scholarly journals Nitrogen or Sulfur Starvation Differentially Affects Phycobilisome Degradation and Expression of the nblA Gene in Synechocystis Strain PCC 6803

2001 ◽  
Vol 183 (10) ◽  
pp. 2989-2994 ◽  
Author(s):  
Catherine Richaud ◽  
Gérald Zabulon ◽  
Annette Joder ◽  
Jean-Claude Thomas
Keyword(s):  
Sequence Data ◽  
Open Reading Frames ◽  
Northern Hybridization ◽  
Sulfur Starvation ◽  
P Limitation ◽  
Kanamycin Cassette ◽  
N Starvation ◽  
Pcc 7942 ◽  
Reading Frames ◽  
Pcc 6803

ABSTRACT Nitrogen (N) limitation in cyanobacteria is well documented: a reduced growth rate is observed, accompanied by a cessation of phycobiliprotein synthesis and an ordered degradation of phycobilisomes (PBS). This leads to a dramatic bleaching phenomenon known as chlorosis. In Synechococcus strain PCC 7942, bleaching due to PBS degradation is also observed under sulfur (S) or phosphorus (P) limitation, and all three are under the control of the nblAgene product, a 59-amino-acid polypeptide which is overexpressed under N, S, and P starvation (J. L. Collier, and A. R. Grossman, EMBO J. 13:1039–1047, 1994). Cyanobase sequence data forSynechocystis strain PCC 6803 indicate the presence of two tandem open reading frames (sll0452 and sll0453) homologous tonblA. We cloned the two genes, identified a unique 5′ mRNA end suggestive of a single transcription start site, and studiednblA expression under conditions of N or S starvation by Northern hybridization: transcripts were detected only under N starvation (no signal is detected in replete medium or with S starvation), whether nblA1 or nblA2 was used as a probe. Mutations in nblA1 and nblA2 were constructed by insertion of a kanamycin cassette; both mutations were nonbleaching under N starvation. Synechocystis strain PCC 6803 does not bleach under S starvation, consistent with the absence ofnblA induction in these conditions. These results were confirmed by analysis of the PBS components: sequential degradation of phycocyanin and associated linkers was observed only under conditions of N starvation. This indicates differences betweenSynechocystis strain PCC 6803 and Synechococcusstrain PCC 7942 in their regulatory and signaling pathways leading to N- and S-starved phenotypes.

scholarly journals Analysis of Expressed Sequence Tags from Uromyces appendiculatus Hyphae and Haustoria and Their Comparison to Sequences from Other Rust Fungi

2008 ◽  
Vol 98 (10) ◽  
pp. 1126-1135 ◽  
Author(s):  
D. P. Puthoff ◽  
A. Neelam ◽  
M. L. Ehrenfried ◽  
B. E. Scheffler ◽  
L. Ballard ◽  
...  
Keyword(s):  
Expressed Sequence Tags ◽  
Sequence Data ◽  
Cellular Function ◽  
Rust Fungi ◽  
Open Reading Frames ◽  
Uromyces Appendiculatus ◽  
Data Set ◽  
Uromyces Fabae ◽  
Expressed Sequence ◽  
Reading Frames

Hyphae, 2 to 8 days postinoculation (dpi), and haustoria, 5 dpi, were isolated from Uromyces appendiculatus infected bean leaves (Phaseolus vulgaris cv. Pinto 111) and a separate cDNA library prepared for each fungal preparation. Approximately 10,000 hyphae and 2,700 haustoria clones were sequenced from both the 5′ and 3′ ends. Assembly of all of the fungal sequences yielded 3,359 contigs and 927 singletons. The U. appendiculatus sequences were compared with sequence data for other rust fungi, Phakopsora pachyrhizi, Uromyces fabae, and Puccinia graminis. The U. appendiculatus haustoria library included a large number of genes with unknown cellular function; however, summation of sequences of known cellular function suggested that haustoria at 5 dpi had fewer transcripts linked to protein synthesis in favor of energy metabolism and nutrient uptake. In addition, open reading frames in the U. appendiculatus data set with an N-terminal signal peptide were identified and compared with other proteins putatively secreted from rust fungi. In this regard, a small family of putatively secreted RTP1-like proteins was identified in U. appendiculatus and P. graminis.

scholarly journals A Single Monooxygenase, Ese, Is Involved in the Metabolism of the Organochlorides Endosulfan and Endosulfate in an Arthrobacter sp

