scholarly journals Involvement of the SppA1 Peptidase in Acclimation to Saturating Light Intensities in Synechocystis sp. Strain PCC 6803

2004 ◽  
Vol 186 (12) ◽  
pp. 3991-3999 ◽  
Author(s):  
E. Pojidaeva ◽  
V. Zinchenko ◽  
S. V. Shestakov ◽  
A. Sokolenko
Keyword(s):  
Light Intensity ◽  
Wild Type Strain ◽  
Wild Type ◽  
Additional Mechanism ◽  
Gene Encoding ◽  
Light Intensities ◽  
In The Wild ◽  
Pcc 6803

ABSTRACT The sll1703 gene, encoding an Arabidopsis homologue of the thylakoid membrane-associated SppA peptidase, was inactivated by interposon mutagenesis in Synechocystis sp. strain PCC 6803. Upon acclimation from a light intensity of 50 to 150 μE m−2 s−1, the mutant preserved most of its phycobilisome content, whereas the wild-type strain developed a bleaching phenotype due to the loss of about 40% of its phycobiliproteins. Using in vivo and in vitro experiments, we demonstrate that the ΔsppA1 strain does not undergo the cleavage of the LR 33 and LCM 99 linker proteins that develops in the wild type exposed to increasing light intensities. We conclude that a major contribution to light acclimation under a moderate light regime in cyanobacteria originates from an SppA1-mediated cleavage of phycobilisome linker proteins. Together with changes in gene expression of the major phycobiliproteins, it contributes an additional mechanism aimed at reducing the content in phycobilisome antennae upon acclimation to a higher light intensity.

scholarly journals Regulation of ppk Expression and In Vivo Function of Ppk in Streptomyces lividans TK24

2006 ◽  
Vol 188 (17) ◽  
pp. 6269-6276 ◽  
Author(s):  
Sofiane Ghorbel ◽  
Aleksey Smirnov ◽  
Hichem Chouayekh ◽  
Brice Sperandio ◽  
Catherine Esnault ◽  
...  
Keyword(s):  
Mutant Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Sufficient Conditions ◽  
Streptomyces Lividans ◽  
Antibiotic Biosynthesis ◽  
Wild Type ◽  
In The Wild

ABSTRACT The ppk gene of Streptomyces lividans encodes an enzyme catalyzing, in vitro, the reversible polymerization of the γ phosphate of ATP into polyphosphate and was previously shown to play a negative role in the control of antibiotic biosynthesis (H. Chouayekh and M. J. Virolle, Mol. Microbiol. 43:919-930, 2002). In the present work, some regulatory features of the expression of ppk were established and the polyphosphate content of S. lividans TK24 and the ppk mutant was determined. In Pi sufficiency, the expression of ppk was shown to be low but detectable. DNA gel shift experiments suggested that ppk expression might be controlled by a repressor using ATP as a corepressor. Under these conditions, short acid-soluble polyphosphates accumulated upon entry into the stationary phase in the wild-type strain but not in the ppk mutant strain. The expression of ppk under Pi-limiting conditions was shown to be much higher than that under Pi-sufficient conditions and was under positive control of the two-component system PhoR/PhoP. Under these conditions, the polyphosphate content of the cell was low and polyphosphates were reproducibly found to be longer and more abundant in the ppk mutant strain than in the wild-type strain, suggesting that Ppk might act as a nucleoside diphosphate kinase. In light of our results, a novel view of the role of this enzyme in the regulation of antibiotic biosynthesis in S. lividans TK24 is proposed.

scholarly journals Expression and function of the cdgD gene, encoding a CHASE–PAS-DGC-EAL domain protein, in Azospirillum brasilense

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
José Francisco Cruz-Pérez ◽  
Roxana Lara-Oueilhe ◽  
Cynthia Marcos-Jiménez ◽  
Ricardo Cuatlayotl-Olarte ◽  
María Luisa Xiqui-Vázquez ◽  
...  
Keyword(s):  
Azospirillum Brasilense ◽  
Type Strain ◽  
Wild Type Strain ◽  
Soil Conditions ◽  
Wild Type ◽  
Gene Encoding ◽  
Wheat Roots ◽  
Genes Encoding ◽  
In The Wild ◽  
Plant Growth Promoting

