scholarly journals Calcium-activated (p)ppGpp Synthetase in Chloroplasts of Land Plants

2007 ◽  
Vol 282 (49) ◽  
pp. 35536-35545 ◽  
Author(s):  
Yuzuru Tozawa ◽  
Akira Nozawa ◽  
Takuya Kanno ◽  
Takakuni Narisawa ◽  
Shinji Masuda ◽  
...  
Keyword(s):  
Stringent Response ◽  
Transit Peptide ◽  
Genetic System ◽  
Land Plants ◽  
Synthetase Activity ◽  
Chloroplast Transit Peptide ◽  
Ef Hand ◽  
Genes Encoding

The genetic system of chloroplasts, including the machinery for transcription, translation, and DNA replication, exhibits substantial similarity to that of eubacteria. Chloroplasts are also thought to possess a system for generating guanosine 5′-triphosphate ((p)ppGpp), which triggers the stringent response in eubacteria, with genes encoding chloroplastic (p)ppGpp synthetase having been identified. We now describe the identification and characterization of genes (OsCRSH1, OsCRSH2, and OsCRSH3) for a novel type of (p)ppGpp synthetase in rice. The proteins encoded by these genes contain a putative chloroplast transit peptide at the NH2 terminus, a central RelA-SpoT-like domain, and two EF-hand motifs at the COOH terminus. The recombinant OsCRSH1 protein was imported into chloroplasts in vitro, and genetic complementation analysis revealed that expression of OsCRSH1 suppressed the phenotype of an Escherichia coli mutant deficient in the RelA and SpoT enzymes. Biochemical analysis showed that the OsCRSH proteins possess (p)ppGpp synthetase activity that is dependent both on Ca2+ and on the EF-hand motifs. A data base search identified a CRSH homolog in the dicotyledon Arabidopsis thaliana, indicating that such genes are conserved among both monocotyledonous and dicotyledonous land plants. CRSH proteins thus likely function as Ca2+-activated (p)ppGpp synthetases in plant chloroplasts, implicating both Ca2+ and (p)ppGpp signaling in regulation of the genetic system of these organelles.

scholarly journals From (p)ppGpp to (pp)pGpp: Characterization of Regulatory Effects of pGpp Synthesized by the Small Alarmone Synthetase of Enterococcus faecalis

2015 ◽  
Vol 197 (18) ◽  
pp. 2908-2919 ◽  
Author(s):  
Anthony O. Gaca ◽  
Pavel Kudrin ◽  
Cristina Colomer-Winter ◽  
Jelena Beljantseva ◽  
Kuanqing Liu ◽  
...  
Keyword(s):  
Stress Tolerance ◽  
Enterococcus Faecalis ◽  
Second Messengers ◽  
Stringent Response ◽  
Synthetase Activity ◽  
Protective Effects ◽  
Content Type ◽  
Regulatory Effects ◽  
Complex Target

ABSTRACTThe bacterial stringent response (SR) is a conserved stress tolerance mechanism that orchestrates physiological alterations to enhance cell survival. This response is mediated by the intracellular accumulation of the alarmones pppGpp and ppGpp, collectively called (p)ppGpp. InEnterococcus faecalis, (p)ppGpp metabolism is carried out by the bifunctional synthetase/hydrolaseE. faecalisRel (RelEf) and the small alarmone synthetase (SAS) RelQEf. Although Rel is the main enzyme responsible for SR activation inFirmicutes, there is emerging evidence that SASs can make important contributions to bacterial homeostasis. Here, we showed that RelQEfsynthesizes ppGpp more efficiently than pppGpp without the need for ribosomes, tRNA, or mRNA. In addition to (p)ppGpp synthesis from GDP and GTP, RelQEfalso efficiently utilized GMP to form GMP 3′-diphosphate (pGpp). Based on this observation, we sought to determine if pGpp exerts regulatory effects on cellular processes affected by (p)ppGpp. We found that pGpp, like (p)ppGpp, strongly inhibits the activity ofE. faecalisenzymes involved in GTP biosynthesis and, to a lesser extent, transcription ofrrnBbyEscherichia coliRNA polymerase. Activation ofE. coliRelA synthetase activity was observed in the presence of both pGpp and ppGpp, while RelQEfwas activated only by ppGpp. Furthermore, enzymatic activity of RelQEfis insensitive to relacin, a (p)ppGpp analog developed as an inhibitor of “long” RelA/SpoT homolog (RSH) enzymes. We conclude that pGpp can likely function as a bacterial alarmone with target-specific regulatory effects that are similar to what has been observed for (p)ppGpp.IMPORTANCEAccumulation of the nucleotide second messengers (p)ppGpp in bacteria is an important signal regulating genetic and physiological networks contributing to stress tolerance, antibiotic persistence, and virulence. Understanding the function and regulation of the enzymes involved in (p)ppGpp turnover is therefore critical for designing strategies to eliminate the protective effects of this molecule. While characterizing the (p)ppGpp synthetase RelQ ofEnterococcus faecalis(RelQEf), we found that, in addition to (p)ppGpp, RelQEfis an efficient producer of pGpp (GMP 3′-diphosphate).In vitroanalysis revealed that pGpp exerts complex, target-specific effects on processes known to be modulated by (p)ppGpp. These findings provide a new regulatory feature of RelQEfand suggest that pGpp may represent a new member of the (pp)pGpp family of alarmones.

