apd1<SUP>+</SUP>, a Gene required for red pigment formation in ade6 mutants of Schizosaccharomyces pombe, encodes an enzyme required for glutathione biosynthesis: a role for glutathione and a glutathione-conjugate pump

Mutants in the adenine biosynthetic pathway of yeasts (ade1 and ade2 of Saccharomyces cerevisiae, ade6 and ade7 of Schizosaccharomyces pombe) accumulate an intense red pigment in their vacuoles when grown under adenine-limiting conditions. The precise events that determine the formation of the pigment are however, still unknown. We have begun a genetic investigation into the nature and cause of pigmentation of ade6 mutants of S. pombe and have discovered that one of these pigmentation defective mutants, apd1 (adenine pigmentation defective), is a strict glutathione auxotroph. The gene apd1+ was found to encode the first enzyme in glutathione biosynthesis, &#947;-glutamylcysteine synthetase, gcs1+. This gene when expressed in the mutant could confer both glutathione prototrophy and the characteristic red pigmentation, and disruption of the gene led to a loss in both phenotypes. Supplementation of glutathione in the medium, however, could only restore growth but not the pigmentation because the cells were unable to achieve sufficient intracellular levels of glutathione. Disruption of the second enzyme in glutathione biosynthesis, glutathione synthetase, gsh2+, also led to glutathione auxotrophy, but only a partial defect in pigment formation. A reevaluation of the major amino acids previously reported to be present in the pigment indicated that the pigment is probably a glutathione conjugate. The ability of vanadate to inhibit pigment formation indicated that the conjugate was transported into the vacuole through a glutathione-conjugate pump. This was further confirmed using strains of S. cerevisiae bearing disruptions in the recently identified glutathione-conjugate pump, YCF1, where a significant reduction in pigment formation was observed. The pump of S. pombe is distinct from the previously identified vacuolar pump, hmt1p, for transporting cadystin peptides into vacuoles of S. pombe

Interaction of Salivary alpha-Amylase and Amylase-Binding-Protein A (AbpA) of Streptococcus gordonii with Glucosyltransferase of S. gordonii and Streptococcus mutans

<p>Abstract</p> <p>Background</p> <p>Glucosyltransferases (Gtfs), enzymes that produce extracellular glucans from dietary sucrose, contribute to dental plaque formation by <it>Streptococcus gordonii </it>and <it>Streptococcus mutans</it>. The alpha-amylase-binding protein A (AbpA) of <it>S. gordonii</it>, an early colonizing bacterium in dental plaque, interacts with salivary amylase and may influence dental plaque formation by this organism. We examined the interaction of amylase and recombinant AbpA (rAbpA), together with Gtfs of <it>S. gordonii </it>and <it>S. mutans</it>.</p> <p>Results</p> <p>The addition of salivary alpha-amylase to culture supernatants of <it>S. gordonii </it>precipitated a protein complex containing amylase, AbpA, amylase-binding protein B (AbpB), and the glucosyltransferase produced by <it>S. gordonii </it>(Gtf-G). rAbpA was expressed from an inducible plasmid, purified from <it>Escherichia coli </it>and characterized. Purified rAbpA, along with purified amylase, interacted with and precipitated Gtfs from culture supernatants of both <it>S. gordonii </it>and <it>S. mutans</it>. The presence of amylase and/or rAbpA increased both the sucrase and transferase component activities of <it>S. mutans </it>Gtf-B. Enzyme-linked immunosorbent assay (ELISA) using anti-Gtf-B antibody verified the interaction of rAbpA and amylase with Gtf-B. A <it>S. gordonii abp</it>A-deficient mutant showed greater biofilm growth under static conditions than wild-type in the presence of sucrose. Interestingly, biofilm formation by every strain was inhibited in the presence of saliva.</p> <p>Conclusion</p> <p>The results suggest that an extracellular protein network of AbpA-amylase-Gtf may influence the ecology of oral biofilms, likely during initial phases of colonization.</p

Salmonella Typhimurium Infection Leads to Colonization of the Mouse Brain and Is Not Completely Cured With Antibiotics

