In Vivo Energy Transfer from Bacteriochlorophyll c,d,e, orfto Bacteriochlorophyll ain Wild-Type and Mutant Cells of the Green Sulfur BacteriumChlorobaculum limnaeum

ChemPhotoChem ◽  
2017 ◽  
Vol 2 (3) ◽  
pp. 190-195 ◽  
Author(s):  
Jiro Harada ◽  
Yutaka Shibata ◽  
Misato Teramura ◽  
Tadashi Mizoguchi ◽  
Yusuke Kinoshita ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Wild Type ◽  
Bacteriochlorophyll C ◽  
Green Sulfur ◽  
Mutant Cells

An alkaline phosphatase mutant of Pseudomonas aeruginosa. 1. Effects of regulatory, structural, and environmental shifts on enzyme function

1978 ◽  
Vol 24 (4) ◽  
pp. 427-432 ◽  
Author(s):  
M. L. Marceau-Day ◽  
D. F Day ◽  
J. M. Ingram
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Alkaline Phosphatase ◽  
Core Region ◽  
Significant Activity ◽  
Wild Type ◽  
Glycerol Phosphate ◽  
The Core ◽  
Nitrophenyl Phosphate ◽  
Mutant Cells

An alkaline phosphatase mutant of Pseudomonas aeruginosa exhibiting both regulatory and catalytic changes was isolated. Under repression conditions (i.e. high inorganic phosphate (Pi)) the mutant culture produced an alkaline phosphatase (APase) displaying significant activity against both β-glycerol phosphate (βGP) and p-nitrophenyl phosphate (pNPP), while the wild type displayed no activity directed towards these substrates under the same conditions. In vivo, the mutant enzyme's ratio of specific activities was 45:1 in favour of βGP versus pNPP, whereas this ratio was reversed to 1:9 βGP versus pNPP for the same enzyme isolated from mutant cells. In addition, the kinetic parameters and stability requirements for the mutant-derived enzyme was altered in comparison with those of the wild type. A study of lipopolysaccharide(LPS) preparations from both the mutant and wild type indicated the mutant to be deficient in the core region of its LPS. The authors propose that the modifications in the catalytic activity of the mutant enzyme, demonstrated in vivo, are due to a change in the enzyme's microenvironment.

scholarly journals Loss of the F-Actin Binding and Vesicle-Associated Protein Comitin Leads to a Phagocytosis Defect

2002 ◽  
Vol 1 (6) ◽  
pp. 906-914 ◽  
Author(s):  
Thomas Schreiner ◽  
Martina R. Mohrs ◽  
Rosemarie Blau-Wasser ◽  
Alfred von Krempelhuber ◽  
Michael Steinert ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Gene Replacement ◽  
Actin Binding ◽  
Wild Type ◽  
Hyperosmotic Shock ◽  
Latex Beads ◽  
Knockout Mutants ◽  
Mutant Cells

ABSTRACT Comitin is an F-actin binding and membrane-associated protein from Dictyostelium discoideum, which is present on Golgi and vesicle membranes and changes its localization in response to agents affecting the cytoskeleton. To investigate its in vivo functions we have generated knockout mutants by gene replacement. Based on comitin's in vitro functions we examined properties related to vesicular transport and microfilament function. Whereas cell growth, pinocytosis, secretion, chemotaxis, motility, and development were unaltered, comitin-lacking cells were impaired in the early steps of phagocytosis of Saccharomyces cerevisiae particles and of Escherichia coli, whereas uptake of latex beads was unaffected. Furthermore, the lack of comitin positively affected survival of pathogenic bacteria. Mutant cells also showed an altered response to hyperosmotic shock in comparison to the wild type. The redistribution of comitin during hyperosmotic shock in wild-type cells and its presence on early phagosomes suggest a direct involvement of comitin in these processes.

scholarly journals Chlorobium tepidum Mutant Lacking Bacteriochlorophyll c Made by Inactivation of the bchK Gene, Encoding Bacteriochlorophyll c Synthase

2002 ◽  
Vol 184 (12) ◽  
pp. 3368-3376 ◽  
Author(s):  
Niels-Ulrik Frigaard ◽  
Ginny D. Voigt ◽  
Donald A. Bryant
Keyword(s):  
Photosynthetic Reaction Center ◽  
Chlorobium Tepidum ◽  
Wild Type ◽  
Bacteriochlorophyll C ◽  
Gene Encoding ◽  
Thin Sections ◽  
Insertional Inactivation ◽  
In The Wild ◽  
Green Sulfur ◽  
Isoprenoid Quinones

