scholarly journals Membrane-Bound, 2-Keto-d-Gluconate-Yielding d-Gluconate Dehydrogenase from “Gluconobacter dioxyacetonicus” IFO 3271: Molecular Properties and Gene Disruption

2007 ◽  
Vol 73 (20) ◽  
pp. 6551-6556 ◽  
Author(s):  
Hirohide Toyama ◽  
Naoko Furuya ◽  
Ittipon Saichana ◽  
Yoshitaka Ano ◽  
Osao Adachi ◽  
...  
Keyword(s):  
Double Mutant ◽  
Culture Medium ◽  
Wild Type Strain ◽  
Reductase Activity ◽  
Pcr Primers ◽  
Open Reading Frames ◽  
Pcr Product ◽  
Mutant Strains ◽  
Membrane Bound ◽  
Reading Frames

ABSTRACT Most Gluconobacter species produce and accumulate 2-keto-d-gluconate (2KGA) and 5KGA simultaneously from d-glucose via GA in culture medium. 2KGA is produced by membrane-bound flavin adenine dinucleotide-containing GA 2-dehydrogenase (FAD-GADH). FAD-GADH was purified from “Gluconobacter dioxyacetonicus” IFO 3271, and N-terminal sequences of the three subunits were analyzed. PCR primers were designed from the N-terminal sequences, and part of the FAD-GADH genes was cloned as a PCR product. Using this PCR product, gene fragments containing whole FAD-GADH genes were obtained, and finally the nucleotide sequence of 9,696 bp was determined. The cloned sequence had three open reading frames (ORFs), gndS, gndL, and gndC, corresponding to small, large, and cytochrome c subunits of FAD-GADH, respectively. Seven other ORFs were also found, one of which showed identity to glucono-δ-lactonase, which might be involved directly in 2KGA production. Three mutant strains defective in either gndL or sldA (the gene responsible for 5KGA production) or both were constructed. Ferricyanide-reductase activity with GA in the membrane fraction of the gndL-defective strain decreased by about 60% of that of the wild-type strain, while in the sldA-defective strain, activity with GA did not decrease and activities with glycerol, d-arabitol, and d-sorbitol disappeared. Unexpectedly, the strain defective in both gndL and sldA (double mutant) still showed activity with GA. Moreover, 2KGA production was still observed in gndL and double mutant strains. 5KGA production was not observed at all in sldA and double mutant strains. Thus, it seems that “G. dioxyacetonicus” IFO 3271 has another membrane-bound enzyme that reacts with GA, producing 2KGA.

scholarly journals Two neuronal peptides encoded from a single transcript regulate mitochondrial function in Drosophila

2020 ◽  
Author(s):  
Justin A. Bosch ◽  
Berrak Ugur ◽  
Israel Pichardo-Casas ◽  
Jorden Rabasco ◽  
Felipe Escobedo ◽  
...  
Keyword(s):  
Mitochondrial Function ◽  
Double Mutant ◽  
Open Reading Frames ◽  
Biological Functions ◽  
Neuronal Function ◽  
Phenotypic Analysis ◽  
Single Transcript ◽  
Sequence Similarities ◽  
Reading Frames ◽  
Small Open Reading Frames

SummaryNaturally produced peptides (<100 amino acids) are important regulators of physiology, development, and metabolism. Recent studies have predicted that thousands of peptides may be translated from transcripts containing small open reading frames (smORFs). Here, we describe two previously uncharacterized peptides in Drosophila encoded by conserved smORFs, Sloth1 and Sloth2. These peptides are translated from the same bicistronic transcript and share sequence similarities, suggesting that they encode paralogs. We provide evidence that Sloth1/2 are highly expressed in neurons, localize to mitochondria, and form a complex. Double mutant analysis in animals and cell culture revealed that sloth1 and sloth2 are not functionally redundant, and their loss causes animal lethality, reduced neuronal function, impaired mitochondrial function, and neurodegeneration. These results suggest that phenotypic analysis of smORF genes in Drosophila can provide a wealth of information on the biological functions of this poorly characterized class of genes.

