scholarly journals Inactivation of a Predicted Leader Peptidase Prevents Photoautotrophic Growth of Synechocystis sp. Strain PCC 6803

2005 ◽  
Vol 187 (9) ◽  
pp. 3071-3078 ◽  
Author(s):  
Maria Zhbanko ◽  
Vladislav Zinchenko ◽  
Michael Gutensohn ◽  
Angelika Schierhorn ◽  
Ralf Bernd Klösgen
Keyword(s):  
Photosynthetic Electron Transport ◽  
Kanamycin Resistance ◽  
Open Reading Frames ◽  
Type I ◽  
Signal Peptides ◽  
Photosynthetic Oxygen Evolution ◽  
Photoautotrophic Growth ◽  
Photosynthetic Machinery ◽  
Leader Peptidase ◽  
Pcc 6803

ABSTRACT To establish the role of the two putative type I leader peptidases (LepB1 and LepB2) encoded in the genome of the cyanobacterium Synechocystis sp. strain PCC 6803, we generated independent knockout mutants for both genes by introducing kanamycin resistance cassettes into the two open reading frames (sll0716 [lepB1] and slr1377 [lepB2], respectively). Although the insertion was successful in both instances, it was not possible to select homozygous mutant cells for lepB2, suggesting that the function of this gene is essential for cell viability. In contrast, LepB1 is apparently essential only for photoautotrophic growth, because homozygous lepB1::Kmr cells could be propagated under heterotrophic conditions. They were even capable to some extent of photosynthetic oxygen evolution. However, the photosynthetic activity decreased gradually with extended incubation in the light and was particularly affected by high light intensities. Both features were indicative of photooxidative damage, which was probably caused by inefficient replacement of damaged components of the photosynthetic machinery due to the lack of a leader peptidase removing the signal peptides from photosynthetic precursor proteins. Indeed, processing of the PsbO precursor polypeptide to the corresponding mature protein was significantly affected in the mutant, and reduced amounts of other proteins that are synthesized as precursors with signal peptides accumulated in the cells. These results strongly suggest that LepB1 is important for removal of the signal peptides after membrane transport of the components of the photosynthetic machinery, which in turn is a prerequisite for the biogenesis of a functional photosynthetic electron transport chain.

scholarly journals Suppressor Mutations in the Study of Photosystem I Biogenesis: sll0088 Is a Previously Unidentified Gene Involved in Reaction Center Accumulation in Synechocystis sp. Strain PCC 6803

2003 ◽  
Vol 185 (13) ◽  
pp. 3878-3887 ◽  
Author(s):  
Jianping Yu ◽  
Gaozhong Shen ◽  
Tao Wang ◽  
Donald A. Bryant ◽  
John H. Golbeck ◽  
...  
Keyword(s):  
Photosystem I ◽  
Point Mutations ◽  
Kanamycin Resistance ◽  
Negative Regulator ◽  
Binding Motif ◽  
Wild Type ◽  
Photoautotrophic Growth ◽  
Suppressor Mutations ◽  
Mutant Strains ◽  
Pcc 6803

ABSTRACT In previous work, some members of our group isolated mutant strains of Synechocystis sp. strain PCC 6803 in which point mutations had been inserted into the psaC gene to alter the cysteine residues to the FA and FB iron-sulfur clusters in the PsaC subunit of photosystem I (J. P. Yu, I. R. Vassiliev, Y. S. Jung, J. H. Golbeck, and L. McIntosh, J. Biol. Chem. 272:8032-8039, 1997). These mutant strains did not grow photoautotrophically due to suppressed levels of chlorophyll a and photosystem I. In the results described here, we show that suppressor mutations produced strains that are capable of photoautotrophic growth at moderate light intensity (20 μmol m−2 s−1). Two separate suppressor strains of C14SPsaC, termed C14SPsaC-R62 and C14SPsaC-R18, were studied and found to have mutations in a previously uncharacterized open reading frame of the Synechocystis sp. strain PCC 6803 genome named sll0088. C14SPsaC-R62 was found to substitute Pro for Arg at residue 161 as the result of a G482→C change in sll0088, and C14SPsaC-R18 was found to have a three-amino-acid insertion of Gly-Tyr-Phe following Cys231 as the result of a TGGTTATTT duplication at T690 in sll0088. These suppressor strains showed near-wild-type levels of chlorophyll a and photosystem I, yet the serine oxygen ligand to FB was retained as shown by the retention of the S ≥ 3/2 spin state of the [4Fe-4S] cluster. The inactivation of sll0088 by insertion of a kanamycin resistance cartridge in the primary C14SPsaC mutant produced an engineered suppressor strain capable of photoautotrophic growth. There was no difference in psaC gene expression or in the amount of PsaC protein assembled in thylakoids between the wild type and an sll0088 deletion mutant. The sll0088 gene encodes a protein predicted to be a transcriptional regulator with sequence similarities to transcription factors in other prokaryotic and eukaryotic organisms, including Arabidopsis thaliana. The protein contains a typical helix-turn-helix DNA-binding motif and can be classified as a negative regulator by phylogenetic analysis. This suggests that the product of sll0088 has a role in regulating the biogenesis of photosystem I.

