The genome and proteome of the Kluyvera bacteriophage Kvp1 – another member of the T7-like Autographivirinae

BACKGROUND: Kluyvera, a genus within the family Enterobacteriaceae, is an infrequent cause of human infections. Bacteriophage Kvp1, the only bacteriophage isolated for one of its species, Kluyvera cryocrescens, is a member of the viral family Podoviridae. RESULTS: The genome of Kvp1, the first Kluyvera cryocrescens-specific bacteriophage, was sequenced using pyrosequencing (454 technology) at the McGill University and Genome Québec Innovation Centre. The two contigs were closed using PCR and the sequence of the terminal repeats completed by primer walking off the phage DNA. The phage structural proteome was investigated by SDS-PAGE and mass spectrometry. CONCLUSION: At 39,472 bp, the annotated genome revealed a closer relationship to coliphage T3 than T7 with Kvp1 containing homologs to T3 early proteins S-adenosyl-L-methionine hydrolase (0.3) and protein kinase (0.7). The quantitative nature of the relationships between Kvp1 and the other members of the T7-like virus genus (T7, T3, φA1122, φYeO3-12, Berlin, K1F, VP4 and gh-1) was confirmed using CoreGenes

In vivo monoubiquitination of anaplerotic phosphoenolpyruvate carboxylase occurs at Lys624 in germinating sorghum seeds

Phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) is an important cytosolic regulatory enzyme that plays a pivotal role in numerous physiological processes in plants, including seed development and germination. Previous studies demonstrated the occurrence of immunoreactive PEPC polypeptides of ~110kDa and 107kDa (p110 and p107, respectively) on immunoblots of clarified extracts of germinating sorghum (Sorghum bicolor) seeds. In order to establish the biochemical basis for this observation, a 460kDa PEPC heterotetramer composed of an equivalent ratio of p110 and p107 subunits was purified to near homogeneity from the germinated seeds. Mass spectrometry established that p110 and p107 are both encoded by the same plant-type PEPC gene (CP21), but that p107 was in vivo monoubiquitinated at Lys624 to form p110. This residue is absolutely conserved in vascular plant PEPCs and is proximal to a PEP-binding/catalytic domain. Anti-ubiquitin IgG immunodetected p110 but not p107, whereas incubation with a deubiquitinating enzyme (USP-2 core) efficiently converted p110 into p107, while relieving the enzyme’s feedback inhibition by l-malate. Partial PEPC monoubiquitination was also detected during sorghum seed development. It is apparent that monoubiquitination at Lys624 is opposed to phosphorylation at Ser7 in terms of regulating the catalytic activity of sorghum seed PEPC. PEPC monoubiquitination is hypothesized to fine-tune anaplerotic carbon flux according to the cell’s immediate physiological requirements for tricarboxylic acid cycle intermediates needed in support of biosynthesis and carbon–nitrogen interactions.España, Ministerio de Economía y Competitividad AGL2012-35708España Junta de Andalucía P06-CVI-02186 BIO29

In vivo regulatory phosphorylation of the phosphoenolpyruvate carboxylase AtPPC1 in phosphate-starved Arabidopsis thaliana

