Molecular analysis of high CO2 requiring mutants: involvement of genes in the region of rbc, including rbcS, in the ability of cyanobacteria to grow under low CO2

1991 ◽  
Vol 69 (5) ◽  
pp. 945-950 ◽  
Author(s):  
Judy Lieman-Hurwitz ◽  
Rakefet Schwarz ◽  
Flor Martinez ◽  
Zeev Maor ◽  
Leonora Reinhold ◽  
...  
Keyword(s):  
Small Subunit ◽  
Kanamycin Resistance ◽  
Genomic Region ◽  
Open Reading Frames ◽  
Chemical Mutagenesis ◽  
Wild Type ◽  
Bisphosphate Carboxylase ◽  
Low Co2 ◽  
In The Wild ◽  
Type Mutant

Modifications of the genomic region near (and including) rbc, the operon that codes for the large and small subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), resulted in cyanobacterial mutants that demand high CO2 for growth. Mutant EK6 originated from the fusion of the 3' end of rbcS (which codes for the small subunit of Rubisco) with 84 nucleotides from the 5′ flanking region of nptII (kanamycin-resistance gene), leading to a 17-kDa small subunit, as compared to 14 kDa in the wild type. Mutant D4 originated from substitution of the 1.4-kb PstI fragment, downstream of rbc, with nptII, inactivating several open reading frames in this region. Mutant O105 was obtained by chemical mutagenesis and the mutation was mapped approximately 9 kb upstream of rbc. Mutants EK6 and O105 exhibited a very low apparent photosynthetic affinity for inorganic carbon, whereas D4 had an affinity similar to that observed in wild-type cells grown under high CO2. These mutants, and the constructs used to raise them, can be used to study the role of the small subunit of Rubisco and the genomic region near rbc in cyanobacterial photosynthesis. We propose that this region contains a cluster of genes involved in the ability of cyanobacteria to grow under low ambient CO2..

Changes in protein and gene expression during induction of the CO2-concentrating mechanism in wild-type and mutant Chlamydomonas

1991 ◽  
Vol 69 (5) ◽  
pp. 1008-1016 ◽  
Author(s):  
Martin H. Spalding ◽  
Thomas L. Winder ◽  
James C. Anderson ◽  
Anne M. Geraghty ◽  
Laura F. Marek
Keyword(s):  
Transcript Abundance ◽  
Small Subunit ◽  
Wild Type ◽  
Bisphosphate Carboxylase ◽  
Phosphoglycolate Phosphatase ◽  
Concentrating Mechanism ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
In The Wild ◽  
Inorganic Carbon Transport

Several changes occur in wild-type Chlamydomonas reinhardtii upon exposure to limiting CO2, including induction of several polypeptides. Polypeptide induction was previously shown to correlate with appearance of the active CO2-concentrating mechanism (CCM) of this alga. In this paper induction of polypeptides by limiting CO2 was investigated in mutants with lesions in the CCM. Mutants with lesions in the ca-1 and pmp-1 loci exhibited alterations in polypeptide induction, but it was concluded that the alterations probably do not represent their primary genetic lesions. Other changes that occur in this alga in response to limiting CO2 were also investigated. Based on a lack of significant change in the transcript abundance of ribulose-1,5-bisphosphate carboxylase/oxygenase large and small subunit genes in the wild type, it was concluded that the previously reported transient decline in synthesis of both subunits is controlled at the translational level. A transient increase in the activity of the photorespiratory enzyme phosphoglycolate phosphatase was observed in the wild type but not in a mutant, cia-5, that lacks induction of the CCM. In addition, changes in expression of genes encoding periplasmic carbonic anhydrase, a 36-kDa membrane-associated protein and a chlorophyll-binding protein occurred in the wild type but not in cia-5 in response to limiting CO2. The absence of these changes in cia-5 was attributed to a lack of either the signal itself or transduction of the signal responsible for adaptation to limiting CO2, which led to speculation that a larger range of responses are regulated by the same signal than was previously recognized. Key words: photosynthesis, photorespiration, algae, inorganic carbon transport, transcription, translation.