2006 ◽  
Vol 72 (5) ◽  
pp. 3524-3530 ◽  
Author(s):  
Kahli M. Weir ◽  
Tara D. Sutherland ◽  
Irene Horne ◽  
Robyn J. Russell ◽  
John G. Oakeshott
Keyword(s):  
Sole Source ◽  
Amino Acid Identity ◽  
Open Reading Frames ◽  
Sulfur Source ◽  
Toxic Metabolite ◽  
16S Rrna Sequence ◽  
Soil Microbial ◽  
Sulfur Starvation ◽  
Soil Microbial Population ◽  
Reading Frames

ABSTRACT In this paper we describe isolation of a bacterium capable of degrading both isomers of the organochloride insecticide endosulfan and its toxic metabolite, endosulfate. The bacterium was isolated from a soil microbial population that was enriched with continuous pressure to use endosulfate as the sole source of sulfur. Analysis of the 16S rRNA sequence of the bacterium indicated that it was an Arthrobacter species. The organochloride-degrading activity was not observed in the presence of sodium sulfite as an alternative sulfur source, suggesting that the activity was part of the sulfur starvation response of the strain. A gene, ese, encoding an enzyme capable of degrading both isomers of endosulfan and endosulfate was isolated from this bacterium. The enzyme belongs to the two-component flavin-dependent monooxygenase family whose members require reduced flavin for activity. Nuclear magnetic resonance analyses identified the metabolite of endosulfan as endosulfan monoalcohol and the metabolite of endosulfate as endosulfan hemisulfate. The ese gene was located in a cluster of 10 open reading frames encoding proteins with low levels of sulfur-containing amino acids. These open reading frames were organized into two apparent divergently orientated operons and a gene encoding a putative LysR-type transcriptional regulator. The operon not containing ese did contain a homologue whose product exhibited 62% amino acid identity to the ese-encoded protein.

scholarly journals Succinate:Quinol Oxidoreductases in the Cyanobacterium Synechocystis sp. Strain PCC 6803: Presence and Function in Metabolism and Electron Transport

2000 ◽  
Vol 182 (3) ◽  
pp. 714-722 ◽  
Author(s):  
Jason W. Cooley ◽  
Crispin A. Howitt ◽  
Wim F. J. Vermaas
Keyword(s):  
Succinate Dehydrogenase ◽  
Dehydrogenase Activity ◽  
Double Mutant ◽  
Sequence Similarity ◽  
Open Reading Frames ◽  
Growth Media ◽  
Wild Type ◽  
Succinate Dehydrogenase Activity ◽  
Reading Frames ◽  
Pcc 6803

ABSTRACT The open reading frames sll1625 andsll0823, which have significant sequence similarity to genes coding for the FeS subunits of succinate dehydrogenase and fumarate reductase, were deleted singly and in combination in the cyanobacterium Synechocystis sp. strain PCC 6803. When the organic acid content in the Δsll1625 and Δsll0823 strains was analyzed, a 100-fold decrease in succinate and fumarate concentrations was observed relative to the wild type. A similar analysis for the Δsll1625 Δsll0823 strain revealed that 17% of the wild-type succinate levels remained, while only 1 to 2% of the wild-type fumarate levels were present. Addition of 2-oxoglutarate to the growth media of the double mutant strain prior to analysis of organic acids in cells caused succinate to accumulate. This indicates that succinate dehydrogenase activity had been blocked by the deletions and that 2-oxoglutarate can be converted to succinate in vivo in this organism, even though a traditional 2-oxoglutarate dehydrogenase is lacking. In addition, reduction of the thylakoid plastoquinone pool in darkness in the presence of KCN was up to fivefold slower in the mutants than in the wild type. Moreover, in vitro succinate dehydrogenase activity observed in wild-type membranes is absent from those isolated from the double mutant and reduced in those from the single mutants, further indicating that the sll1625 and sll0823 open reading frames encode subunits of succinate dehydrogenase complexes that are active in the thylakoid membrane of the cyanobacterium.

scholarly journals The Protein Phosphatases of Synechocystis sp. Strain PCC 6803: Open Reading Frames sll1033 and sll1387 Encode Enzymes That Exhibit both Protein-Serine and Protein-Tyrosine Phosphatase Activity In Vitro

2005 ◽  
Vol 187 (17) ◽  
pp. 5877-5884 ◽  
Author(s):  
Renhui Li ◽  
M. Ben Potters ◽  
Liang Shi ◽  
Peter J. Kennelly
Keyword(s):  
Inductively Coupled Plasma ◽  
Metal Ion ◽  
Protein Phosphatases ◽  
Tyrosine Phosphatase ◽  
Maximal Activity ◽  
Open Reading Frames ◽  
Inductively Coupled ◽  
Reading Frames ◽  
Pcc 6803

