Expression studies of two paralogous ppa genes encoding distinct Family I pyrophosphatases in marine unicellular cyanobacteria reveal inactivation of the typical cyanobacterial gene

2002 ◽  
Vol 295 (4) ◽  
pp. 890-897 ◽  
Author(s):  
Marı́a R Gómez-Garcı́a ◽  
Aurelio Serrano
Keyword(s):  
Expression Studies ◽  
Cyanobacterial Gene ◽  
Genes Encoding

scholarly journals CAU-1, a Subclass B3 Metallo-β-Lactamase of Low Substrate Affinity Encoded by an Ortholog Present in the Caulobacter crescentus Chromosome

2002 ◽  
Vol 46 (6) ◽  
pp. 1823-1830 ◽  
Author(s):  
Jean-Denis Docquier ◽  
Fabrizio Pantanella ◽  
Francesco Giuliani ◽  
Maria Cristina Thaller ◽  
Gianfranco Amicosante ◽  
...  
Keyword(s):  
Caulobacter Crescentus ◽  
Hypothetical Protein ◽  
Exchange Chromatography ◽  
Substrate Affinity ◽  
Gene Encoding ◽  
Native Form ◽  
Significant Similarity ◽  
Expression Studies ◽  
Genes Encoding ◽  
Isoelectric Ph

ABSTRACT The sequenced chromosome of Caulobacter crescentus CB15 encodes a hypothetical protein that exhibits significant similarity (30 to 35% identical residues) to metallo-β-lactamases of subclass B3. An allelic variant of this gene (divergent by 3% of its nucleotides) was cloned in Escherichia coli from C. crescentus type strain DSM4727. Expression studies confirmed the metallo-β-lactamase activity of its product, CAU-1. The enzyme produced in E. coli was purified by two ion-exchange chromatography steps. CAU-1 contains a 29-kDa polypeptide with an alkaline isoelectric pH (>9), and unlike the L1 enzyme of Stenotrophomonas maltophilia, the native form is monomeric. Kinetic analysis revealed a preferential activity toward penicillins, carbapenems, and narrow-spectrum cephalosporins, while oxyimino cephalosporins were poorly or not hydrolyzed. Affinities for the various β-lactams were poor overall (Km values were always >100 μM and often >400 μM). The interaction with divalent ion chelators appeared to occur by a mechanism similar to that prevailing in other members of subclass B3. In C. crescentus, the CAU-1 enzyme is produced independently of β-lactam exposure and, interestingly, the bla CAU determinant is bracketed by three other genes, including two genes encoding enzymes involved in methionine biosynthesis and a gene encoding a putative transcriptional regulator, in an operon-like structure. The CAU-1 enzyme is the first example of a metallo-β-lactamase in a member of the α subdivision of the class Proteobacteria.

scholarly journals Prediction of the structure of the common perimitochondrial localization signal of nuclear transcripts in yeast.

2007 ◽  
Vol 54 (1) ◽  
pp. 55-61 ◽  
Author(s):  
Radoslaw K Ejsmont ◽  
Pawel Golik ◽  
Piotr P Stepien
Keyword(s):  
Subcellular Localization ◽  
Mitochondrial Proteins ◽  
Consensus Structure ◽  
Stem Loop ◽  
Cis Acting ◽  
Stem Loop Structure ◽  
Expression Studies ◽  
Genes Encoding ◽  
Specific Localization ◽  
Localization Signal

Many nuclear genes encoding mitochondrial proteins require specific localization of their mRNAs to the vicinity of mitochondria for proper expression. Studies in Saccharomyces cerevisiae have shown that the cis-acting signal responsible for subcellular localization of mRNAs is localized in the 3' UTR of the transcript. In this paper we present an in silico approach for prediction of a common perimitochondrial localization signal of nuclear transcripts encoding mitochondrial proteins. We computed a consensus structure for this signal by comparison of 3' UTR models for about 3000 yeast transcripts with known localization. Our studies show a short stem-loop structure which appears in most mRNAs localized to the vicinity of mitochondria. The degree of similarity of a given 3' UTR to our consensus structure strongly correlates with experimentally determined perimitochondrial localization of the mRNA, therefore we believe that the structure we predicted acts as a subcellular localization signal. Since our algorithm operates on structures, it seems to be more reliable than sequence-based algorithms. The good predictive value of our model is supported by statistical analysis.