Salmonella systemic infections claim thousands of lives worldwide even today. Certain cases lead to an infection in the brain culminating in meningitis and associated neurological abnormalities. Multiple reports have indicated neurological manifestations in patients suffering from typhoid fever during the course of infection and afterwards. While the meanderings of Salmonella systemic infections are fairly well studied, the flow of events in the brain is very poorly understood. We investigated the colonization of various brain parts by Salmonella in mice. It was observed that the bacterium is frequently able to invade various brain parts in mice. Selected mutants namely deletion mutants of key proteins encoded by the Salmonella pathogenicity islands (SPIs) 1 and 2 and ompA gene were also used to decipher the roles of specific genes in establishing an infection in the brain. Our results suggest roles for the Salmonella pathogenicity island (SPI) 1 and outer membrane protein A gene in enabling blood-brain barrier penetration by the pathogen. We further investigated behavioral abnormalities in infected mice and used an antibiotic treatment regime in an attempt to reverse the same. Results show some mice still display behavioral abnormalities and a high bacterial burden in brain despite clearance from spleen and liver. Overall, our study provides novel insights into S. Typhimurium's capacity to invade the mouse brain and the effectiveness of antibiotic treatment on behavioral manifestations due to infection. These observations could have important implications in understanding reported neurological manifestations in typhoid patients

Human DNA replication initiation factors, ORC and MCM, associate with oriP of Epstein–Barr virus

The 165-kb chromosome of Epstein–Barr virus (EBV) is replicated by cellular enzymes only once per cell cycle in human cells that are latently infected. Here, we report that the human origin recognition complex, ORC, can be detected in association with an EBV replication origin, oriP, in cells by using antibodies against three different subunits of human ORC to precipitate crosslinked chromatin. Mcm2, a subunit of the MCM replication licensing complex, was found to associate with oriP during G(1) and to dissociate from it during S phase. The detection of ORC and Mcm2 at oriP was shown to require the presence of the 120-bp replicator of oriP. Licensing and initiation of replication at oriP of EBV thus seem to be mediated by ORC. This is an example of a virus apparently using ORC and associated factors for the propagation of its genome

Image_1_Salmonella Typhimurium Infection Leads to Colonization of the Mouse Brain and Is Not Completely Cured With Antibiotics.TIF

<p>Salmonella systemic infections claim thousands of lives worldwide even today. Certain cases lead to an infection in the brain culminating in meningitis and associated neurological abnormalities. Multiple reports have indicated neurological manifestations in patients suffering from typhoid fever during the course of infection and afterwards. While the meanderings of Salmonella systemic infections are fairly well studied, the flow of events in the brain is very poorly understood. We investigated the colonization of various brain parts by Salmonella in mice. It was observed that the bacterium is frequently able to invade various brain parts in mice. Selected mutants namely deletion mutants of key proteins encoded by the Salmonella pathogenicity islands (SPIs) 1 and 2 and ompA gene were also used to decipher the roles of specific genes in establishing an infection in the brain. Our results suggest roles for the Salmonella pathogenicity island (SPI) 1 and outer membrane protein A gene in enabling blood-brain barrier penetration by the pathogen. We further investigated behavioral abnormalities in infected mice and used an antibiotic treatment regime in an attempt to reverse the same. Results show some mice still display behavioral abnormalities and a high bacterial burden in brain despite clearance from spleen and liver. Overall, our study provides novel insights into S. Typhimurium's capacity to invade the mouse brain and the effectiveness of antibiotic treatment on behavioral manifestations due to infection. These observations could have important implications in understanding reported neurological manifestations in typhoid patients.</p

Amylase-Binding Protein B of Streptococcus gordonii Is an Extracellular Dipeptidyl-Peptidase▿

The oral commensal bacterium Streptococcus gordonii interacts with salivary amylase via two amylase-binding proteins, AbpA and AbpB. Based on sequence analysis, the 20-kDa AbpA protein is unique to S. gordonii, whereas the 82-kDa AbpB protein appears to share sequence homology with other bacterial dipeptidases. The aim of this study was to verify the peptidase activity of AbpB and further explore its potential functions. The abpB gene was cloned, and histidine-tagged AbpB (His-AbpB) was expressed in Escherichia coli and purified. Its amylase-binding activity was verified in an amylase ligand binding assay, and its cross-reactivity was verified with an anti-AbpB antibody. Both recombinant His-AbpB and partially purified native AbpB displayed dipeptidase activity and degraded human type VI collagen and fibrinogen, but not salivary amylase. Salivary amylase precipitates not only AbpA and AbpB but also glucosyltransferase G (Gtf-G) from S. gordonii supernatants. Since Streptococcus mutans also releases Gtf enzymes that could also be involved in multispecies plaque interactions, the effect of S. gordonii AbpB on S. mutans Gtf-B activity was also tested. Salivary amylase and/or His-AbpB caused a 1.4- to 2-fold increase of S. mutans Gtf-B sucrase activity and a 3- to 6-fold increase in transferase activity. An enzyme-linked immunosorbent assay verified the interaction of His-AbpB and amylase with Gtf-B. In summary, AbpB demonstrates proteolytic activity and interacts with and modulates Gtf activity. These activities may help explain the crucial role AbpB appears to play in S. gordonii oral colonization