scholarly journals Increased Production of Zeaxanthin and Other Pigments by Application of Genetic Engineering Techniques toSynechocystis sp. Strain PCC 6803

2000 ◽  
Vol 66 (1) ◽  
pp. 64-72 ◽  
Author(s):  
Delphine Lagarde ◽  
Laurent Beuf ◽  
Wim Vermaas
Keyword(s):  
Mutant Strain ◽  
Fold Increase ◽  
Carotenoid Biosynthesis ◽  
Carotenoid Content ◽  
Wild Type ◽  
Gene Encoding ◽  
Genes Encoding ◽  
In The Wild ◽  
Β Carotene ◽  
Pcc 6803

ABSTRACT The psbAII locus was used as an integration platform to overexpress genes involved in carotenoid biosynthesis inSynechocystis sp. strain PCC 6803 under the control of the strong psbAII promoter. The sequences of the genes encoding the yeast isopentenyl diphosphate isomerase (ipi) and theSynechocystis β-carotene hydroxylase (crtR) and the linked Synechocystis genes coding for phytoene desaturase and phytoene synthase (crtP andcrtB, respectively) were introduced intoSynechocystis, replacing the psbAII coding sequence. Expression of ipi, crtR, andcrtP and crtB led to a large increase in the corresponding transcript levels in the mutant strains, showing that the psbAII promoter can be used to drive transcription and to overexpress various genes in Synechocystis. Overexpression of crtP and crtB led to a 50% increase in the myxoxanthophyll and zeaxanthin contents in the mutant strain, whereas the β-carotene and echinenone contents remained unchanged. Overexpression of crtR induced a 2.5-fold increase in zeaxanthin accumulation in the corresponding overexpressing mutant compared to that in the wild-type strain. In this mutant strain, zeaxanthin becomes the major pigment (more than half the total amount of carotenoid) and the β-carotene and echinenone amounts are reduced by a factor of 2. However, overexpression of ipi did not result in a change in the carotenoid content of the mutant. To further alter the carotenoid content of Synechocystis, the crtOgene, encoding β-carotene ketolase, which converts β-carotene to echinenone, was disrupted in the wild type and in the overexpressing strains so that they no longer produced echinenone. In this way, by a combination of overexpression and deletion of particular genes, the carotenoid content of cyanobacteria can be altered significantly.

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 Insights into an alternative pathway for glycerol metabolism in a glycerol kinase deficientPseudomonas putidaKT2440

2019 ◽  
Author(s):  
Meg Walsh ◽  
William Casey ◽  
Shane T. Kenny ◽  
Tanja Narancic ◽  
Lars M. Blank ◽  
...  
Keyword(s):  
Mutant Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Alternative Pathway ◽  
Glycerol Kinase ◽  
Glycerol Metabolism ◽  
Wild Type ◽  
Short Chain Length ◽  
Genes Encoding ◽  
In The Wild

AbstractPseudomonas putidaKT2440 is known to metabolise glycerol via glycerol-3-phosphate using glycerol kinase an enzyme previously described as critical for glycerol metabolism (1). However, when glycerol kinase was knocked out inP. putidaKT2440 it retained the ability to use glycerol as the sole carbon source, albeit with a much-extended lag period and 2 fold lower final biomass compared to the wild type strain. A metabolomic study identified glycerate as a major and the most abundant intermediate in glycerol metabolism in this mutated strain with levels 21-fold higher than wild type. Erythrose-4-phosphate was detected in the mutant strain, but not in the wild type strain. Glyceraldehyde and glycraldehyde-3-phosphate were detected at similar levels in the mutant strain and the wild type. Transcriptomic studies identified 191 genes that were more than 2-fold upregulated in the mutant compared to the wild type and 175 that were down regulated. The genes involved in short chain length fatty acid metabolism were highly upregulated in the mutant strain. The genes encoding 3-hydroxybutyrate dehydrogenase were 5.8-fold upregulated and thus the gene was cloned, expressed and purified to reveal it can act on glyceraldehyde but not glycerol as a substrate.

scholarly journals Substrate Specificities and Availability of Fucosyltransferase and β-Carotene Hydroxylase for Myxol 2′-Fucoside Synthesis in Anabaena sp. Strain PCC 7120 Compared with Synechocystis sp. Strain PCC 6803