Identification and preliminary characterization of cell-wall-anchored proteins of Staphylococcus epidermidis

Microbiology ◽  
2005 ◽  
Vol 151 (5) ◽  
pp. 1453-1464 ◽  
Author(s):  
M. Gabriela Bowden ◽  
Wei Chen ◽  
Jenny Singvall ◽  
Yi Xu ◽  
Sharon J. Peacock ◽  
...  
Keyword(s):  
Cell Wall ◽  
Staphylococcus Epidermidis ◽  
Tertiary Structure ◽  
Genomic Sequence ◽  
Human Infection ◽  
Pcr Analysis ◽  
Genes Encoding ◽  
Antibody Titres

Staphylococcus epidermidis is a ubiquitous human skin commensal that has emerged as a major cause of foreign-body infections. Eleven genes encoding putative cell-wall-anchored proteins were identified by computer analysis of the publicly available S. epidermidis unfinished genomic sequence. Four genes encode previously described proteins (Aap, Bhp, SdrF and SdrG), while the remaining seven have not been characterized. Analysis of primary sequences of the Staphylococcus epidermidis surface (Ses) proteins indicates that they have a structural organization similar to the previously described cell-wall-anchored proteins from S. aureus and other Gram-positive cocci. However, not all of the Ses proteins are direct homologues of the S. aureus proteins. Secondary and tertiary structure predictions suggest that most of the Ses proteins are composed of several contiguous subdomains, and that the majority of these predicted subdomains are folded into β-rich structures. PCR analysis indicates that certain genes may be found more frequently in disease isolates compared to strains isolated from healthy skin. Patients recovering from S. epidermidis infections had higher antibody titres against some Ses proteins, implying that these proteins are expressed during human infection. Western blot analyses of early-logarithmic and late-stationary in vitro cultures suggest that different regulatory mechanisms control the expression of the Ses proteins.

scholarly journals Characterization of an L-Ascorbate Catabolic Pathway with Unprecedented Enzymatic Transformations

2019 ◽  
Author(s):  
Tyler Stack ◽  
Katelyn Morrison ◽  
Thomas Dettmer ◽  
Brendan Wille ◽  
Chan Kim ◽  
...  
Keyword(s):  
Ralstonia Eutropha ◽  
Sequence Similarity ◽  
Catabolic Genes ◽  
Genes Encoding ◽  
Benzilic Acid ◽  
Amidohydrolase Superfamily ◽  
Sequence Similarity Networks

<p>L-Ascorbate (vitamin C) is ubiquitous in both our diet and the environment. <i>Ralstonia eutropha </i>H16 (<i>Cupriavidus necator </i>ATCC 17699) uses L-ascorbate as sole carbon source but lacks the genes encoding the known catabolic pathways. RNAseq identified eight candidate catabolic genes. Sequence similarity networks and genome neighborhood networks guided predictions for function of the encoded proteins; the predictions were confirmed by <i>in vitro</i> assays and <i>in vivo</i> growth phenotypes of gene deletion mutants. L-Ascorbate, a lactone, is oxidized and ring-opened by enzymes in the cytochrome b<sub>561</sub> and gluconolactonase families, respectively, to form 2,3-diketo-L-gulonate. A protein predicted to have a WD40-like fold catalyzes an unprecedented benzilic acid rearrangement involving migration of a carboxylate group to form 2-carboxy-L-lyxonolactone; the lactone is hydrolyzed by a member of the amidohydrolase superfamily to yield 2-carboxy-L-lyxonate. A member of the PdxA family of oxidative decarboxylases catalyzes a novel decarboxylation that uses NAD<sup>+</sup> catalytically. The product, L-lyxonate, is catabolized to alpha-ketoglutarate by a previously characterized pathway.</p>

scholarly journals Contractility parameters of human β-cardiac myosin with the hypertrophic cardiomyopathy mutation R403Q show loss of motor function