PEPC [PEP(phosphoenolpyruvate) carboxylase] is a tightly controlled cytosolic enzyme situated at a major branchpoint in plant metabolism. Accumulating evidence indicates important functions for PEPC and PPCK (PEPC kinase) in plant acclimation to nutritional Pi deprivation. However, little is known about the genetic origin or phosphorylation status of native PEPCs from −Pi (Pi-deficient) plants. The transfer of Arabidopsis suspension cells or seedlings to −Pi growth media resulted in: (i) the marked transcriptional upregulation of genes encoding the PEPC isoenzyme AtPPC1 (Arabidopsis thaliana PEPC1), and PPCK isoenzymes AtPPCK1 and AtPPCK2; (ii) >2-fold increases in PEPC specific activity and in the amount of an immunoreactive 107-kDa PEPC polypeptide (p107); and (iii) In vivo p107 phosphorylation as revealed by immunoblotting of clarified extracts with phosphosite-specific antibodies to Ser-11 (which could be reversed following Pi resupply). Approx. 1.3 mg of PEPC was purified 660-fold from −Pi suspension cells to apparent homogeneity with a specific activity of 22.3 units · mg−1 of protein. Gel filtration, SDS/PAGE and immunoblotting demonstrated that purified PEPC exists as a 440-kDa homotetramer composed of identical p107 subunits. Sequencing of p107 tryptic and Asp-N peptides by tandem MS established that this PEPC is encoded by AtPPC1. Pi-affinity PAGE coupled with immunoblotting indicated stoichiometric phosphorylation of the p107 subunits of AtPPC1 at its conserved Ser-11 phosphorylation site. Phosphorylation activated AtPPC1 at pH 7.3 by lowering its Km(PEP) and its sensitivity to inhibition by L-malate and L-aspartate, while enhancing activation by glucose 6-phosphate. Our results indicate that the simultaneous induction and In vivo phosphorylation activation of AtPPC1 contribute to the metabolic adaptations of −Pi Arabidopsis

New insights into the post-translational modification of multiple phosphoenolpyruvate carboxylase isoenzymes by phosphorylation and monoubiquitination during sorghum seed development and germination

Phosphoenolpyruvate carboxylase (PEPC; E.C. 4.1.1.31) was characterized in developing and germinating sorghum seeds, focusing on the transcript and polypeptide abundance of multiple plant-type phosphoenolpyruvate carboxylase (PTPC) genes, and the post-translational modification of each isoenzyme by phosphorylation versus monoubiquitination during germination. We observed high levels of SbPPC4 (Sb07g014960) transcripts during early development (stage I), and extensive transcript abundance of SbPPC2 (Sb02g021090) and SbPPC3 (Sb04g008720) throughout the entire life cycle of the seed. Although tandem mass spectrometry (MS) analysis of immunopurified PTPC indicated that four different PTPC isoenzymes were expressed in the developing and germinating seeds, SbPPC3 was the most abundant isozyme of the developing seed, and of the embryo and the aleurone layer of germinating seeds. In vivo phosphorylation of the different PTPC isoenzymes at their conserved N-terminal seryl phosphorylation site during germination was also established by MS/MS analysis. Furthermore, three of the four isoenzymes were partially monoubiquitinated, with MS/MS pinpointing SbPPC2 and SbPPC3 monoubiquitination at the conserved Lys-630 and Lys-624 residues, respectively. Our results demonstrate that monoubiquitination and phosphorylation simultaneously occur in vivo with different PTPC isozymes during seed germination. In addition, we show that PTPC monoubiquitination in germinating sorghum seeds always increases at stage II (emergence of the radicle), is maintained during the aerobic period of rapid cell division and reserve mobilization, and remains relatively constant until stage IV-V when coleoptiles initiate the formation of the photosynthetic tissues

The genome and proteome of a virulent Escherichia coli O157:H7 bacteriophage closely resembling Salmonella phage Felix O1

Based upon whole genome and proteome analysis, Escherichia coli O157:H7-specific bacteriophage (phage) wV8 belongs to the new myoviral genus, "the Felix O1-like viruses" along with Salmonella phage Felix O1 and Erwinia amylovora phage φEa21-4. The genome characteristics of phage wV8 (size 88.49 kb, mol%G+C 38.9, 138 ORFs, 23 tRNAs) are very similar to those of phage Felix O1 (86.16 kb, 39.0 mol%G+C, 131 ORFs and 22 tRNAs) and, indeed most of the proteins have their closest homologs within Felix O1. Approximately one-half of the Escherichia coli O157:H7 mutants resistant to phage wV8 still serotype as O157:H7 indicating that this phage may recognize, like coliphage T4, two different surface receptors: lipopolysaccharide and, perhaps, an outer membrane protein