scholarly journals Manual Annotation, Transcriptional Analysis, and Protein Expression Studies Reveal Novel Genes in the agl Cluster Responsible for N Glycosylation in the Halophilic Archaeon Haloferax volcanii

2009 ◽  
Vol 191 (9) ◽  
pp. 3068-3075 ◽  
Author(s):  
Sophie Yurist-Doutsch ◽  
Jerry Eichler
Keyword(s):  
Protein Expression ◽  
Gene Cluster ◽  
Present Report ◽  
Haloferax Volcanii ◽  
Transcriptional Analysis ◽  
Halophilic Archaeon ◽  
Expression Studies ◽  
Genes Encoding ◽  
Computer Based ◽  
Automated Tools

ABSTRACT While Eukarya, Bacteria, and Archaea are all capable of protein N glycosylation, the archaeal version of this posttranslational modification is the least understood. To redress this imbalance, recent studies of the halophilic archaeon Haloferax volcanii have identified a gene cluster encoding the Agl proteins involved in the assembly and attachment of a pentasaccharide to select Asn residues of the surface layer glycoprotein in this species. However, because the automated tools used for rapid annotation of genome sequences, including that of H. volcanii, are not always accurate, a reannotation of the agl cluster was undertaken in order to discover genes not previously recognized. In the present report, reanalysis of the gene cluster that includes aglB, aglE, aglF, aglG, aglI, and aglJ, which are known components of the H. volcanii protein N-glycosylation machinery, was undertaken. Using computer-based tools or visual inspection, together with transcriptional analysis and protein expression approaches, genes encoding AglP, AglQ, and AglR are now described.

scholarly journals To have neighbour's fare: extending the molecular toolbox for Streptococcus pneumoniae

Microbiology ◽  
2006 ◽  
Vol 152 (2) ◽  
pp. 351-359 ◽  
Author(s):  
Tomas G. Kloosterman ◽  
Jetta J. E. Bijlsma ◽  
Jan Kok ◽  
Oscar P. Kuipers
Keyword(s):  
Streptococcus Pneumoniae ◽  
Transcriptional Activation ◽  
Expression System ◽  
Inducible Promoter ◽  
Defined Medium ◽  
Reporter System ◽  
Two Component System ◽  
Over Expression ◽  
Expression Studies ◽  
Genes Encoding

In past years, several useful genetic tools have been developed to study the molecular biology of Streptococcus pneumoniae. In order to extend the existing spectrum of tools, advantage was taken of the toolbox originally developed for the closely related bacterium Lactococcus lactis, which was adapted for the manipulation of S. pneumoniae. The modified tools are as follows. (i) An improved nisin-inducible (over)expression system (NICE). The nisRK genes, encoding a two-component system essential for transcriptional activation in response to nisin, were integrated into the bgaA locus of S. pneumoniae D39. In this strain, D39nisRK, addition of nisin resulted in the overexpression of several genes placed under the control of the nisin-inducible promoter, while no detectable expression was observed in the absence of nisin. (ii) A lacZ reporter system. Using strain D39nisRK, which lacks endogenous β-galactosidase activity, the usefulness of the lacZ reporter vector pORI13 for the generation of chromosomal transcriptional fusions was demonstrated. In addition, the repA gene, necessary for the replication of pORI13, was introduced into the bgaA locus, thereby generating a background for plasmid-based promoter expression studies. (iii) A simplified chemically defined medium, which supports growth of all sequenced S. pneumoniae strains to a level comparable to that in complex medium. (iv) A system for the introduction of unmarked deletions and mutations into the chromosome, which is independent of the genotype of the target strain. Most of these systems were successfully applied in strains R6 and TIGR4 as well. In addition, the tools offer several improvements and advantages compared to existing ones. Thus, the molecular toolbox for S. pneumoniae has been successfully extended.

fur-independent regulation of the Pasteurella multocida hbpA gene encoding a haemin-binding protein

Microbiology ◽  
2003 ◽  
Vol 149 (8) ◽  
pp. 2273-2281 ◽  
Author(s):  
M. Elena Garrido ◽  
Montserrat Bosch ◽  
Ricardo Medina ◽  
Anna Bigas ◽  
Montserrat Llagostera ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Pasteurella Multocida ◽  
Outer Membrane Proteins ◽  
Protein Composition ◽  
Electrophoretic Analysis ◽  
Gene Encoding ◽  
Expression Studies ◽  
Genes Encoding ◽  
Fur Protein ◽  
Iron Manganese