2015 ◽  
Vol 1 (9) ◽  
pp. e1500511 ◽  
Author(s):  
Suman Nag ◽  
Ruth F. Sommese ◽  
Zoltan Ujfalusi ◽  
Ariana Combs ◽  
Stephen Langer ◽  
...  
Keyword(s):  
Hypertrophic Cardiomyopathy ◽  
Actin Filaments ◽  
Duty Ratio ◽  
Cardiac Myosin ◽  
Wild Type ◽  
Genes Encoding ◽  
New Methodologies ◽  
Unloaded Velocity

Hypertrophic cardiomyopathy (HCM) is the most frequently occurring inherited cardiovascular disease. It is caused by mutations in genes encoding the force-generating machinery of the cardiac sarcomere, including human β-cardiac myosin. We present a detailed characterization of the most debated HCM-causing mutation in human β-cardiac myosin, R403Q. Despite numerous studies, most performed with nonhuman or noncardiac myosin, there is no consensus about the mechanism of action of this mutation on the function of the enzyme. We use recombinant human β-cardiac myosin and new methodologies to characterize in vitro contractility parameters of the R403Q myosin compared to wild type. We extend our studies beyond pure actin filaments to include the interaction of myosin with regulated actin filaments containing tropomyosin and troponin. We find that, with pure actin, the intrinsic force generated by R403Q is ~15% lower than that generated by wild type. The unloaded velocity is, however, ~10% higher for R403Q myosin, resulting in a load-dependent velocity curve that has the characteristics of lower contractility at higher external loads compared to wild type. With regulated actin filaments, there is no increase in the unloaded velocity and the contractility of the R403Q myosin is lower than that of wild type at all loads. Unlike that with pure actin, the actin-activated adenosine triphosphatase activity for R403Q myosin with Ca2+-regulated actin filaments is ~30% lower than that for wild type, predicting a lower unloaded duty ratio of the motor. Overall, the contractility parameters studied fit with a loss of human β-cardiac myosin contractility as a result of the R403Q mutation.

scholarly journals Functional and Phylogenetic Divergence of Fungal Adenylate-Forming Reductases

2014 ◽  
Vol 80 (19) ◽  
pp. 6175-6183 ◽  
Author(s):  
Daniel Kalb ◽  
Gerald Lackner ◽  
Dirk Hoffmeister
Keyword(s):  
Lysine Biosynthesis ◽  
Reduction Step ◽  
Content Type ◽  
Ceriporiopsis Subvermispora ◽  
Major Class ◽  
Genes Encoding ◽  
Short Chain Dehydrogenase ◽  
Phylogenetic Divergence

ABSTRACTA key step in fungall-lysine biosynthesis is catalyzed by adenylate-formingl-α-aminoadipic acid reductases, organized in domains for adenylation, thiolation, and the reduction step. However, the genomes of numerous ascomycetes and basidiomycetes contain an unexpectedly large number of additional genes encoding similar but functionally distinct enzymes. Here, we describe the functionalin vitrocharacterization of four reductases which were heterologously produced inEscherichia coli. TheCeriporiopsis subvermisporaserine reductase Nps1 features a terminal ferredoxin-NADP+reductase (FNR) domain and thus belongs to a hitherto undescribed class of fungal multidomain enzymes. The second major class is characterized by the canonical terminal short-chain dehydrogenase/reductase domain and represented byCeriporiopsis subvermisporaNps3 as the first biochemically characterizedl-α-aminoadipic acid reductase of basidiomycete origin.Aspergillus flavusl-tyrosine reductases LnaA and LnbA are members of a distinct phylogenetic clade. Phylogenetic analysis supports the view that fungal adenylate-forming reductases are more diverse than previously recognized and belong to four distinct classes.