AbstractThe plant growth-promoting bacterium Azospirillum brasilense contains several genes encoding proteins involved in the biosynthesis and degradation of the second messenger cyclic-di-GMP, which may control key bacterial functions, such as biofilm formation and motility. Here, we analysed the function and expression of the cdgD gene, encoding a multidomain protein that includes GGDEF-EAL domains and CHASE and PAS domains. An insertional cdgD gene mutant was constructed, and analysis of biofilm and extracellular polymeric substance production, as well as the motility phenotype indicated that cdgD encoded a functional diguanylate protein. These results were correlated with a reduced overall cellular concentration of cyclic-di-GMP in the mutant over 48 h compared with that observed in the wild-type strain, which was recovered in the complemented strain. In addition, cdgD gene expression was measured in cells growing under planktonic or biofilm conditions, and differential expression was observed when KNO3 or NH4Cl was added to the minimal medium as a nitrogen source. The transcriptional fusion of the cdgD promoter with the gene encoding the autofluorescent mCherry protein indicated that the cdgD gene was expressed both under abiotic conditions and in association with wheat roots. Reduced colonization of wheat roots was observed for the mutant compared with the wild-type strain grown in the same soil conditions. The Azospirillum-plant association begins with the motility of the bacterium towards the plant rhizosphere followed by the adsorption and adherence of these bacteria to plant roots. Therefore, it is important to study the genes that contribute to this initial interaction of the bacterium with its host plant.

scholarly journals Topoisomerase I involvement in illegitimate recombination in Saccharomyces cerevisiae.

1996 ◽  
Vol 16 (4) ◽  
pp. 1805-1812 ◽  
Author(s):  
J Zhu ◽  
R H Schiestl
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Aberrations ◽  
Topoisomerase I ◽  
Wild Type Strain ◽  
Hot Spot ◽  
Illegitimate Recombination ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae

Chromosome aberrations may cause cancer and many heritable diseases. Topoisomerase I has been suspected of causing chromosome aberrations by mediating illegitimate recombination. The effects of deletion and of overexpression of the topoisomerase I gene on illegitimate recombination in the yeast Saccharomyces cerevisiae have been studied. Yeast transformations were carried out with DNA fragments that did not have any homology to the genomic DNA. The frequency of illegitimate integration was 6- to 12-fold increased in a strain overexpressing topoisomerase I compared with that in isogenic control strains. Hot spot sequences [(G/C)(A/T)T] for illegitimate integration target sites accounted for the majority of the additional events after overexpression of topoisomerase I. These hot spot sequences correspond to sequences previously identified in vitro as topoisomerase I preferred cleavage sequences in other organisms. Furthermore, such hot spot sequences were found in 44% of the integration events present in the TOP1 wild-type strain and at a significantly lower frequency in the top1delta strain. Our results provide in vivo evidence that a general eukaryotic topoisomerase I enzyme nicks DNA and ligates nonhomologous ends, leading to illegitimate recombination.

scholarly journals Identification and molecular characterization of a Chlamydomonas reinhardtii mutant that shows a light intensity dependent progressive chlorophyll deficiency

F1000Research ◽  
2013 ◽  
Vol 2 ◽  
pp. 142
Author(s):  
Phillip B Grovenstein ◽  
Darryel A Wilson ◽  
Kathryn D Lankford ◽  
Kelsey A Gaston ◽  
Surangi Perera ◽  
...  
Keyword(s):  
Light Intensity ◽  
Insertional Mutagenesis ◽  
Protoporphyrin Ix ◽  
Model Organism ◽  
Genomic Region ◽  
Oxygenic Photosynthesis ◽  
Autotrophic Growth ◽  
Wild Type ◽  
Light Intensities ◽  
In The Wild

The green micro-alga Chlamydomonas reinhardtii is an elegant model organism to study all aspects of oxygenic photosynthesis. Chlorophyll (Chl) and heme are major tetrapyrroles that play an essential role in energy metabolism in photosynthetic organisms. These tetrapyrroles are synthesized via a common branched pathway that involves mainly nuclear encoded enzymes. One of the enzymes in the pathway is Mg chelatase (MgChel) which inserts Mg2+ into protoporphyrin IX (PPIX, proto) to form Magnesium-protoporphyrin IX (MgPPIX, Mgproto), the first biosynthetic intermediate in the Chl branch. The GUN4 (genomes uncoupled 4) protein is not essential for the MgChel activity but has been shown to significantly stimulate its activity. We have isolated a light sensitive mutant, 6F14, by random DNA insertional mutagenesis. 6F14 cannot tolerate light intensities higher than 90-100 μmol photons m-2 s-1. It shows a light intensity dependent progressive photo-bleaching. 6F14 is incapable of photo-autotrophic growth under light intensity higher than 100 μmol photons m-2 s-1. PCR based analyses show that in 6F14 the insertion of the plasmid outside the GUN4 locus has resulted in a genetic rearrangement of the GUN4 gene and possible deletions in the genomic region flanking the GUN4 gene. Our gun4 mutant has a Chl content very similar to that in the wild type in the dark and is very sensitive to fluctuations in the light intensity in the environment unlike the earlier identified Chlamydomonas gun4 mutant. Complementation with a functional copy of the GUN4 gene restored light tolerance, Chl biosynthesis and photo-autotrophic growth under high light intensities in 6F14. 6F14 is the second gun4 mutant to be identified in C. reinhardtii. Additionally, we show that our two gun4 complements over-express the GUN4 protein and show a higher Chl content per cell compared to that in the wild type strain.