Complete Genomic Sequence of Bacteriophage Felix O1†

Bacteriophage O1 is a Myoviridae A1 group member used historically for identifying Salmonella. Sequencing revealed a single, linear, 86,155-base-pair genome with 39% average G+C content, 131 open reading frames, and 22 tRNAs. Closest protein homologs occur in Erwinia amylovora phage φEa21-4 and Escherichia coli phage wV8. Proteomic analysis indentified structural proteins: Gp23, Gp36 (major tail protein), Gp49, Gp53, Gp54, Gp55, Gp57, Gp58 (major capsid protein), Gp59, Gp63, Gp64, Gp67, Gp68, Gp69, Gp73, Gp74 and Gp77 (tail fiber). Based on phage-host codon differences, 7 tRNAs could affect translation rate during infection. Introns, holin-lysin cassettes, bacterial toxin homologs and host RNA polymerase-modifying genes were absent

Identification of endogenous small peptides involved in rice immunity through transcriptomics- and proteomics-based screening

Small signalling peptides, generated from larger protein precursors, are important components to orchestrate various plant processes such as development and immune responses. However, small signalling peptides involved in plant immunity remain largely unknown. Here, we developed a pipeline using transcriptomics- and proteomics-based screening to identify putative precursors of small signalling peptides: small secreted proteins (SSPs) in rice, induced by rice blast fungus Magnaporthe oryzae and its elicitor, chitin. We identified 236 SSPs including members of two known small signalling peptide families, namely rapid alkalinization factors and phytosulfokines, as well as many other protein families that are known to be involved in immunity, such as proteinase inhibitors and pathogenesis-related protein families. We also isolated 52 unannotated SSPs and among them, we found one gene which we named immune response peptide (IRP) that appeared to encode the precursor of a small signalling peptide regulating rice immunity. In rice suspension cells, the expression of IRP was induced by bacterial peptidoglycan and fungal chitin. Overexpression of IRP enhanced the expression of a defence gene, PAL1 and induced the activation of the MAPKs in rice suspension cells. Moreover, the IRP protein level increased in suspension cell medium after chitin treatment. Collectively, we established a simple and efficient pipeline to discover SSP candidates that probably play important roles in rice immunity and identified 52 unannotated SSPs that may be useful for further elucidation of rice immunity. Our method can be applied to identify SSPs that are involved not only in immunity but also in other plant functions

Propuesta de implementación de un sistema de gestión de seguridad y salud en el trabajo bajo la norma Ohsas 18001, para minimizar los factores de riesgo en la Institución Educativa Experimental Antonio Guillermo Urrelo

El autor no autoriza la publicación de la tesi

Assessing trends and predictors of tuberculosis in Taiwan

<p>Abstract</p> <p>Background</p> <p>Variety of environmental and individual factors can cause tuberculosis (TB) incidence change. The purpose of this study was to assess the characteristics of TB trends in the period 2004 - 2008 in Taiwan by month, year, gender, age, temperature, seasonality, and aborigines.</p> <p>Methods</p> <p>The generalized regression models were used to examine the potential predictors for the monthly TB incidence in regional and national scales.</p> <p>Results</p> <p>We found that (<it>i</it>) in Taiwan the average TB incidence was 68 per 100,000 population with mortality rate of 0.036 person^-1yr^-1, (<it>ii</it>) the highest TB incidence rate was found in eastern Taiwan (116 per 100,000 population) with the largest proportion of TB relapse cases (8.17%), (<it>iii</it>) seasonality, aborigines, gender, and age had a consistent and dominant role in constructing TB incidence patterns in Taiwan, and (<it>iv</it>) gender, time trend, and 2-month lag maximum temperature showed strong association with TB trends in aboriginal subpopulations.</p> <p>Conclusions</p> <p>The proposed Poisson regression model is capable of forecasting patterns of TB incidence at regional and national scales. This study suggested that assessment of TB trends in eastern Taiwan presents an important opportunity for understanding the time-series dynamics and control of TB infections, given that this is the typical host demography in regions where these infections remain major public health problems.</p