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 Overexpression of the MexEF-OprN Multidrug Efflux System Affects Cell-to-Cell Signaling in Pseudomonas aeruginosa

2001 ◽  
Vol 183 (18) ◽  
pp. 5213-5222 ◽  
Author(s):  
Thilo Köhler ◽  
Christian van Delden ◽  
Lasta Kocjancic Curty ◽  
Mehri Michea Hamzehpour ◽  
Jean-Claude Pechere
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Efflux Pump ◽  
Homoserine Lactone ◽  
Wild Type ◽  
Multidrug Efflux ◽  
Efflux System ◽  
Sensing Systems ◽  
In The Wild ◽  
Type Mutant ◽  
Regulator Genes

ABSTRACT Intrinsic and acquired antibiotic resistance of the nosocomial pathogen Pseudomonas aeruginosa is mediated mainly by the expression of several efflux pumps of broad substrate specificity. Here we report that nfxC type mutants, overexpressing the MexEF-OprN efflux system, produce lower levels of extracellular virulence factors than the susceptible wild type. These include pyocyanin, elastase, and rhamnolipids, three factors controlled by the las and rhl quorum-sensing systems of P. aeruginosa. In agreement with these observations are the decreased transcription of the elastase genelasB and the rhamnosyltransferase genesrhlAB measured in nfxC type mutants. Expression of the lasR and rhlR regulator genes was not affected in the nfxC type mutant. In contrast, transcription of the C4-homoserine lactone (C4-HSL) autoinducer synthase gene rhlI was reduced by 50% in the nfxC type mutant relative to that in the wild type. This correlates with a similar decrease in C4-HSL levels detected in supernatants of the nfxC type mutant. Transcription of an rhlAB-lacZ fusion could be partially restored by the addition of synthetic C4-HSL andPseudomonas quinolone signal (PQS). It is proposed that the MexEF-OprN efflux pump affects intracellular PQS levels.

Analysis of high CO2 requiring mutants indicates a central role for the 5′ flanking region of rbc and for the carboxysomes in cyanobacterial photosynthesis

1990 ◽  
Vol 68 (6) ◽  
pp. 1303-1310 ◽  
Author(s):  
Aaron Kaplan
Keyword(s):  
Inorganic Carbon ◽  
Quantitative Model ◽  
Photosynthetic Performance ◽  
Kanamycin Resistance ◽  
Open Reading Frames ◽  
Bisphosphate Carboxylase ◽  
Insertional Inactivation ◽  
Flanking Region ◽  
Inorganic Carbon Transport ◽  
Reading Frames

The mutants E1 and O221, isolated from Synechococcus sp. PCC7942, exhibit a very low apparent photosynthetic affinity for both extracellular and intracellular inorganic carbon and hence require high CO2 concentrations for growth. These mutants possess defective carboxysomes, but the activity of ribulose 1,5-bisphosphate carboxylase is normal. The mutations in these mutants have been mapped to the 5′-flanking region of rbc, and two open reading frames, the functions of which are not yet known, have been identified in this region. Insertional inactivation (by inserting a kanamycin-resistance cartridge) of one of these open reading frames, where the mutation in O221 is located, resulted in a new high CO2 requiring phenotype. This mutant contains defective carboxysomes similar to those of O221. The role of the rbc and its 5′-flanking region in the photosynthetic performance of cyanobacteria and the structural organization of the carboxysomes are discussed in view of our recently proposed quantitative model for inorganic carbon transport and photosynthesis in cyanobacteria.

scholarly journals Screening System for Xenosiderophores as Potential Drug Delivery Agents in Mycobacteria

2001 ◽  
Vol 45 (5) ◽  
pp. 1317-1322 ◽  
Author(s):  
Gisbert Schumann ◽  
Ute Möllmann
Keyword(s):  
Mycobacterium Smegmatis ◽  
Ligand Exchange ◽  
Growth Promotion ◽  
Iron Acquisition ◽  
Gene Replacement ◽  
Kanamycin Resistance ◽  
Chemical Mutagenesis ◽  
Screening System ◽  
Wild Type ◽  
Kanamycin Resistance Gene

ABSTRACT In order to establish a screening system for xenosiderophores which can be utilized by mycobacteria, we generated a set of mutants ofMycobacterium smegmatis that are blocked in different steps of the well-known iron acquisition system. One mutant with a block in mycobactin biosynthesis was generated from strain mc2155 by chemical mutagenesis. The exochelin biosynthesis gene fxbAand the ferric exochelin uptake gene fxuA, previously identified by Fiss et al. (E. H. Fiss, S. Yu, and W. R. Jacobs, Jr., Mol. Microbiol. 14:557–559, 1994), were knocked out by gene replacement. Adjacent chromosomal fragments were used for homologous recombination in order to replace wild-type genes by the kanamycin resistance gene from transposon Tn903. Gene replacement was confirmed by PCR. The isolated mutants show the expected phenotype: fxbA mutants are defective in exochelin biosynthesis, whereas fxuA mutants excrete a significantly larger amount of exochelin compared to the amount excreted by the parent strain. This is due to their defectiveness in ferriexochelin uptake, as demonstrated in growth promotion assays. This new set of mutants allows differentiation of siderophores that supply mycobacteria with iron by ligand exchange with exochelin or mycobactin, by the use of separate siderophore uptake routes, or by the use of the exochelin permease. All these types of iron uptake routes were identified with 25 exogenous siderophores as test substances. Siderophores that act without ligand exchange are potential candidates as drug vectors that can be used to overcome permeability-mediated resistance.