Treatment of bacterial cultures with chelating agents such as 2,2′-dipyridyl (DPD) induces expression of iron-regulated genes. It is known that in the γ-Proteobacteria, the Fur protein is the major regulator of genes encoding haem- or haemoglobin-binding proteins. Electrophoretic analysis of outer-membrane proteins of the γ-proteobacterium Pasteurella multocida has revealed the induction of two proteins of 60 and 40 kDa in DPD-treated cultures in both wild-type and fur-defective strains. These two proteins have the same N-terminal amino acid sequence, which identifies this protein as the product of the PM0592 ORF. Analysis of the sequence of this ORF, which encodes a protein of 60 kDa, revealed the presence of a hexanucleotide (AAAAAA) at which a programmed translational frameshift can occur giving rise to a 40 kDa protein. Analyses conducted in Escherichia coli, using the complete PM0592 ORF and a derivative truncated at the hexanucleotide position, have shown that both polypeptides bind haemin. For this reason, the PM0592 ORF product has been designated HbpA (for haemin-binding protein). Expression studies using both RT-PCR and lacZ fusions, as well as electrophoretic profiles of outer-membrane protein composition, have demonstrated that the hbpA gene is negatively regulated by iron, manganese and haemin through a fur-independent pathway. Despite the fact that serum of mice infected with P. multocida contained antibodies that reacted with both the 60 and 40 kDa products of the hbpA gene, these proteins did not offer protection when used in immunization assays against this micro-organism.

scholarly journals σB Activation under Environmental and Energy Stress Conditions in Listeria monocytogenes

2006 ◽  
Vol 72 (8) ◽  
pp. 5197-5203 ◽  
Author(s):  
Soraya Chaturongakul ◽  
Kathryn J. Boor
Keyword(s):  
Listeria Monocytogenes ◽  
Cumene Hydroperoxide ◽  
Stress Conditions ◽  
Wild Type ◽  
Regulation Of Expression ◽  
Transcript Levels ◽  
Energy Stress ◽  
Expression Studies ◽  
Genes Encoding ◽  
Gene Expression Studies

ABSTRACT To measure σB activation in Listeria monocytogenes under environmental or energy stress conditions, quantitative reverse transcriptase PCR (TaqMan) was used to determine the levels of transcripts for the σB-dependent opuCA and clpC genes in strains having null mutations in genes encoding regulator of sigma B proteins (rsbT and rsbV) and sigma B (sigB) and in the L. monocytogenes wild-type 10403S strain under different stress conditions. The ΔsigB, ΔrsbT, and ΔrsbV strains previously exhibited increased hemolytic activities compared to the hemolytic activity of the wild-type strain; therefore, transcript levels for hly were also determined. RsbT, RsbV, and σB were all required for opuCA expression during growth under carbon-limiting conditions or following exposure to pH 4.5, salt, ethanol, or the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). Expression of clpC was RsbT, RsbV, and σB dependent in the presence of CCCP but not under the other conditions. hly expression was not RsbT, RsbV, or σB dependent in the presence of either CCCP or salt. opuCA transcript levels did not increase in the presence of rapidly lethal stresses (i.e., pH 2.5 or 13 mM cumene hydroperoxide) despite the enhanced survival of the wild type compared with the survival of the mutant strains under these conditions. These findings highlight the importance of complementing phenotypic characterizations with gene expression studies to identify direct and indirect effects of null mutations in regulatory genes, such as sigB. Overall, our data show that while σB activation occurs through a single pathway under both environmental and energy stress conditions, regulation of expression of some stress response and virulence genes in the σB regulon (e.g., clpC) appears to require networks involving multiple transcriptional regulators.

scholarly journals Discovery of Anthocyanin Biosynthetic Pathway in Cosmos caudatus Kunth. Using Omics Analysis

Agronomy ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 661
Author(s):  
Darvien Gunasekaran ◽  
Noor Idayu Tahir ◽  
Muhamad Afiq Akbar ◽  
Syazwani Basir ◽  
Ismanizan Ismail ◽  
...  
Keyword(s):  
Biosynthetic Pathway ◽  
Anthocyanin Biosynthesis ◽  
Natural Colorant ◽  
Gene Pathway ◽  
Cosmos Caudatus ◽  
Anthocyanin Biosynthetic Pathway ◽  
Medicinal Value ◽  
Expression Studies ◽  
Genes Encoding ◽  
Nutritional Compounds