ABSTRACT The open reading frames (ORFs) encoding two potential protein-serine/threonine phosphatases from the cyanobacterium Synechocystis sp. strain PCC 6803 were cloned and their protein products expressed in Escherichia coli cells. The product of ORF sll1033, SynPPM3, is a homologue of the PPM family of protein-serine/threonine phosphatases found in all eukaryotes as well as many members of the Bacteria. Surprisingly, the recombinant protein phosphatase dephosphorylated phosphotyrosine- as well as phosphoserine-containing proteins in vitro. While kinetic analyses indicate that the enzyme was more efficient at dephosphorylating the latter, replacement of Asp608 by asparagine enhanced activity toward a phosphotyrosine-containing protein fourfold. The product of ORF sll1387, SynPPP1, is the sole homolog of the PPP family of protein phosphatases encoded by the genome of Synechocystis sp. strain PCC 6803. Like many other bacterial PPPs, the enzyme dephosphorylated phosphoserine- and phosphotyrosine-containing proteins with comparable efficiencies. However, while previously described PPPs from prokaryotic organisms required the addition of exogenous metal ion cofactors, such as Mg2+ or Mn2+, for activity, recombinantly produced SynPPP1 displayed near-maximal activity in the absence of added metals. Inductively coupled plasma mass spectrometry indicated that recombinant SynPPP1 contained significant quantities, 0.32 to 0.44 mol/mole total, of Mg and Mn. In this respect, the cyanobacterial enzyme resembled eukaryotic members of the PPP family, which are metalloproteins. mRNA encoding SynPPP1 or SynPPM3 could be detected in cells grown under many, but not all, environmental conditions.

scholarly journals Cloning and Genetic Analyses of the Bacteriocin 41 Determinant Encoded on the Enterococcus faecalis Pheromone-Responsive Conjugative Plasmid pYI14: a Novel Bacteriocin Complemented by Two Extracellular Components (Lysin and Activator)

2008 ◽  
Vol 190 (6) ◽  
pp. 2075-2085 ◽  
Author(s):  
Haruyoshi Tomita ◽  
Elizabeth Kamei ◽  
Yasuyoshi Ike
Keyword(s):  
Enterococcus Faecalis ◽  
Sequence Data ◽  
Open Reading Frames ◽  
Conjugative Plasmid ◽  
Cell Surface Receptor ◽  
Bacteriocin Activity ◽  
Lytic Enzymes ◽  
Repeat Structure ◽  
Surface Receptor ◽  
Reading Frames

ABSTRACT The conjugative plasmid pYI14 (61 kbp) was isolated from Enterococcus faecalis YI714, a clinical isolate. pYI14 conferred a pheromone response on its host and encoded bacteriocin 41 (bac41). Bacteriocin 41 (Bac41) only showed activity against E. faecalis. Physical mapping of pYI14 showed that it consisted of EcoRI fragments A to P. The clone pHT1100, containing EcoRI fragments A (12.6 kbp) and H (3.5 kbp), conferred the bacteriocin activity on E. faecalis strains. Genetic analysis showed that the determinant was located in a 6.6-kbp region within the EcoRI AH fragments. Six open reading frames (ORFs) were identified in this region and designated ORF7 (bacL1 ) ORF8 (bacL2 ), ORF9, ORF10, ORF11 (bacA), and ORF12 (bacI). They were aligned in this order and oriented in the same direction. ORFs bacL1 , bacL2 , bacA, and bacI were essential for expression of the bacteriocin in E. faecalis. Extracellular complementation of bacteriocin expression was possible for bacL1 and -L2 and bacA mutants. bacL1 and -L2 and bacA encoded bacteriocin component L and activator component A, respectively. The products of these genes are secreted into the culture medium and extracellularly complement bacteriocin expression. bacI encoded immunity, providing the host with resistance to its own bacteriocin activity. The bacL1 -encoded protein had significant homology with lytic enzymes that attack the gram-positive bacterial cell wall. Sequence data for the deduced bacL1 -encoded protein suggested that it has a domain structure consisting of an N-terminal signal peptide, a second domain with the enzymatic activity, and a third domain with a three-repeat structure directing the proenzyme to its cell surface receptor.

scholarly journals Identification and analysis of proton-translocating pyrophosphatases in the methanogenic archaeonMethanosarcina mazei