scholarly journals In VivoEfficacy of Human Simulated Regimens of Carbapenems and Comparator Agents against NDM-1-Producing Enterobacteriaceae

2013 ◽  
Vol 58 (3) ◽  
pp. 1671-1677 ◽  
Author(s):  
Dora E. Wiskirchen ◽  
Patrice Nordmann ◽  
Jared L. Crandon ◽  
David P. Nicolau
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Similar Efficacy ◽  
Infection Model ◽  
High Dose ◽  
Wild Type ◽  
Prolonged Infusion ◽  
Content Type

ABSTRACTDoripenem and ertapenem have demonstrated efficacy against several NDM-1-producing isolatesin vivo, despite having high MICs. In this study, we sought to further characterize the efficacy profiles of humanized regimens of standard (500 mg given every 8 h) and high-dose, prolonged infusion of doripenem (2 g given every 8 h, 4-h infusion) and 1 g of ertapenem given intravenously every 24 h and the comparator regimens of ceftazidime at 2 g given every 8 h (2-h infusion), levofloxacin at 500 mg every 24 h, and aztreonam at 2 g every 6 h (1-h infusion) against a wider range of isolates in a murine thigh infection model. An isogenic wild-type strain and NDM-1-producingKlebsiella pneumoniaeand eight clinical NDM-1-producing members of the familyEnterobacteriaceaewere tested in immunocompetent- and neutropenic-mouse models. The wild-type strain was susceptible to all of the agents, while the isogenic NDM-1-producing strain was resistant to ceftazidime, doripenem, and ertapenem. Clinical NDM-1-producing strains were resistant to nearly all five of the agents (two were susceptible to levofloxacin). In immunocompetent mice, all of the agents produced ≥1-log10CFU reductions of the isogenic wild-type and NDM-1-producing strains after 24 h. Minimal efficacy of ceftazidime, aztreonam, and levofloxacin against the clinical NDM-1-producing strains was observed. However, despitein vitroresistance, ≥1-log10CFU reductions of six of eight clinical strains were achieved with high-dose, prolonged infusion of doripenem and ertapenem. Slight enhancements of doripenem activity over the standard doses were obtained with high-dose, prolonged infusion for three of the four isolates tested. Similar efficacy observations were noted in neutropenic mice. These data suggest that carbapenems are a viable treatment option for infections caused by NDM-1-producingEnterobacteriaceae.

scholarly journals Involvement of H-NS in Transpositional Recombination Mediated by IS1

2001 ◽  
Vol 183 (8) ◽  
pp. 2476-2484 ◽  
Author(s):  
Yasuyuki Shiga ◽  
Yasuhiko Sekine ◽  
Yasunobu Kano ◽  
Eiichi Ohtsubo
Keyword(s):  
Dna Binding ◽  
Wild Type Strain ◽  
Integration Host Factor ◽  
Deficient Mutant ◽  
Wild Type ◽  
Transposase Gene ◽  
In The Wild ◽  
Transposition Reaction ◽  
Target Plasmid

ABSTRACT IS1, the smallest active transposable element in bacteria, encodes a transposase that promotes inter- and intramolecular transposition. Host-encoded factors, e.g., histone-like proteins HU and integration host factor (IHF), are involved in the transposition reactions of some bacterial transposable elements. Host factors involved in the IS1 transposition reaction, however, are not known. We show that a plasmid with an IS1 derivative that efficiently produces transposase did not generate miniplasmids, the products of intramolecular transposition, in mutants deficient in a nucleoid-associated DNA-binding protein, H-NS, but did generate them in mutants deficient in histone-like proteins HU, IHF, Fis, and StpA. Nor did IS1 transpose intermolecularly to the target plasmid in the H-NS-deficient mutant. The hns mutation did not affect transcription from the indigenous promoter of IS1 for the expression of the transposase gene. These findings show that transpositional recombination mediated by IS1 requires H-NS but does not require the HU, IHF, Fis, or StpA protein in vivo. Gel retardation assays of restriction fragments of IS1-carrying plasmid DNA showed that no sites were bound preferentially by H-NS within the IS1 sequence. The central domain of H-NS, which is involved in dimerization and/or oligomerization of the H-NS protein, was important for the intramolecular transposition of IS1, but the N- and C-terminal domains, which are involved in the repression of certain genes and DNA binding, respectively, were not. The SOS response induced by the IS1 transposase was absent in the H-NS-deficient mutant strain but was present in the wild-type strain. We discuss the possibility that H-NS promotes the formation of an active IS1 DNA-transposase complex in which the IS1 ends are cleaved to initiate transpositional recombination through interaction with IS1 transposase.