scholarly journals Sustained Photoevolution of Molecular Hydrogen in a Mutant of Synechocystis sp. Strain PCC 6803 Deficient in the Type I NADPH-Dehydrogenase Complex

2004 ◽  
Vol 186 (6) ◽  
pp. 1737-1746 ◽  
Author(s):  
Laurent Cournac ◽  
Geneviève Guedeney ◽  
Gilles Peltier ◽  
Paulette M. Vignais
Keyword(s):  
Photosynthetic Electron Transport ◽  
Type I ◽  
Adaptation Period ◽  
Hydrogen Metabolism ◽  
Whole Cells ◽  
Time Period ◽  
Nadph Dehydrogenase ◽  
Dehydrogenase Complex ◽  
Pcc 6803

ABSTRACT The interaction between hydrogen metabolism, respiration, and photosynthesis was studied in vivo in whole cells of Synechocystis sp. strain PCC 6803 by continuously monitoring the changes in gas concentrations (H2, CO2, and O2) with an online mass spectrometer. The in vivo activity of the bidirectional [NiFe]hydrogenase [H2:NAD(P) oxidoreductase], encoded by the hoxEFUYH genes, was also measured independently by the proton-deuterium (H-D) exchange reaction in the presence of D2. This technique allowed us to demonstrate that the hydrogenase was insensitive to light, was reversibly inactivated by O2, and could be quickly reactivated by NADH or NADPH (+H2). H2 was evolved by cells incubated anaerobically in the dark, after an adaptation period. This dark H2 evolution was enhanced by exogenously added glucose and resulted from the oxidation of NAD(P)H produced by fermentation reactions. Upon illumination, a short (less than 30-s) burst of H2 output was observed, followed by rapid H2 uptake and a concomitant decrease in CO2 concentration in the cyanobacterial cell suspension. Uptake of both H2 and CO2 was linked to photosynthetic electron transport in the thylakoids. In the ndhB mutant M55, which is defective in the type I NADPH-dehydrogenase complex (NDH-1) and produces only low amounts of O2 in the light, H2 uptake was negligible during dark-to-light transitions, allowing several minutes of continuous H2 production. A sustained rate of photoevolution of H2 corresponding to 6 μmol of H2 mg of chlorophyll−1 h−1 or 2 ml of H2 liter−1 h−1 was observed over a longer time period in the presence of glucose and was slightly enhanced by the addition of the O2 scavenger glucose oxidase. By the use of the inhibitors DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea] and DBMIB (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone), it was shown that two pathways of electron supply for H2 production operate in M55, namely photolysis of water at the level of photosystem II and carbohydrate-mediated reduction of the plastoquinone pool.

Essential roles of iron superoxide dismutase in photoautotrophic growth of Synechocystis sp. PCC 6803 and heterogeneous expression of marine Synechococcus sp. CC9311 copper/zinc superoxide dismutase within its sodB knockdown mutant

Microbiology ◽  
2014 ◽  
Vol 160 (1) ◽  
pp. 228-241 ◽  
Author(s):  
Wen-Ting Ke ◽  
Guo-Zheng Dai ◽  
Hai-Bo Jiang ◽  
Rui Zhang ◽  
Bao-Sheng Qiu
Keyword(s):  
Superoxide Dismutase ◽  
Growth Conditions ◽  
Kanamycin Resistance ◽  
Photoautotrophic Growth ◽  
Iron Superoxide Dismutase ◽  
Copper Zinc Superoxide Dismutase ◽  
Kanamycin Resistance Cassette ◽  
Bg11 Medium ◽  
Gene Copies ◽  
Pcc 6803