scholarly journals Dual Role of Cysteine 172 in Redox Regulation of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase Activity and Degradation

2003 ◽  
Vol 185 (5) ◽  
pp. 1509-1517 ◽  
Author(s):  
Yehouda Marcus ◽  
Hagit Altman-Gueta ◽  
Aliza Finkler ◽  
Michael Gurevitz
Keyword(s):  
Redox Regulation ◽  
Inhibitory Effect ◽  
Cysteine Residues ◽  
Wild Type ◽  
Bisphosphate Carboxylase ◽  
Enzyme Form ◽  
Oxygenase Activity ◽  
In The Wild ◽  
Cross Links

ABSTRACT Alkylation and oxidation of cysteine residues significantly decrease the catalytic activity and stimulate the degradation of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO). We analyzed the role of vicinal cysteine residues in redox regulation of RuBisCO from Synechocystis sp. strain PCC 6803. Cys172 and Cys192, which are adjacent to the catalytic site, and Cys247, which cross-links two large subunits, were replaced by alanine. Whereas all mutant cells (C172A, C192A, C172A-C192A, and C247A) and the wild type grew photoautotrophically at similar rates, the maximal photosynthesis rates of C172A mutants decreased 10 to 20% as a result of 40 to 60% declines in RuBisCO turnover number. Replacement of Cys172, but not replacement of Cys192, prominently decreased the effect of cysteine alkylation or oxidation on RuBisCO. Oxidants that react with vicinal thiols had a less inhibitory effect on the activity of either the C172A or C192A enzyme variants, suggesting that a disulfide bond was formed upon oxidation. Thiol oxidation induced RuBisCO dissociation into subunits. This effect was either reduced in the C172A and C192A mutant enzymes or eliminated by carboxypentitol bisphosphate (CPBP) binding to the activated enzyme form. The CPBP effect presumably resulted from a conformational change in the carbamylated CPBP-bound enzyme, as implied from an alteration in the electrophoretic mobility. Stress conditions, provoked by nitrate deprivation, decreased the RuBisCO contents and activities in the wild type and in the C192A and C247A mutants but not in the C172A and C172A-C192A mutants. These results suggest that although Cys172 does not participate in catalysis, it plays a role in redox regulation of RuBisCO activity and degradation.

scholarly journals Stable Accumulation of σ54 in Helicobacter pylori Requires the Novel Protein HP0958

2005 ◽  
Vol 187 (13) ◽  
pp. 4463-4469 ◽  
Author(s):  
Lara Pereira ◽  
Timothy R. Hoover
Keyword(s):  
Helicobacter Pylori ◽  
Hypothetical Protein ◽  
Kanamycin Resistance ◽  
Wild Type ◽  
Protein Protein Interaction ◽  
In The Wild ◽  
Interaction Map ◽  
H Pylori ◽  
A Minor ◽  
Flagellar Biogenesis

ABSTRACT Several flagellar genes in Helicobacter pylori are dependent on σ54 (RpoN) for their expression. These genes encode components of the basal body, the hook protein, and a minor flagellin, FlaB. A protein-protein interaction map for H. pylori constructed from a high-throughput screen of a yeast two-hybrid assay (http://pim.hybrigenics.com/pimrider ext/common/) revealed interactions between σ54 and the conserved hypothetical protein HP0958. To see if HP0958 influences σ54 function, the corresponding gene was disrupted with a kanamycin resistance gene (aphA3) in H. pylori ATCC 43504 and the resulting mutant was analyzed. The hp0958:aphA3 mutant was nonmotile and failed to produce flagella. Introduction of a functional copy of hp0958 into the genome of the hp0958:aphA3 mutant restored flagellar biogenesis and motility. The hp0958:aphA3 mutant was deficient in expressing two σ54-dependent reporter genes, flaB′-′xylE and hp1120′-′xylE. Levels of σ54 in the hp0958 mutant were substantially lower than those in the parental strain, suggesting that the failure of the mutant to express the genes in the RpoN regulon and produce flagella was due to reduced σ54 levels. Expressing σ54 at high levels by putting rpoN under the control of the ureA promoter restored flagellar biogenesis and motility in the hp0958:aphA3 mutant. Turnover of σ54 was more rapid in the hp0958:aphA3 mutant than it was in the wild-type strain, suggesting that HP0958 supports wild-type σ54 levels in H. pylori by protecting it from proteolysis.