Functional characterization of chloroplast transit peptide in the small subunit of Rubisco in maize

2019 ◽  
Vol 237 ◽  
pp. 12-20
Author(s):  
Lifen Chen ◽  
Ximeng Wang ◽  
Lei Wang ◽  
Yuan Fang ◽  
Xiucai Pan ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Transit Peptide ◽  
Small Subunit ◽  
Chloroplast Transit Peptide

scholarly journals Genetic and mutational characterization of the small alarmone synthetase gene relV of Vibrio cholerae

Microbiology ◽  
2014 ◽  
Vol 160 (9) ◽  
pp. 1855-1866 ◽  
Author(s):  
Shreya Dasgupta ◽  
Pallabi Basu ◽  
Ritesh Ranjan Pal ◽  
Satyabrata Bag ◽  
Rupak K. Bhadra
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Small Molecules ◽  
Vibrio Cholerae ◽  
Stringent Response ◽  
Synthetase Activity ◽  
Gram Negative Bacteria ◽  
Amino Acid Residues ◽  
Gram Negative

In Vibrio cholerae, the causative agent of cholera, products of three genes, relA, spoT and relV, govern nutritional stress related stringent response (SR). SR in bacteria is critically regulated by two intracellular small molecules, guanosine 3′-diphosphate 5′-triphosphate (pppGpp) and guanosine 3′,5′-bis(diphosphate) (ppGpp), collectively called (p)ppGpp or alarmone. Evolution of relV is unique in V. cholerae because other Gram-negative bacteria carry only relA and spoT genes. Recent reports suggest that RelV is needed for pathogenesis. RelV carries a single (p)ppGpp synthetase or RelA-SpoT domain (SYNTH/RSD) and belongs to the small alarmone synthetase (SAS) family of proteins. Here, we report extensive functional characterizations of the relV gene by constructing several deletion and site-directed mutants followed by their controlled expression in (p)ppGpp0 cells of Escherichia coli or V. cholerae. Substitution analysis indicated that the amino acid residues K107, D129, R132, L150 and E188 of the RSD region of RelV are essential for its activity. While K107, D129 and E188 are highly conserved in RelA and SAS proteins, L150 appears to be conserved in the latter group of enzymes, and the R132 residue was found to be unique in RelV. Extensive progressive deletion analysis indicated that the amino acid residues at positions 59 and 248 of the RelV protein are the functional N- and C-terminal boundaries, respectively. Since the minimal functional length of RelV was found to be 189 aa, which includes the 94 aa long RSD region, it seems that the flanking residues of the RSD are also important for maintaining the (p)ppGpp synthetase activity.

scholarly journals Structural and Biological Characterization of a Capsular Polysaccharide Produced by Staphylococcus haemolyticus

2007 ◽  
Vol 190 (5) ◽  
pp. 1649-1657 ◽  
Author(s):  
Sigrid Flahaut ◽  
Evgeny Vinogradov ◽  
Kathryn A. Kelley ◽  
Shannon Brennan ◽  
Keiichi Hiramatsu ◽  
...  
Keyword(s):  
Capsular Polysaccharide ◽  
In Vitro Assays ◽  
Sugar Residue ◽  
Biological Characterization ◽  
Staphylococcus Haemolyticus ◽  
Acyl Substituent ◽  
Genes Encoding ◽  
Opsonophagocytic Killing

ABSTRACT The DNA sequence of the genome of Staphylococcus haemolyticus JCSC1435 revealed a putative capsule operon composed of 13 genes in tandem. The first seven genes (capABCDEFGSh ) showed ≥57% similarity with the Staphylococcus aureus cap5 or cap8 locus. However, the capHIJKLMSh genes are unique to S. haemolyticus and include genes encoding a putative flippase, an aminotransferase, two glycosyltransferases, and a transcriptional regulator. Capsule-like material was readily apparent by immunoelectron microscopy on bacteria harvested in the postexponential phase of growth. Electron micrographs of a JCSC1435 mutant with a deleted cap region lacked the capsule-like material. Both strains produced small amounts of surface-associated material that reacted with antibodies to polyglutamic acid. S. haemolyticus cap genes were amplified from four of seven clinical isolates of S. haemolyticus from humans, and three of these strains produced a serologically cross-reactive capsular polysaccharide. In vitro assays demonstrated that the acapsular mutant strain showed greater biofilm formation but was more susceptible to complement-mediated opsonophagocytic killing than the parent strain. Structural characterization of capsule purified from S. haemolyticus strain JCSC1435 showed a trisaccharide repeating unit: −3-α-l-FucNAc-3-(2-NAc-4-N-Asp-2,4,6-trideoxy-β-d-Glc)-4-α-d-GlcNAc-. This structure is unique among staphylococcal polysaccharides in that its composition includes a trideoxy sugar residue with aspartic acid as an N-acyl substituent.

scholarly journals Polynucleotide Phosphorylase Functions as Both an Exonuclease and a Poly(A) Polymerase in Spinach Chloroplasts