Synechocystis sp. PCC 6803 possesses only one sod gene, sodB, encoding iron superoxide dismutase (FeSOD). It could not be knocked out completely by direct insertion of the kanamycin resistance cassette. When the promoter of sodB in WT Synechocystis was replaced with the copper-regulated promoter PpetE, a completely segregated PpetE–sodB strain could be obtained. When this strain was cultured in copper-starved BG11 medium, the chlorophyll a content was greatly reduced, growth was seriously inhibited and the strain was nearly dead during the 8 days of growth, whilst the WT strain grew well under the same growth conditions. These results indicated that sodB was essential for photoautotrophic growth of Synechocystis. The reduction of sodB gene copies in the Synechocystis genome rendered the cells more sensitive to oxidative stress produced by methyl viologen and norflurazon. sodB still could not be knocked out completely after active expression of sodC (encoding Cu/ZnSOD) from Synechococcus sp. CC9311 in the neutral site slr0168 under the control of the psbAII promoter, which means the function of FeSOD could not be complemented completely by Cu/ZnSOD. Heterogeneously expressed sodC increased the oxidation and photoinhibition tolerance of the Synechocystis sodB knockdown mutant. Membrane fractionation followed by immunoblotting revealed that FeSOD was localized in the cytoplasm, and Cu/ZnSOD was localized in the soluble and thylakoid membrane fractions of the transformed Synechocystis. Cu/ZnSOD has a predicted N-terminal signal peptide, so it is probably a lumen protein. The different subcellular localization of these two SODs may have resulted in the failure of substitution of sodC for sodB.

scholarly journals Vaccinia Virus F13L Protein with a Conserved Phospholipase Catalytic Motif Induces Colocalization of the B5R Envelope Glycoprotein in Post-Golgi Vesicles

2001 ◽  
Vol 75 (16) ◽  
pp. 7528-7542 ◽  
Author(s):  
Matloob Husain ◽  
Bernard Moss
Keyword(s):  
Vaccinia Virus ◽  
Fluorescent Protein ◽  
Plasmid Vector ◽  
Recombinant Vaccinia Virus ◽  
Open Reading Frames ◽  
Type I ◽  
Golgi Vesicles ◽  
Infected Cells ◽  
Catalytic Motif ◽  
Green Fluorescent

ABSTRACT The wrapping of intracellular mature vaccinia virions by modifiedtrans-Golgi or endosomal cisternae to form intracellular enveloped virions is dependent on at least two viral proteins encoded by the B5R and F13L open reading frames. B5R is a type I integral membrane glycoprotein, whereas F13L is an unglycosylated, palmitylated protein with a motif that is conserved in a superfamily of phospholipid-metabolizing enzymes. Microscopic visualization of the F13L protein was achieved by fusing it to the enhanced green fluorescent protein (GFP). F13L-GFP was functional when expressed by a recombinant vaccinia virus in which it replaced the wild-type F13L gene or by transfection of uninfected cells with a plasmid vector followed by infection with an F13L deletion mutant. In uninfected or infected cells, F13L-GFP was associated with Golgi cisternae and post-Golgi vesicles containing the LAMP 2 late endosomal-lysosomal marker. Association of F13L-GFP with vesicles was dependent on an intact phospholipase catalytic motif and sites of palmitylation. The B5R protein was also associated with LAMP2-containing vesicles when F13L-GFP was coexpressed, but was largely restricted to Golgi cisternae in the absence of F13L-GFP or when the F13L moiety was mutated. We suggest that the F13L protein, like its human phospholipase D homolog, regulates vesicle formation and that this process is involved in intracellular enveloped virion membrane formation.

scholarly journals Targeted Random Mutagenesis To Identify Functionally Important Residues in the D2 Protein of Photosystem II in Synechocystis sp. Strain PCC 6803

2001 ◽  
Vol 183 (1) ◽  
pp. 145-154 ◽  
Author(s):  
Svetlana Ermakova-Gerdes ◽  
Zhenbao Yu ◽  
Wim Vermaas
Keyword(s):  
Photosystem Ii ◽  
Screening Method ◽  
Random Mutagenesis ◽  
Terminal Part ◽  
Photoautotrophic Growth ◽  
Intragenic Complementation ◽  
Novel Method ◽  
Α Helix ◽  
D2 Protein ◽  
Pcc 6803