Archaea ◽  
2002 ◽  
Vol 1 (1) ◽  
pp. 1-7 ◽  
Author(s):  
Sebastian Bäumer ◽  
Sabine Lentes ◽  
Gerhard Gottschalk ◽  
Uwe Deppenmeier
Keyword(s):  
Specific Activity ◽  
Sequence Data ◽  
Sequence Similarity ◽  
Open Reading Frames ◽  
Inorganic Pyrophosphatase ◽  
Amino Acid Sequence Similarity ◽  
Sequence Alignments ◽  
Multiple Sequence ◽  
Inorganic Pyrophosphate ◽  
Reading Frames

Analysis of genome sequence data from the methanogenic archaeonMethanosarcina mazeiGö1 revealed the existence of two open reading frames encoding proton-translocating pyrophosphatases (PPases). These open reading frames are linked by a 750-bp intergenic region containing TC-rich stretches and are transcribed in opposite directions. The corresponding polypeptides are referred to as Mvp1 and Mvp2 and consist of 671 and 676 amino acids, respectively. Both enzymes represent extremely hydrophobic, integral membrane proteins with 15 predicted transmembrane segments and an overall amino acid sequence similarity of 50.1%. Multiple sequence alignments revealed that Mvp1 is closely related to eukaryotic PPases, whereas Mvp2 shows highest homologies to bacterial PPases. Northern blot experiments with RNA from methanol-grown cells harvested in the mid-log growth phase indicated that only Mvp2 was produced under these conditions. Analysis of washed membranes showed that Mvp2 had a specific activity of 0.34 U mg (protein)–1. Proton translocation experiments with inverted membrane vesicles prepared from methanol-grown cells showed that hydrolysis of 1 mol of pyrophosphate was coupled to the translocation of about 1 mol of protons across the cytoplasmic membrane. Appropriate conditions formvp1 expression could not be determined yet. The pyrophosphatases ofM. mazeiGö1 represent the first examples of this enzyme class in methanogenic archaea and may be part of their energy-conserving system. Abbreviations: DCCD,N,N′-dicyclohexylcarbodiimide; PPase, inorganic pyrophosphatase; PPi, inorganic pyrophosphate; Δp, proton motive force.

scholarly journals A Eukaryotic-Type Protein Kinase, SpkA, Is Required for Normal Motility of the Unicellular CyanobacteriumSynechocystis sp. Strain PCC 6803

2001 ◽  
Vol 183 (5) ◽  
pp. 1505-1510 ◽  
Author(s):  
Ayako Kamei ◽  
Takashi Yuasa ◽  
Kumi Orikawa ◽  
Xiao Xing Geng ◽  
Masahiko Ikeuchi
Keyword(s):  
Protein Kinase ◽  
Structural Integrity ◽  
Open Reading Frames ◽  
Cellular Motility ◽  
Sequence Determination ◽  
Unicellular Cyanobacterium ◽  
Type Protein ◽  
Glucose Tolerant ◽  
Reading Frames ◽  
Pcc 6803

ABSTRACT The genome of the unicellular cyanobacteriumSynechocystis sp. strain PCC 6803 comprises many open reading frames (ORFs) which putatively encode eukaryotic-type protein kinase and protein phosphatase. Based on gene disruption analysis, a region of the hypothetical ORF sll1575, which retained a part of the protein kinase motif, was found to be required for normal motility in the original isolate of strain PCC 6803. Sequence determination revealed that in this strain sll1575 was part of a gene (designated spkA) which harbored an entire eukaryotic-type Ser/Thr protein kinase motif. Strain ATCC 27184 and a glucose-tolerant strain derived from the same isolate as the PCC strain had a frameshift mutation dividing spkA into ORFssll1574 and sll1575. The structural integrity of spkA agreed well with the motility phenotype, determined by colony morphology on agar plates. The spkA gene was expressed in Escherichia coli as a His-tagged protein, which was purified by Ni2+ affinity chromatography. With [γ-32P]ATP, SpkA was autophosphorylated and transferred the phosphate group to casein, myelin basic protein, and histone. SpkA also phosphorylated several proteins in the membrane fraction ofSynechocystis cells. These results suggest that SpkA is a eukaryotic-type Ser/Thr protein kinase and regulates cellular motility via phosphorylation of the membrane proteins inSynechocystis.

scholarly journals Target enrichment of long open reading frames and ultraconserved elements to link microevolution and macroevolution in non-model organisms

2021 ◽  
Author(s):  
Claudia Ortiz-Sepulveda ◽  
Mathieu Genete ◽  
Christelle Blassiau ◽  
Cécile Godé ◽  
Christian Albrecht ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Sequence Data ◽  
Early Stage ◽  
Phylogenetic Analyses ◽  
Open Reading Frames ◽  
Model Organisms ◽  
Recent Common Ancestor ◽  
Ultraconserved Elements ◽  
Most Recent Common Ancestor ◽  
Reading Frames