2008 ◽  
Vol 190 (20) ◽  
pp. 6726-6733 ◽  
Author(s):  
Mari Mochimaru ◽  
Hajime Masukawa ◽  
Takashi Maoka ◽  
Hatem E. Mohamed ◽  
Wim F. J. Vermaas ◽  
...  
Keyword(s):  
High Performance ◽  
Carotenoid Biosynthesis ◽  
Biosynthetic Pathways ◽  
Wild Type ◽  
Substrate Specificities ◽  
Field Desorption Mass Spectrometry ◽  
Anabaena Sp ◽  
Pcc 7120 ◽  
Β Carotene ◽  
Pcc 6803

ABSTRACT To elucidate the biosynthetic pathways of carotenoids, especially myxol 2′-glycosides, in cyanobacteria, Anabaena sp. strain PCC 7120 (also known as Nostoc sp. strain PCC 7120) and Synechocystis sp. strain PCC 6803 deletion mutants lacking selected proposed carotenoid biosynthesis enzymes and GDP-fucose synthase (WcaG), which is required for myxol 2′-fucoside production, were analyzed. The carotenoids in these mutants were identified using high-performance liquid chromatography, field desorption mass spectrometry, and 1H nuclear magnetic resonance. The wcaG (all4826) deletion mutant of Anabaena sp. strain PCC 7120 produced myxol 2′-rhamnoside and 4-ketomyxol 2′-rhamnoside as polar carotenoids instead of the myxol 2′-fucoside and 4-ketomyxol 2′-fucoside produced by the wild type. Deletion of the corresponding gene in Synechocystis sp. strain PCC 6803 (sll1213; 79% amino acid sequence identity with the Anabaena sp. strain PCC 7120 gene product) produced free myxol instead of the myxol 2′-dimethyl-fucoside produced by the wild type. Free myxol might correspond to the unknown component observed previously in the same mutant (H. E. Mohamed, A. M. L. van de Meene, R. W. Roberson, and W. F. J. Vermaas, J. Bacteriol. 187:6883-6892, 2005). These results indicate that in Anabaena sp. strain PCC 7120, but not in Synechocystis sp. strain PCC 6803, rhamnose can be substituted for fucose in myxol glycoside. The β-carotene hydroxylase orthologue (CrtR, Alr4009) of Anabaena sp. strain PCC 7120 catalyzed the transformation of deoxymyxol and deoxymyxol 2′-fucoside to myxol and myxol 2′-fucoside, respectively, but not the β-carotene-to-zeaxanthin reaction, whereas CrtR from Synechocystis sp. strain PCC 6803 catalyzed both reactions. Thus, the substrate specificities or substrate availabilities of both fucosyltransferase and CrtR were different in these species. The biosynthetic pathways of carotenoids in Anabaena sp. strain PCC 7120 are discussed.

scholarly journals Disruption of sucA, Which Encodes the E1 Subunit of α-Ketoglutarate Dehydrogenase, Affects the Survival of Nitrosomonas europaea in Stationary Phase

2006 ◽  
Vol 188 (1) ◽  
pp. 343-347 ◽  
Author(s):  
Norman G. Hommes ◽  
Elizabeth G. Kurth ◽  
Luis A. Sayavedra-Soto ◽  
Daniel J. Arp
Keyword(s):  
Stationary Phase ◽  
Mutant Strain ◽  
Nitrosomonas Europaea ◽  
Wild Type ◽  
Late Stationary Phase ◽  
Gene Encoding ◽  
Hydroxylamine Oxidoreductase ◽  
Genes Encoding ◽  
Knockout Mutation ◽  
Ketoglutarate Dehydrogenase

ABSTRACT Although Nitrosomonas europaea lacks measurable α-ketoglutarate dehydrogenase activity, the recent completion of the genome sequence revealed the presence of the genes encoding the enzyme. A knockout mutation was created in the sucA gene encoding the E1 subunit. Compared to wild-type cells, the mutant strain showed an accelerated loss of ammonia monooxygenase and hydroxylamine oxidoreductase activities upon entering stationary phase. In addition, unlike wild-type cells, the mutant strain showed a marked lag in the ability to resume growth in response to pH adjustments in late stationary phase.