ABSTRACT The gene encoding bacteriochlorophyll (BChl) c synthase was identified by insertional inactivation in the photosynthetic green sulfur bacterium Chlorobium tepidum and was named bchK. The bchK mutant of C. tepidum was rusty-orange in color and completely lacked BChl c. Because of the absence of the BChl c antenna, the mutant grew about seven times slower than the wild type at light intensities that were limiting to the wild type (<90 μmol m−2 s−1). Various pheophorbides, which probably represent precursors of BChl c which had lost magnesium, accumulated in the mutant cells. A small fraction of these pheophorbides were apparently esterified by the remaining chlorophyll (Chl) a and BChl a synthases in cells. The amounts of BChl a, Chl a, isoprenoid quinones, carotenoids, Fenna-Matthews-Olson protein, and chlorosome envelope protein CsmA were not significantly altered on a cellular basis in the mutant compared to in the wild type. This suggests that the BChl a antennae, photosynthetic reaction centers, and remaining chlorosome components were essentially unaffected in the mutant. Electron microscopy of thin sections revealed that the mutant lacked normal chlorosomes. However, a fraction containing vestigial chlorosomes, denoted “carotenosomes,” was partly purified by density centrifugation; these structures contained carotenoids, isoprenoid quinones, and a 798-nm-absorbing BChl a species that is probably protein associated. Because of the absence of the strong BChl c absorption found in the wild type, the bchK mutant should prove valuable for future analyses of the photosynthetic reaction center and of the roles of BChl a in photosynthesis in green bacteria. An evolutionary implication of our findings is that the photosynthetic ancestor of green sulfur bacteria could have evolved without chlorosomes and BChl c and instead used only BChl a-containing proteins as the major light-harvesting antennae.

scholarly journals Cse1p Is Involved in Export of Yeast Importin α from the Nucleus

1998 ◽  
Vol 18 (11) ◽  
pp. 6805-6815 ◽  
Author(s):  
Jens Solsbacher ◽  
Patrick Maurer ◽  
F. Ralf Bischoff ◽  
Gabriel Schlenstedt
Keyword(s):  
Nuclear Export ◽  
Fluorescent Protein ◽  
Nuclear Pore ◽  
Wild Type ◽  
Importin Α ◽  
Localization Signal ◽  
Green Fluorescent ◽  
Mutant Cells

ABSTRACT Proteins bearing a nuclear localization signal (NLS) are targeted to the nucleus by the heterodimeric transporter importin. Importin α binds to the NLS and to importin β, which carries it through the nuclear pore complex (NPC). Importin disassembles in the nucleus, evidently by binding of RanGTP to importin β. The importin subunits are exported separately. We investigated the role of Cse1p, theSaccharomyces cerevisiae homologue of human CAS, in nuclear export of Srp1p (yeast importin α). Cse1p is located predominantly in the nucleus but also is present in the cytoplasm and at the NPC. We analyzed the in vivo localization of the importin subunits fused to the green fluorescent protein in wild-type and cse1-1 mutant cells. Srp1p but not importin β accumulated in nuclei ofcse1-1 mutants, which are defective in NLS import but not defective in NLS-independent import pathways. Purified Cse1p binds with high affinity to Srp1p only in the presence of RanGTP. The complex is dissociated by the cytoplasmic RanGTP-binding protein Yrb1p. Combined with the in vivo results, this suggests that a complex containing Srp1p, Cse1p, and RanGTP is exported from the nucleus and is subsequently disassembled in the cytoplasm by Yrb1p. The formation of the trimeric Srp1p-Cse1p-RanGTP complex is inhibited by NLS peptides, indicating that only NLS-free Srp1p will be exported to the cytoplasm.

scholarly journals Clathrin heavy chain functions in sorting and secretion of lysosomal enzymes in Dictyostelium discoideum.

1994 ◽  
Vol 126 (2) ◽  
pp. 343-352 ◽  
Author(s):  
T Ruscetti ◽  
J A Cardelli ◽  
M L Niswonger ◽  
T J O'Halloran
Keyword(s):  
Dictyostelium Discoideum ◽  
Heavy Chain ◽  
Lysosomal Enzyme ◽  
Lysosomal Enzymes ◽  
Secretory Pathway ◽  
Chain Gene ◽  
Wild Type ◽  
Clathrin Heavy Chain ◽  
Mutant Cells