Despite the increasing accessibility of high-throughput sequencing, obtaining high-quality genomic data on non-model organisms without proximate well-assembled and annotated genomes remains challenging. Here we describe a workflow that takes advantage of distant genomic resources and ingroup transcriptomes to select and jointly enrich long open reading frames (ORFs) and ultraconserved elements (UCEs) from genomic samples for integrative studies of microevolutionary and macroevolutionary dynamics. This workflow is applied to samples of the African unionid bivalve tribe Coelaturini (Parreysiinae) at basin and continent-wide scales. Our results indicate that ORFs are efficiently captured without prior identification of intron-exon boundaries. The enrichment of UCEs was less successful, but nevertheless produced a substantial dataset. Exploratory continent-wide phylogenetic analyses with ORF supercontigs (>515,000 parsimony informative sites) resulted in a fully resolved phylogeny, the backbone of which was also retrieved with UCEs (>11,000 informative sites), although some branches lack support in the latter case. Variant calling on the exome of Coelaturini from the Malawi Basin produced ~2,000 SNPs per population pair. Nucleotide diversity and population differentiation was low compared to previous estimates in mollusks, but comparable to those in recently diversifying Malawi cichlids and other taxa at an early stage of speciation. Skimming non-specific sequence data obtained for Coelaturini of the Malawi Basin, we reconstructed the maternally-inherited mitogenome, which displays an identical gene order to that of the most recent common ancestor of Unionidae. Overall, our workflow and results provide exciting perspectives for the development of integrative genomic studies on micro- and macroevolutionary dynamics in non-model organisms.

Incorporation of iron-sulphur clusters in membrane-bound proteins

2001 ◽  
Vol 29 (4) ◽  
pp. 418-421 ◽  
Author(s):  
A. Seidler ◽  
K. Jaschkowitz ◽  
M. Wollenberg
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Synechocystis Pcc 6803 ◽  
Membrane Bound ◽  
Nif Proteins ◽  
Reading Frames ◽  
Pcc 6803

The completely sequenced genome of the cyano-bacterium Synechocystis PCC 6803 contains several open reading frames, of which the deduced amino acid sequences show similarities to proteins known to be involved in FeS cluster synthesis of nitrogenase (Nif proteins) and other FeS proteins (Isc proteins). In this article, the results of our studies on these proteins are summarized and discussed with respect to their relevance in FeS cluster incorporation in chloroplasts. In cyanobacteria, there appears to exist several pathways for FeS cluster synthesis.

scholarly journals Characterization of the Multiple-Copy Host-Selective Toxin Gene, ToxB, in Pathogenic and Nonpathogenic Isolates of Pyrenophora tritici-repentis

2004 ◽  
Vol 17 (5) ◽  
pp. 467-474 ◽  
Author(s):  
J. Patrick Martinez ◽  
Nicholas W. Oesch ◽  
Lynda M. Ciuffetti
Keyword(s):  
Sequence Data ◽  
Pathogenic Fungi ◽  
Single Copy ◽  
Culture Conditions ◽  
Open Reading Frames ◽  
Toxin Gene ◽  
Pyrenophora Tritici Repentis ◽  
Race 4 ◽  
Reading Frames

ToxB, a gene that encodes a 6.6-kDa host-selective toxin (HST), is present in several races of the wheat pathogen Pyrenophora tritici-repentis. To learn more about the multiple ToxB open reading frames (ORFs), six of the estimated nine copies from a race 5 isolate were cloned and analyzed. All six copies of ToxB have identical 261-bp ORFs and thus encode the same form of Ptr ToxB. Sequence analysis of regions flanking the cloned ToxB loci revealed that the majority of loci are associated with portions of retrotransposons and a transposon-like sequence. Data indicate that ToxB loci reside on two chromosomes, 3.5 and 2.7 Mb, with the majority of copies located on the 2.7 Mb chromosome. A related gene, referred to as toxb, from a nonpathogenic race 4 isolate was also cloned and characterized. This is interesting because, until now, HST genes have only been found in toxin-producing, pathogenic isolates of plant pathogenic fungi. The toxb gene from nonpathogenic isolates is 86% similar to ToxB, and data suggest that toxb is a single-copy gene. No toxb transcript was detected under culture conditions that favor the expression of ToxB; therefore, these genes differ in their transcriptional regulation.

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