scholarly journals The Nonessential H2A N-Terminal Tail Can Function as an Essential Charge Patch on the H2A.Z Variant N-Terminal Tail

2003 ◽  
Vol 23 (8) ◽  
pp. 2778-2789 ◽  
Author(s):  
Qinghu Ren ◽  
Martin A. Gorovsky
Keyword(s):  
Tetrahymena Thermophila ◽  
Gene Replacement ◽  
In Vitro Mutagenesis ◽  
Wild Type ◽  
Wild Type Protein ◽  
Lysine Residues ◽  
In The Wild ◽  
Essential Function

ABSTRACT Tetrahymena thermophila cells contain three forms of H2A: major H2A.1 and H2A.2, which make up ∼80% of total H2A, and a conserved variant, H2A.Z. We showed previously that acetylation of H2A.Z was essential (Q. Ren and M. A. Gorovsky, Mol. Cell 7:1329-1335, 2001). Here we used in vitro mutagenesis of lysine residues, coupled with gene replacement, to identify the sites of acetylation of the N-terminal tail of the major H2A and to analyze its function in vivo. Tetrahymena cells survived with all five acetylatable lysines replaced by arginines plus a mutation that abolished acetylation of the N-terminal serine normally found in the wild-type protein. Thus, neither posttranslational nor cotranslational acetylation of major H2A is essential. Surprisingly, the nonacetylatable N-terminal tail of the major H2A was able to replace the essential function of the acetylation of the H2A.Z N-terminal tail. Tail-swapping experiments between H2A.1 and H2A.Z revealed that the nonessential acetylation of the major H2A N-terminal tail can be made to function as an essential charge patch in place of the H2A.Z N-terminal tail and that while the pattern of acetylation of an H2A N-terminal tail is determined by the tail sequence, the effects of acetylation on viability are determined by properties of the H2A core and not those of the N-terminal tail itself.

scholarly journals A Single Amino Acid at Residue 188 of Hexon Protein is Responsible for the Pathogenicity of the Emerging Novel Fowl Adenovirus 4

2021 ◽  
Author(s):  
Yu Zhang ◽  
Aijing Liu ◽  
Yanan Wang ◽  
Hongyu Cui ◽  
Yulong Gao ◽  
...  
Keyword(s):  
Amino Acid ◽  
Mutant Strain ◽  
Molecular Basis ◽  
Type Strain ◽  
Wild Type Strain ◽  
Wild Type ◽  
Live Attenuated Vaccine ◽  
Hexon Protein

Since 2015, severe hydropericardium-hepatitis syndrome (HHS) associated with a novel fowl adenovirus 4 (FAdV-4) has emerged in China, representing a new challenge for the poultry industry. Although various highly pathogenic FAdV-4 strains have been isolated, the virulence factor and the pathogenesis of novel FAdV-4 are unclear. In our previous studies, we reported that a large genomic deletion (1966 bp) is not related to increased virulence. In this study, two recombinant chimeric viruses, rHN20 strain and rFB2 strain, were generated from a highly pathogenic FAdV-4 strain by replacing hexon or fiber-2 gene of a non-pathogenic FAdV-4, respectively. Both chimeric strains showed similar titers to the wild type strain in vitro . Notably, rFB2 and the wild type strain induced 100% mortality, while no mortality or clinical signs appeared in chickens inoculated with rHN20, indicating that hexon, but not fiber-2, determines the novel FAdV-4 virulence. Furthermore, an R188I mutation in the hexon protein identified residue 188 as the key amino acid for the reduced pathogenicity. The rR188I mutant strain was significantly neutralized by chicken serum in vitro and in vivo , whereas the wild type strain was able to replicate efficiently. Finally, the immunogenicity of the rescued rR188I was investigated. Non-pathogenic rR188I provided full protection against lethal FAdV-4 challenge. Collectively, these findings provide an in-depth understanding of the molecular basis of novel FAdV-4 pathogenicity and present rR188I as a potential live attenuated vaccine candidate or a novel vaccine vector for HHS vaccines. Importance HHS associated with a novel FAdV-4 infection in chickens has caused huge economic losses to the poultry industry in China since 2015. The molecular basis for the increased virulence remains largely unknown. Here, we demonstrate that the hexon gene is vital for FAdV-4 pathogenicity. Furthermore, we show that the amino acid residue at position 188 of the hexon protein is responsible for pathogenicity. Importantly, the rR188I mutant strain was neutralized by chicken serum in vitro and in vivo , whereas the wild type strain was not. Further, the rR188I mutant strain provided complete protection against FAdV-4 challenge. Our results provide a molecular basis of the increased virulence of novel FAdV-4. We propose that the rR188I mutant is a potential live attenuated vaccine against HHS and a new vaccine vector for HHS-combined vaccines.