scholarly journals Uterus-specific transcriptional regulation underlies eggshell pigment production in Japanese quail

2021 ◽  
Author(s):  
Satoshi Ishishita ◽  
Shumpei Kitahara ◽  
Mayuko Takahashi ◽  
Sakura Iwasaki ◽  
Shoji Tatsumoto ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Japanese Quail ◽  
Aminolevulinic Acid ◽  
Protoporphyrin Ix ◽  
Genomic Region ◽  
Pigment Production ◽  
Wild Type ◽  
Its Gene ◽  
Uterine Mucosa ◽  
In The Wild

The precursor of heme, protoporphyrin IX (PPIX), accumulates abundantly in the uterus of birds, such as Japanese quail, Coturnix japonica, resulting in brown-speckled eggshells. The molecular basis of PPIX production in the uterus remains largely unknown. Here, we investigated the cause of low PPIX production in a classical Japanese quail mutant exhibiting white eggshells by comparing its gene expression in the uterus with that of the wild type using transcriptome analysis and performed genetic linkage mapping to identify the causative genomic region of the white eggshell phenotype. We showed that 11 genes, including the 5-aminolevulinic acid synthase 1 (ALAS1) and ferroxidase hephaestin-like 1 (HEPHL1) genes, were specifically upregulated in the wild-type uterus and downregulated in the mutant. We mapped the 172 kb candidate genomic region on chromosome 6, which contains several genes, including a part of the paired-like homeodomain 3 (PITX3), which encodes a transcription factor. ALAS1, HEPHL1, and PITX3 were expressed in the apical cells of the luminal epithelium and lamina propria cells of the uterine mucosa of the wild-type quail, and their expression was downregulated in these cells of the mutant quail. Biochemical analysis using uterine homogenates indicated that the restricted availability of 5-aminolevulinic acid is the main cause of low PPIX production. These results suggest that uterus-specific transcriptional regulation of heme-biosynthesis-related genes is an evolutionarily acquired mechanism of eggshell pigment production in Japanese quail.

scholarly journals Identification and molecular characterization of the second Chlamydomonas gun4 mutant, gun4-II

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 oxygenic photosynthesis. Chlorophyll (Chl) and heme are major tetrapyrroles that play an essential role in photosynthesis and respiration. These tetrapyrroles are synthesized via a common branched pathway that involves mainly enzymes, encoded by nuclear genes. One of the enzymes in the pathway is Mg chelatase (MgChel). MgChel catalyzes insertion of 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 Structural and Functional Complexity of the Genomic Region Controlling AK-Toxin Biosynthesis and Pathogenicity in the Japanese Pear Pathotype of Alternaria alternata

2000 ◽  
Vol 13 (9) ◽  
pp. 975-986 ◽  
Author(s):  
Aiko Tanaka ◽  
Takashi Tsuge
Keyword(s):  
Blot Analysis ◽  
Alternaria Alternata ◽  
Black Spot ◽  
Genomic Region ◽  
Cosmid Clone ◽  
Japanese Pear ◽  
Wild Type ◽  
Single Chromosome ◽  
In The Wild ◽  
Functional Complexity

The Japanese pear pathotype of Alternaria alternata produces host-specific AK-toxin and causes black spot of Japanese pear. Previously, a cosmid clone, pcAKT-1, was isolated that contains two genes, AKT1 and AKT2, within a 5.0-kb region required for AK-toxin biosynthesis. The wild-type strain has multiple, nonfunctional copies of these genes. In the present study, two additional genes, AKTR-1 and AKT3-1, downstream of AKT2 were identified. Transformation of the wild type with AKTR-1- and AKT3-1-targeting vectors produced toxin-deficient (Tox¯), nonpathogenic mutants. DNA gel blot analysis, however, demonstrated that the fragments targeted in Tox¯ mutants were different from those containing AKTR-1 and AKT3-1 on the transforming vectors. A cosmid clone, pcAKT-2, containing the targeted DNA was isolated and shown to carry two genes, AKTR-2 and AKT3-2, with high similarity to AKTR-1 and AKT3-1, respectively. Transcripts from not only AKTR-2 and AKT3-2 but also AKTR-1 and AKT3-1 were found in the wild type. DNA gel blot analysis with pulsed-field gel electrophoresis showed that AKT1, AKT2, AKT3, and AKTR and their homologues are on a single chromosome. These results indicate the structural and functional complexity of the genomic region controlling AK-toxin biosynthesis.

Sign in / Sign up

Export Citation Format

Share Document