The clathrin heavy chain is a major component of clathrin-coated vesicles that function in selective membrane traffic in eukaryotic cells. We disrupted the clathrin heavy chain gene (chcA) in Dictyostelium discoideum to generate a stable clathrin heavy chain-deficient cell line. Measurement of pinocytosis in the clathrin-minus mutant revealed a four-to five-fold deficiency in the internalization of fluid-phase markers. Once internalized, these markers recycled to the cell surface of mutant cells at wild-type rates. We also explored the involvement of clathrin heavy chain in the trafficking of lysosomal enzymes. Pulse chase analysis revealed that clathrin-minus cells processed most alpha-mannosidase to mature forms, however, approximately 20-25% of the precursor molecules remained uncleaved, were missorted, and were rapidly secreted by the constitutive secretory pathway. The remaining intracellular alpha-mannosidase was successfully targeted to mature lysosomes. Standard secretion assays showed that the rate of secretion of alpha-mannosidase was significantly less in clathrin-minus cells compared to control cells in growth medium. Interestingly, the secretion rates of another lysosomal enzyme, acid phosphatase, were similar in clathrin-minus and wild-type cells. Like wild-type cells, clathrin-minus mutants responded to starvation conditions with increased lysosomal enzyme secretion. Our study of the mutant cells provide in vivo evidence for roles for the clathrin heavy chain in (a) the internalization of fluid from the plasma membrane; (b) sorting of hydrolase precursors from the constitutive secretory pathway to the lysosomal pathway; and (c) secretion of mature hydrolases from lysosomes to the extracellular space.

scholarly journals Mutations in RNA Polymerase II and Elongation Factor SII Severely Reduce mRNA Levels in Saccharomyces cerevisiae

1998 ◽  
Vol 18 (10) ◽  
pp. 5771-5779 ◽  
Author(s):  
J. Cale Lennon ◽  
Megan Wind ◽  
Laura Saunders ◽  
M. Benjamin Hock ◽  
Daniel Reines
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Genetic Interaction ◽  
Elongation Factor ◽  
Mrna Levels ◽  
Wild Type ◽  
Mutant Cells ◽  
Rna Levels

ABSTRACT Elongation factor SII interacts with RNA polymerase II and enables it to transcribe through arrest sites in vitro. The set of genes dependent upon SII function in vivo and the effects on RNA levels of mutations in different components of the elongation machinery are poorly understood. Using yeast lacking SII and bearing a conditional allele of RPB2, the gene encoding the second largest subunit of RNA polymerase II, we describe a genetic interaction between SII and RPB2. An SII gene disruption or therpb2-10 mutation, which yields an arrest-prone enzyme in vitro, confers sensitivity to 6-azauracil (6AU), a drug that depresses cellular nucleoside triphosphates. Cells with both mutations had reduced levels of total poly(A)+ RNA and specific mRNAs and displayed a synergistic level of drug hypersensitivity. In cells in which the SII gene was inactivated, rpb2-10 became dominant, as if template-associated mutant RNA polymerase II hindered the ability of wild-type polymerase to transcribe. Interestingly, while 6AU depressed RNA levels in both wild-type and mutant cells, wild-type cells reestablished normal RNA levels, whereas double-mutant cells could not. This work shows the importance of an optimally functioning elongation machinery for in vivo RNA synthesis and identifies an initial set of candidate genes with which SII-dependent transcription can be studied.

scholarly journals A stealth adhesion factor contributes toVibrio vulnificuspathogenicity: Flp pili play roles in host invasion, survival in the blood stream and resistance to complement activation

2019 ◽  
Author(s):  
Tra–My Duong–Nu ◽  
Kwangjoon Jeong ◽  
Soo Young Kim ◽  
Wenzhi Tan ◽  
Sao Puth ◽  
...  
Keyword(s):  
Host Cell ◽  
Complement Activation ◽  
Vibrio Vulnificus ◽  
Alternative Pathway ◽  
Molecular Genetic ◽  
Wild Type ◽  
Triple Mutant ◽  
Mutant Cells