ABSTRACT To identify important residues in the D2 protein of photosystem II (PSII) in the cyanobacterium Synechocystis sp. strain PCC 6803, we randomly mutagenized a region of psbDI (coding for a 96-residue-long C-terminal part of D2) with sodium bisulfite. Mutagenized plasmids were introduced into a Synechocystissp. strain PCC 6803 mutant that lacks both psbD genes, and mutants with impaired PSII function were selected. Nine D2 residues were identified that are important for PSII stability and/or function, as their mutation led to impairment of photoautotrophic growth. Five of these residues are likely to be involved in the formation of the QA-binding niche; these are Ala249, Ser254, Gly258, Ala260, and His268. Three others (Gly278, Ser283, and Gly288) are in transmembrane α-helix E, and their alteration leads to destabilization of PSII but not to major functional alterations of the remaining centers, indicating that they are unlikely to interact directly with cofactors. In the C-terminal lumenal tail of D2, only one residue (Arg294) was identified as functionally important for PSII. However, from the number of mutants generated it is likely that most or all of the 70 residues that are susceptible to bisulfite mutagenesis have been altered at least once. The fact that mutations in most of these residues have not been picked up by our screening method suggests that these mutations led to a normal photoautotrophic phenotype. A novel method of intragenic complementation in Synechocystissp. strain PCC 6803 was developed to facilitate genetic analysis ofpsbDI mutants containing several amino acid changes in the targeted domain. Recombination between genome copies in the same cell appears to be much more prevalent in Synechocystis sp. strain PCC 6803 than was generally assumed.

scholarly journals Complete Genome Sequence of Brevibacterium linens SMQ-1335

2016 ◽  
Vol 4 (6) ◽  
Author(s):  
Alessandra G. de Melo ◽  
Simon J. Labrie ◽  
Jeannot Dumaresq ◽  
Richard J. Roberts ◽  
Denise M. Tremblay ◽  
...  
Keyword(s):  
Complete Genome Sequence ◽  
Open Reading Frames ◽  
Type I ◽  
Industrial Strain ◽  
Modification System ◽  
Brevibacterium Linens ◽  
Restriction Modification System ◽  
New Type ◽  
Restriction Modification ◽  
Reading Frames

Brevibacterium linens is one of the main bacteria found in the smear of surface-ripened cheeses. The genome of the industrial strain SMQ-1335 was sequenced using PacBio. It has 4,209,935 bp, a 62.6% G+C content, 3,848 open reading frames, and 61 structural RNAs. A new type I restriction-modification system was identified.

scholarly journals Escherichia coli Mutants Lacking All Possible Combinations of Eight Penicillin Binding Proteins: Viability, Characteristics, and Implications for Peptidoglycan Synthesis

1999 ◽  
Vol 181 (13) ◽  
pp. 3981-3993 ◽  
Author(s):  
Sylvia A. Denome ◽  
Pamela K. Elf ◽  
Thomas A. Henderson ◽  
David E. Nelson ◽  
Kevin D. Young
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Binding Proteins ◽  
Kanamycin Resistance ◽  
Open Reading Frames ◽  
Penicillin Binding Proteins ◽  
E Coli ◽  
Peptidoglycan Biosynthesis ◽  
Kanamycin Resistance Cassette ◽  
Genes Encoding

ABSTRACT The penicillin binding proteins (PBPs) synthesize and remodel peptidoglycan, the structural component of the bacterial cell wall. Much is known about the biochemistry of these proteins, but little is known about their biological roles. To better understand the contributions these proteins make to the physiology ofEscherichia coli, we constructed 192 mutants from which eight PBP genes were deleted in every possible combination. The genes encoding PBPs 1a, 1b, 4, 5, 6, and 7, AmpC, and AmpH were cloned, and from each gene an internal coding sequence was removed and replaced with a kanamycin resistance cassette flanked by two ressites from plasmid RP4. Deletion of individual genes was accomplished by transferring each interrupted gene onto the chromosome of E. coli via λ phage transduction and selecting for kanamycin-resistant recombinants. Afterwards, the kanamycin resistance cassette was removed from each mutant strain by supplying ParA resolvase in trans, yielding a strain in which a long segment of the original PBP gene was deleted and replaced by an 8-bpres site. These kanamycin-sensitive mutants were used as recipients in further rounds of replacement mutagenesis, resulting in a set of strains lacking from one to seven PBPs. In addition, thedacD gene was deleted from two septuple mutants, creating strains lacking eight genes. The only deletion combinations not produced were those lacking both PBPs 1a and 1b because such a combination is lethal. Surprisingly, all other deletion mutants were viable even though, at the extreme, 8 of the 12 known PBPs had been eliminated. Furthermore, when both PBPs 2 and 3 were inactivated by the β-lactams mecillinam and aztreonam, respectively, several mutants did not lyse but continued to grow as enlarged spheres, so that one mutant synthesized osmotically resistant peptidoglycan when only 2 of 12 PBPs (PBPs 1b and 1c) remained active. These results have important implications for current models of peptidoglycan biosynthesis, for understanding the evolution of the bacterial sacculus, and for interpreting results derived by mutating unknown open reading frames in genome projects. In addition, members of the set of PBP mutants will provide excellent starting points for answering fundamental questions about other aspects of cell wall metabolism.