Cosmos caudatus Kunth. or “king’s salad” contains high values of nutritional compounds that act as health promoters. Although widely consumed for its medicinal value, information on phytochemical contents and their biosynthesis in the species is scarce. Among the interesting compounds are the anthocyanins that possess a dual role; an antioxidant and natural colorant. A complete anthocyanin biosynthetic pathway in C. caudatus was elucidated using transcriptomics, metabolomics, and anatomical approaches in this study. The transcriptomic analysis revealed genes encoding enzymes in the anthocyanin biosynthetic pathway and the genes encoding the transcription factors relevant to the latter pathway. A total of 11 anthocyanins of cyanidin, pelargonidin, and delphinidin derivatives that are significantly abundant in the species were identified, correlating with the anthocyanin mainstream gene pathway. The occurrence of anthocyanin was further validated by light microscopy. Anthocyanin pigments in C. caudatus were detected at the epidermal layer of the leaf, stem, and flower, and at the cortex of stem and root. To our knowledge, this is the first work that has delineated the complete anthocyanin biosynthetic pathway in Malaysia’s underutilized plant, C. caudatus Kunth. This study correlated multi-omics data that will help integrate systems biology and synthetic biology, for a detailed understanding of the molecular mechanism and characterization of the anthocyanin biosynthesis using heterologous expression studies.

scholarly journals Genes encoding membrane proteins showed stable expression in rice under aerobic condition: novel set of reference genes for expression studies

3 Biotech ◽  
2018 ◽  
Vol 8 (9) ◽  
Author(s):  
Amol S. Phule ◽  
Kalyani M. Barbadikar ◽  
M. S. Madhav ◽  
P. Senguttuvel ◽  
M. B. B. Prasad Babu ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Reference Genes ◽  
Aerobic Condition ◽  
Stable Expression ◽  
Expression Studies ◽  
Genes Encoding

scholarly journals Thioredoxin Is Involved in U(VI) and Cr(VI) Reduction in Desulfovibrio desulfuricans G20

2009 ◽  
Vol 191 (15) ◽  
pp. 4924-4933 ◽  
Author(s):  
Xiangkai Li ◽  
Lee R. Krumholz
Keyword(s):  
Specific Inhibitor ◽  
Thioredoxin Reductase ◽  
Desulfovibrio Desulfuricans ◽  
Receptor Protein ◽  
Insertion Mutant ◽  
Gene Encoding ◽  
Transposon Insertion ◽  
Expression Studies ◽  
Genes Encoding ◽  
Camp Receptor

ABSTRACT A transposon insertion mutant has been identified in a Desulfovibrio desulfuricans G20 mutant library that does not grow in the presence of 2 mM U(VI) in lactate-sulfate medium. This mutant has also been shown to be deficient in the ability to grow with 100 μM Cr(VI) and 20 mM As(V). Experiments with washed cells showed that this mutant had lost the ability to reduce U(VI) or Cr(VI), providing an explanation for the lower tolerance. A gene encoding a cyclic AMP (cAMP) receptor protein (CRP) was identified as the site of the transposon insertion. The remainder of the mre operon (metal reduction) contains genes encoding a thioredoxin, thioredoxin reductase, and an additional oxidoreductase whose substrate has not been predicted. Expression studies showed that in the mutant, the entire operon is downregulated, suggesting that the CRP may be involved in regulating expression of the whole operon. Exposure of the cells to U(VI) resulted in upregulation of the entire operon. CdCl2, a specific inhibitor of thioredoxin activity, inhibits U(VI) reduction by washed cells and inhibits growth of cells in culture when U(VI) is present, confirming a role for thioredoxin in U(VI) reduction. The entire mre operon was cloned into Escherichia coli JM109 and the transformant developed increased U(VI) resistance and the ability to reduce U(VI) to U(IV). The oxidoreductase protein (MreG) from this operon was expressed and purified from E. coli. In the presence of thioredoxin, thioredoxin reductase, and NADPH, this protein was shown to reduce both U(VI) and Cr(VI), providing a mechanism for the cytoplasmic reduction of these metals.

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