NADPH Cytochrome P-450 Oxidoreductase and Susceptibility to Ketoconazole

1998 ◽  
Vol 42 (7) ◽  
pp. 1756-1761 ◽  
Author(s):  
K. Venkateswarlu ◽  
Diane E. Kelly ◽  
Nigel J. Manning ◽  
Steven L. Kelly
Keyword(s):  
Ergosterol Biosynthesis ◽  
Squalene Epoxidase ◽  
Wild Type ◽  
Gene Encoding ◽  
Nadph Cytochrome ◽  
Cytochrome P 450 ◽  
Different Levels ◽  
Donor System

ABSTRACT The phenotype of a strain of Saccharomyces cerevisiaecontaining a disruption of the gene encoding NADPH cytochrome P-450 oxidoreductase (CPR) was quantified biochemically and microbiologically, as were those of various transformants of this strain after expression of native CPR, cytochrome P-45051 (CYP51), and a fusion protein of CYP51-CPR (FUS). Only a 4-fold decrease in ergosterol biosynthesis was observed for the cpr strain, but ketoconazole sensitivity increased 200-fold, indicating hypersensitivity to the alternative electron donor system incpr strains. Both phenotypes could be reversed in transformants expressing the CPR and FUS, indicating the availability of the CPR in FUS as well as the expressed native CPR for monoxygenase-associated reactions. The complementation of function was observed both in vitro and in vivo for the monoxygenases squalene epoxidase, CYP51, and CYP61 in the ergosterol biosynthesis pathway with which CPR is coupled. Overexpression of CYP51 and FUS produced different levels of ketoconazole resistance in wild-type cells, indicating that the availability of CPR may limit the potential of overproduction of CYP51 as a mechanism of resistance to azole antifungal agents.

A new adenylyl cyclase, putative disease-resistance RPP13-like protein 3, participates in abscisic acid-mediated resistance to heat stress in maize

2020 ◽  
Author(s):  
Hao Yang ◽  
Yulong Zhao ◽  
Ning Chen ◽  
Yanpei Liu ◽  
Shaoyu Yang ◽  
...  
Keyword(s):  
Heat Stress ◽  
Disease Resistance ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Wild Type ◽  
In Vivo Experiments ◽  
In The Wild ◽  
Shock Proteins

Abstract In plants, 3´,5´-cyclic adenosine monophosphate (cAMP) is an important second messenger with varied functions; however, only a few adenylyl cyclases (ACs) that synthesize cAMP have been identified. Moreover, the biological roles of ACs/cAMP in response to stress remain largely unclear. In this study, we used quantitative proteomics techniques to identify a maize heat-induced putative disease-resistance RPP13-like protein 3 (ZmRPP13-LK3), which has three conserved catalytic AC centres. The AC activity of ZmRPP13-LK3 was confirmed by in vitro enzyme activity analysis, in vivo RNAi experiments, and functional complementation in the E. coli cyaA mutant. ZmRPP13-LK3 is located in the mitochondria. The results of in vitro and in vivo experiments indicated that ZmRPP13-LK3 interacts with ZmABC2, a possible cAMP exporter. Under heat stress, the concentrations of ZmRPP13-LK3 and cAMP in the ABA-deficient mutant vp5 were significantly less than those in the wild-type, and treatment with ABA and an ABA inhibitor affected ZmRPP13-LK3 expression in the wild-type. Application of 8-Br-cAMP, a cAMP analogue, increased heat-induced expression of heat-shock proteins in wild-type plants and alleviated heat-activated oxidative stress. Taken together, our results indicate that ZmRPP13-LK3, a new AC, can catalyse ATP for the production of cAMP and may be involved in ABA-regulated heat resistance.

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