scholarly journals TIP family aquaporins play role in chloroplast osmoregulation and photosynthesis

2020 ◽  
Author(s):  
Azeez Beebo ◽  
Ahmad Zia ◽  
Christopher R. Kinzel ◽  
Andrei Herdean ◽  
Karim Bouhidel ◽  
...  
Keyword(s):  
Electron Transport ◽  
Oxygen Evolution ◽  
Photosynthetic Electron Transport ◽  
Chloroplast Envelope ◽  
Photosynthetic Oxygen Evolution ◽  
Wild Type ◽  
Thylakoid Lumen ◽  
Photosynthetic Oxygen ◽  
Osmotic Treatment ◽  
Envelope Membranes

SUMMARYPhotosynthetic oxygen evolution by photosystem II requires water supply into the chloroplast to reach the thylakoid lumen. A rapid water flow is also required into the chloroplast for optimal oxygen evolution and to overcome osmotic stress. The mechanisms governing water transport in chloroplasts are largely unexplored. Previous proteomics indicated the presence of three aquaporins from the tonoplast intrinsic protein (TIP) family, TIP1;1, TIP1;2 and TIP2;1, in chloroplast membranes of Arabidopsis thaliana. Here we revisited their location and studied their role in chloroplasts. Localization experiments indicated that TIP2;1 resides in the thylakoid, whereas TIP1;2 is present in both thylakoid and envelope membranes. Mutants lacking TIP1;2 and/or TIP2;1 did not display a macroscopic phenotype when grown under standard conditions. The mutant chloroplasts and thylakoids underwent less volume changes than the corresponding wild type preparations upon osmotic treatment and in the light. Significantly reduced rates of photosynthetic electron transport were obtained in the mutant leaves, with implications on the CO2 fixation rates. However, electron transport rates did not significantly differ between mutants and wild type when isolated thylakoids were examined. Less acidification of the thylakoid lumen was measured in mutants thylakoids, resulting in a slower induction of delta pH-dependent photoprotective mechanisms. These results identify TIP1;2 and TIP2;1 as chloroplast proteins and highlight their importance for osmoregulation and optimal photosynthesis. A third aquaporin, TIP1;1, is present in the chloroplast envelope, and may play role in photosynthesis under excessive light conditions, as based on the weak photosynthetic phenotype of its mutant.

Covid-19 Mutations and How the Vaccine Enhances Immune Intelligence

2021 ◽  
Author(s):  
Xanya Sofra
Keyword(s):  
Amino Acid ◽  
Viral Entry ◽  
Open Reading Frames ◽  
Spike Protein ◽  
Single Amino Acid ◽  
Immune Memory ◽  
Type I ◽  
Specific Amino Acid ◽  
Adaptive Efficiency ◽  
Acid Exchange

We traced the coronavirus classification and evolution, analyzed the Covid-19 composition and its distinguishing characteristics when compared to SARS-CoV and MERS-CoV. Despite their close kinship, SARS-CoV and Covid-19 display significant structural differences, including 380 amino acid substitutions, and variable homology between certain open reading frames that are bound to diversify the pathogenesis and virulence of the two viral compounds. A single amino acid substitution such as replacing Aspartate (D) with Glycine (G) composes the D614G mutation that is around 20% more infectious than its predecessor 614D. The B117 variant, that exhibits a 70% transmissibility rate, harbours 23 mutants, each reflecting one amino acid exchange. We examined several globally spreading mutations, 501.V2, B1351, P1, and others, with respect to the specific amino acid conversions involved. Unlike previous versions of coronavirus, where random mutations eventually precipitate extinction, the multiplicity of over 300,000 mutations appears to have rendered Covid-19 more contagious, facilitating its ability to evade detection, thus challenging the effectiveness of a large variety of emerging vaccines. Vaccination enhances immune memory and intelligence to combat or obstruct viral entry by generating antibodies that will prohibit the cellular binding and fusion with the Spike protein, ultimately debilitating the virus from releasing its contents into the cell. Developing antibodies during the innate response, appears to be the most compelling solution in light of the hypothesis that Covid-19 inhibits the production of Interferon type I, compromising adaptive efficiency to recognize the virus, possibly provoking a cytokine storm that injures vital organs. With respect to that perspective, the safety and effectiveness of different vaccines is evaluated and compared, including the Spike protein mRNA version, the Adenovirus DNA, Spike protein subunits, the deactivated virus genres, or, finally, the live attenuated coronavirus that appears to demonstrate the greatest effectiveness, yet, encompass a relatively higher risk.

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