AbstractThe tad operons encode the machinery required for adhesive Flp (fimbrial low-molecular-weight protein) pili biogenesis.Vibrio vulnificus, an opportunistic pathogen, harbors three distincttadloci. Among them, onlytad1locus was highly upregulated inin vivogrowing bacteria compared toin vitroculture condition. To understand the pathogenic roles of the threetadloci during infection, we constructed single, double and triple tad loci deletion mutants. Interestingly, only theΔtad123triple mutant cells exhibited significantly decreased lethality in mice. Ultrastructural observations revealed short, thin filamentous projections disappeared on theΔtad123mutant cells. Since the pilin was paradoxically non-immunogenic, a V5 tag was fused to Flp to visualize the pilin protein by using immunogold EM and immunofluorescence microscopy. TheΔtad123mutant cells showed attenuated host cell adhesion, delayed RtxA1 exotoxin secretion and subsequently impaired translocation across the intestinal epithelium compared to wild type, which could be partially complemented with each wild type operon. TheΔtad123mutant was susceptible to complement-mediated bacteriolysis, predominantly via the alternative pathway, suggesting stealth hiding role of the Tad pili. Taken together, all threetadloci cooperate to confer successful invasion ofV. vulnificusinto deeper tissue and evasion from host defense mechanisms, ultimately resulting in septicemia.Author SummaryTo understand the roles of the three Tad operons in the pathogenesis ofV. vulnificusinfection, we constructed mutant strain with single, double and triple Tad loci deletions. Employing a variety of mouse infection models coupled with molecular genetic analyses, we demonstrate here that all three Tad operons are required forV. vulnificuspathogenicity as the cryptic pili contribute to host cell and tissue invasion, survival in the blood, and resistance to complement activation.

scholarly journals Tightly centromere-linked gene (SPO15) essential for meiosis in the yeast Saccharomyces cerevisiae

1986 ◽  
Vol 6 (1) ◽  
pp. 158-167
Author(s):  
E Yeh ◽  
J Carbon ◽  
K Bloom
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Product ◽  
Transcriptional Activity ◽  
Intragenic Recombination ◽  
Wild Type ◽  
Base Pairs ◽  
Yeast Saccharomyces Cerevisiae ◽  
Linked Gene ◽  
Mutant Cells

We used DNA fragments from the centromere regions of yeast (Saccharomyces cerevisiae) chromosomes III and XI to examine the transcriptional activity within this chromosomal domain. DNA transcripts were found 200 to 300 base pairs from the 250-base-pair centromere core and lie within an ordered chromatin array. No transcripts were detected from the functional centromere region. We examined the cellular function of one of these tightly centromere-linked transcripts. (CEN11)L, by disrupting the coding sequences in vivo and analyzing the phenotype of the mutant yeast cell. Diploids heterozygous for the (CEN11)L disruption sporulated at wild-type levels, and the absence of the (CEN11)L gene product had no effect on the viability or mitotic growth of haploid cells. Diploids homozygous for the (CEN11)L disruption were unable to sporulate when induced by the appropriate nutritional cues. The mutant cells were competent for intragenic recombination and appeared to be blocked at the mononucleate stage. The temporal ordering of (CEN11)L function with respect to the sporulation mutant spo13 suggests that the (CEN11)L gene product may be required at both the first and second meiotic cell divisions. This new sporulation gene has been termed SPO15.

The in vivo role of annexin VII (synexin): characterization of an annexin VII-deficient Dictyostelium mutant indicates an involvement in Ca(2+)-regulated processes

1995 ◽  
Vol 108 (5) ◽  
pp. 2065-2076 ◽  
Author(s):  
V. Doring ◽  
F. Veretout ◽  
R. Albrecht ◽  
B. Muhlbauer ◽  
C. Schlatterer ◽  
...  
Keyword(s):  
Developmental Cycle ◽  
Cell Fractionation ◽  
Wild Type ◽  
Camp Phosphodiesterase ◽  
In The Wild ◽  
Extracellular Camp ◽  
Mutant Cells

Dictyostelium discoideum cells harbor two annexin VII isoforms of 47 and 51 kDa which are present throughout development. In immunofluorescence and cell fractionation studies annexin VII was found in the cytoplasm and on the plasma membrane. In gene disruption mutants lacking both annexin VII isoforms growth, pinocytosis, phagocytosis, chemotaxis and motility were not significantly impaired under routine laboratory conditions, and the cells were able to complete the developmental cycle on bacterial plates. On non-nutrient agar plates development was delayed by three to four hours and a significant number of aggregates was no longer able to form fruiting bodies. Exocytosis as determined by measuring extracellular cAMP phosphodiesterase, alpha-fucosidase and alpha-mannosidase activity was unaltered, the total amounts of these enzymes were however lower in the mutant than in the wild type. The mutant cells were markedly impaired when they were exposed to low Ca2+ concentrations by adding EGTA to the nutrient medium. Under these conditions growth, motility and chemotaxis were severely affected. The Ca2+ concentrations were similar in mutant and wild-type cells both under normal and Ca2+ limiting conditions; however, the distribution was altered under low Ca2+ conditions in SYN-cells. The data suggest that annexin VII is not required for membrane fusion events but rather contributes to proper Ca2+ homeostasis in the cell.

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