protease treatment. An inhibition enzyme-linked immunosorbent assay (ELISA) indicated that McAb Bf4 did not cross react with the sixteen Bacteroides species and strains tested. Cells of B. fragilis NCTC 9343 recovered from the various interfaces of a Percoll discontinuous density gradient were tested in the inhibition ELISA. Bacteria from the 0-20%, 20-40% and 40-60% interfaces inhibited the ELISA; however, cells from the 60-80% interface did not. Electron microscopy with immunogold labelling showed that McAb Bf4 did not react with the extracellular fibrous network on bacteria recovered from the 0-20% interface, or the extracellular electron dense layer on cells from the 60-80% interface; however, it was associated with a surface structure on cells from the 20-40% interface. Growth in vivo did not enrich for bacteria with this structure.