2001 ◽  
Vol 21 (16) ◽  
pp. 5408-5416 ◽  
Author(s):  
Shlomit Yehudai-Resheff ◽  
Merav Hirsh ◽  
Gadi Schuster
Keyword(s):  
Transit Peptide ◽  
Rna Degradation ◽  
Polynucleotide Phosphorylase ◽  
Chloroplast Transit Peptide ◽  
Stem Loop ◽  
Rna Molecules ◽  
Spinach Chloroplasts ◽  
Endonucleolytic Cleavage ◽  
Exonucleolytic Degradation

ABSTRACT The molecular mechanism of mRNA degradation in the chloroplast consists of sequential events including endonucleolytic cleavage, the addition of poly(A)-rich sequences to the endonucleolytic cleavage products, and exonucleolytic degradation by polynucleotide phosphorylase (PNPase). In Escherichia coli,polyadenylation is performed mainly by poly(A)-polymerase (PAP) I or by PNPase in its absence. While trying to purify the chloroplast PAP by following in vitro polyadenylation activity, it was found to copurify with PNPase and indeed could not be separated from it. Purified PNPase was able to polyadenylate RNA molecules with an activity similar to that of lysed chloroplasts. Both activities use ADP much more effectively than ATP and are inhibited by stem-loop structures. The activity of PNPase was directed to RNA degradation or polymerization by manipulating physiologically relevant concentrations of Piand ADP. As expected of a phosphorylase, Pi enhanced degradation, whereas ADP inhibited degradation and enhanced polymerization. In addition, searching the completeArabidopsis genome revealed several putative PAPs, none of which were preceded by a typical chloroplast transit peptide. These results suggest that there is no enzyme similar to E. coli PAP I in spinach chloroplasts and that polyadenylation and exonucleolytic degradation of RNA in spinach chloroplasts are performed by one enzyme, PNPase.

scholarly journals Genetic and Biochemical Characterization of the Phosphoenolpyruvate:Glucose/Mannose Phosphotransferase System of Streptococcus thermophilus

2003 ◽  
Vol 69 (9) ◽  
pp. 5423-5432 ◽  
Author(s):  
Armelle Cochu ◽  
Christian Vadeboncoeur ◽  
Sylvain Moineau ◽  
Michel Frenette
Keyword(s):  
Biochemical Characterization ◽  
Sugar Transport ◽  
Streptococcus Thermophilus ◽  
Phosphate Group ◽  
Phosphotransferase System ◽  
Whole Cells ◽  
Genes Encoding ◽  
Regulatory Functions

ABSTRACT In most streptococci, glucose is transported by the phosphoenolpyruvate (PEP):glucose/mannose phosphotransferase system (PTS) via HPr and IIABMan, two proteins involved in regulatory mechanisms. While most strains of Streptococcus thermophilus do not or poorly metabolize glucose, compelling evidence suggests that S. thermophilus possesses the genes that encode the glucose/mannose general and specific PTS proteins. The purposes of this study were to determine (i) whether these PTS genes are expressed, (ii) whether the PTS proteins encoded by these genes are able to transfer a phosphate group from PEP to glucose/mannose PTS substrates, and (iii) whether these proteins catalyze sugar transport. The pts operon is made up of the genes encoding HPr (ptsH) and enzyme I (EI) (ptsI), which are transcribed into a 0.6-kb ptsH mRNA and a 2.3-kb ptsHI mRNA. The specific glucose/mannose PTS proteins, IIABMan, IICMan, IIDMan, and the ManO protein, are encoded by manL, manM, manN, and manO, respectively, which make up the man operon. The man operon is transcribed into a single 3.5-kb mRNA. To assess the phosphotransfer competence of these PTS proteins, in vitro PEP-dependent phosphorylation experiments were conducted with purified HPr, EI, and IIABMan as well as membrane fragments containing IICMan and IIDMan. These PTS components efficiently transferred a phosphate group from PEP to glucose, mannose, 2-deoxyglucose, and (to a lesser extent) fructose, which are common streptococcal glucose/mannose PTS substrates. Whole cells were unable to catalyze the uptake of mannose and 2-deoxyglucose, demonstrating the inability of the S. thermophilus PTS proteins to operate as a proficient transport system. This inability to transport mannose and 2-deoxyglucose may be due to a defective IIC domain. We propose that in S. thermophilus, the general and specific glucose/mannose PTS proteins are not involved in glucose transport but might have regulatory functions associated with the phosphotransfer properties of HPr and IIABMan.

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