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 Inactivation of Ca2+/H+Exchanger in Synechocystis sp. Strain PCC 6803 Promotes Cyanobacterial Calcification by Upregulating CO2-Concentrating Mechanisms

2013 ◽  
Vol 79 (13) ◽  
pp. 4048-4055 ◽  
Author(s):  
Hai-Bo Jiang ◽  
Hui-Min Cheng ◽  
Kun-Shan Gao ◽  
Bao-Sheng Qiu
Keyword(s):  
Transcript Level ◽  
Wild Type ◽  
Content Type ◽  
Protein Levels ◽  
Genes Encoding ◽  
In The Wild ◽  
Co2 Concentrating Mechanisms ◽  
Mutant Cells ◽  
Global Carbon ◽  
Pcc 6803

ABSTRACTCyanobacteria are important players in the global carbon cycle, accounting for approximately 25% of global CO2fixation. Their CO2-concentrating mechanisms (CCMs) are thought to play a key role in cyanobacterial calcification, but the mechanisms are not completely understood. InSynechocystissp. strain PCC 6803, a single Ca2+/H+exchanger (Slr1336) controls the Ca2+/H+exchange reaction. We knocked out the exchanger and investigated the effects on cyanobacterial calcification and CCMs. Inactivation ofslr1336significantly increased the calcification rate and decreased the zeta potential, indicating a relatively stronger Ca2+-binding ability. Some genes encoding CCM-related components showed increased expression levels, including thecmpAgene, which encodes the Ca2+-dependent HCO3−transporter BCT1. The transcript level ofcmpAin the mutant was 30 times that in wild type. A Western blot analysis further confirmed that protein levels of CmpA were higher in the mutant than the wild type. Measurements of inorganic carbon fluxes and O2evolution proved that both the net HCO3−uptake rate and the BCT1 transporter supported photosynthetic rate in theslr1336mutant were significantly higher than in the wild type. This would cause the mutant cells to liberate more OH−ions out of the cell and stimulate CaCO3precipitation in the microenvironment. We conclude that the mutation of the Ca2+/H+exchanger inSynechocystispromoted the cyanobacterial calcification process by upregulating CCMs, especially the BCT1 HCO3−transporter. These results shed new light on the mechanism by which CCM-facilitated photosynthesis promotes cyanobacterial calcification.

scholarly journals Identification of Gene Products Involved in the Oxidative Stress Response ofMoraxella catarrhalis

2010 ◽  
Vol 79 (2) ◽  
pp. 745-755 ◽  
Author(s):  
Todd C. Hoopman ◽  
Wei Liu ◽  
Stephanie N. Joslin ◽  
Christine Pybus ◽  
Chad A. Brautigam ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Parent Strain ◽  
Dilution Effect ◽  
Dependent Manner ◽  
Wild Type ◽  
Gene Encoding ◽  
Reverse Transcription Pcr ◽  
Genes Encoding ◽  
In The Wild ◽  
Wild Type Parent

ABSTRACTMoraxella catarrhalisis subjected to oxidative stress from both internal and environmental sources. A previous study (C. D. Pericone, K. Overweg, P. W. Hermans, and J. N. Weiser, Infect. Immun.68:3990-3997, 2000) indicated that a wild-type strain ofM. catarrhaliswas very resistant to killing by exogenous hydrogen peroxide (H2O2). The gene encoding OxyR, a LysR family transcriptional regulator, was identified and inactivated inM. catarrhalisstrain O35E, resulting in an increase in sensitivity to killing by H2O2in disk diffusion assays and a concomitant aerobic serial dilution effect. Genes encoding a predicted catalase (KatA) and an alkyl hydroperoxidase (AhpCF) showed dose-dependent upregulation in wild-type cells exposed to H2O2. DNA microarray and real-time reverse transcription-PCR (RT-PCR) analyses identifiedM. catarrhalisgenes whose expression was affected by oxidative stress in an OxyR-dependent manner. Testing ofM. catarrhalisO35EkatAandahpCmutants for their abilities to scavenge exogenous H2O2showed that the KatA catalase was responsible for most of this activity in the wild-type parent strain. The introduction of the same mutations intoM. catarrhalisstrain ETSU-4 showed that the growth of a ETSU-4katAmutant was markedly inhibited by the addition of 50 mM H2O2but that this mutant could still form a biofilm equivalent to that produced by its wild-type parent strain.

scholarly journals Carotenoid Biosynthesis in the Primitive Red Alga Cyanidioschyzon merolae

2006 ◽  
Vol 6 (3) ◽  
pp. 533-545 ◽  
Author(s):  
Francis X. Cunningham ◽  
Hansel Lee ◽  
Elisabeth Gantt
Keyword(s):  
Carotenoid Biosynthesis ◽  
Red Alga ◽  
Land Plants ◽  
Carotenoid Content ◽  
Cyanidioschyzon Merolae ◽  
Origin And Evolution ◽  
Lycopene Cyclase ◽  
Genes Encoding ◽  
Photosynthetic Organism ◽  
Β Carotene

ABSTRACT Cyanidioschyzon merolae is considered to be one of the most primitive of eukaryotic photosynthetic organisms. To obtain insights into the origin and evolution of the pathway of carotenoid biosynthesis in eukaryotic plants, the carotenoid content of C. merolae was ascertained, genes encoding enzymes of carotenoid biosynthesis in this unicellular red alga were identified, and the activities of two candidate pathway enzymes of particular interest, lycopene cyclase and β-carotene hydroxylase, were examined. C. merolae contains perhaps the simplest assortment of chlorophylls and carotenoids found in any eukaryotic photosynthetic organism: chlorophyll a, β-carotene, and zeaxanthin. Carotenoids with ε-rings (e.g., lutein), found in many other red algae and in green algae and land plants, were not detected, and the lycopene cyclase of C. merolae quite specifically produced only β-ringed carotenoids when provided with lycopene as the substrate in Escherichia coli. Lycopene β-ring cyclases from several bacteria, cyanobacteria, and land plants also proved to be high-fidelity enzymes, whereas the structurally related ε-ring cyclases from several plant species were found to be less specific, yielding products with β-rings as well as ε-rings. C. merolae lacks orthologs of genes that encode the two types of β-carotene hydroxylase found in land plants, one a nonheme diiron oxygenase and the other a cytochrome P450. A C. merolae chloroplast gene specifies a polypeptide similar to members of a third class of β-carotene hydroxylases, common in cyanobacteria, but this gene did not produce an active enzyme when expressed in E. coli. The identity of the C. merolae β-carotene hydroxylase therefore remains uncertain.

scholarly journals Oxygen Regulation of the ccoN Gene Encoding a Component of the cbb3 Oxidase inRhodobacter sphaeroides 2.4.1T: Involvement of the FnrL Protein

1998 ◽  
Vol 180 (8) ◽  
pp. 2228-2231 ◽  
Author(s):  
Nigel J. Mouncey ◽  
Samuel Kaplan
Keyword(s):  
Rhodobacter Sphaeroides ◽  
Type Strain ◽  
Wild Type Strain ◽  
Growth Conditions ◽  
Anaerobic Growth ◽  
Wild Type ◽  
Transcriptional Fusion ◽  
Gene Encoding ◽  
Genes Encoding ◽  
In The Wild

ABSTRACT The ccoNOQP gene cluster of Rhodobacter sphaeroides 2.4.1T encodes acbb 3 cytochrome oxidase which is utilized in oxygen-limited conditions for aerobic respiration. The β-galactosidase activity of accoN::lacZ transcriptional fusion was low under high (30%)-oxygen and anaerobic growth conditions. Maximal ccoN::lacZexpression was observed when the oxygen concentration was lowered to 2%. In an FnrL mutant,ccoN::lacZ expression was significantly lower than in the wild-type strain, suggesting that FnrL is a positive regulator of genes encoding thecbb 3 oxidase.

scholarly journals Controlled Transcription of Regulator Gene carS by Tet-on or by a Strong Promoter Confirms Its Role as a Repressor of Carotenoid Biosynthesis in Fusarium fujikuroi

2020 ◽  
Vol 9 (1) ◽  
pp. 71
Author(s):  
Julia Marente ◽  
Javier Avalos ◽  
M. Carmen Limón
Keyword(s):  
Wild Strain ◽  
Ring Finger ◽  
Carotenoid Biosynthesis ◽  
Negative Regulator ◽  
Fusarium Fujikuroi ◽  
Structural Genes ◽  
Gene Encoding ◽  
In The Wild ◽  
Set Up

Carotenoid biosynthesis is a frequent trait in fungi. In the ascomycete Fusarium fujikuroi, the synthesis of the carboxylic xanthophyll neurosporaxanthin (NX) is stimulated by light. However, the mutants of the carS gene, encoding a protein of the RING finger family, accumulate large NX amounts regardless of illumination, indicating the role of CarS as a negative regulator. To confirm CarS function, we used the Tet-on system to control carS expression in this fungus. The system was first set up with a reporter mluc gene, which showed a positive correlation between the inducer doxycycline and luminescence. Once the system was improved, the carS gene was expressed using Tet-on in the wild strain and in a carS mutant. In both cases, increased carS transcription provoked a downregulation of the structural genes of the pathway and albino phenotypes even under light. Similarly, when the carS gene was constitutively overexpressed under the control of a gpdA promoter, total downregulation of the NX pathway was observed. The results confirmed the role of CarS as a repressor of carotenogenesis in F. fujikuroi and revealed that its expression must be regulated in the wild strain to allow appropriate NX biosynthesis in response to illumination.

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