scholarly journals Genetic Characterization of DNA Region Containing the trh and ure Genes of Vibrio parahaemolyticus

2000 ◽  
Vol 68 (10) ◽  
pp. 5742-5748 ◽  
Author(s):  
Kwon-Sam Park ◽  
Tetsuya Iida ◽  
Yoshiharu Yamaichi ◽  
Tomohito Oyagi ◽  
Koichiro Yamamoto ◽  
...  
Keyword(s):  
Vibrio Parahaemolyticus ◽  
Open Reading Frames ◽  
The Other ◽  
Close Proximity ◽  
Mutant Strains ◽  
Thermostable Direct Hemolysin ◽  
Urease Production ◽  
Reading Frames ◽  
Type Nickel

ABSTRACT We have demonstrated that possession of the gene for thermostable direct hemolysin-related hemolysin (trh) coincides with the presence of the urease gene among clinical Vibrio parahaemolyticus strains and that the location of the two genes are in close proximity on the chromosome. Here, we cloned and sequenced the 15,754-bp DNA region containing the trh gene and the gene cluster for urease production from the chromosome of clinicalV. parahaemolyticus (TH3996). We found 16 open reading frames (ORFs) and a lower G+C content (41%) compared with the total genome of this bacterium (46 to 47%). The ure cluster consisted of eight genes, namely, ureDABCEFG andureR. ureR was located 5.2 kb upstream of the other seven genes in the opposite direction. The genetic organization and sequences of the ure genes resembled those found in Proteus mirabilis. Between ureR and the other uregenes, there were five ORFs, which are homologous with the nickel transport operon (nik) of Escherichia coli. We disrupted each of the ureR, ureC, andnikD genes in TH3996 by homologous recombination and analyzed the phenotype of the mutants. In the presence of urea these mutant strains had dramatically less urease activity than the strain they were derived from. Disruption of ureR,nikD, or ureC, however, had no effect on TRH production. The DNA region containing the trh,nik, and ure genes was found in onlytrh-positive strains and not in Kanagawa phenomenon-positive and environmental V. parahaemolyticusstrains. At the end of the region, an insertion sequence-like element existed. These results suggest that the DNA region was introduced intoV. parahaemolyticus in the past through a mechanism mediated by insertion sequences. This is the first reported case that the genes for an ATP-binding cassette-type nickel transport system, which may play a role in nickel transport through bacterial cytoplasmic membrane, are located adjacent to the ure cluster on the genome of an organism.

scholarly journals Phloem Loading of Sorbitol in Apple (Malus domestica Borkh.): Cloning and Sequence Analysis of Potential H+/Sorbitol Symporters from a Mature Leaf cDNA Library

HortScience ◽  
2004 ◽  
Vol 39 (4) ◽  
pp. 756B-756
Author(s):  
Edwin J. Reidel* ◽  
Brian G. Ayre ◽  
E. Robert Turgeon ◽  
Lailiang Cheng
Keyword(s):  
Cdna Library ◽  
Malus Domestica ◽  
Phloem Loading ◽  
Open Reading Frames ◽  
Sugar Transporter ◽  
Sugar Transporters ◽  
Apple Leaves ◽  
Complete Sequencing ◽  
Membrane Bound ◽  
Reading Frames

Sorbitol (d-glucitol) is the major end product of photosynthesis in apple (Malus domestica Borkh.), as well as the predominant phloem-translocated carbohydrate. The mechanism by which sorbitol is phloem-loaded for transport to heterotrophic sink tissues is unknown. We hypothesized that a plasma membrane-bound H+/sorbitol symporter mediates apoplastic phloem-loading of sorbitol. To discover genes potentially encoding sorbitol transporters, a cDNA library was constructed from mature `Gala' apple leaves. A homologous probe was synthesized via PCR with primers were designed against the cherry fruit sorbitol transporter, PcSot1, and using library lysate as template. From an initial plating of approximately 5 × 105 clones, twelve positives were identified after three rounds of hybridization screening. Following single-pass, 5' end sequencing, the clones were sorted into four contiguous sequences. One clone was chosen from each contig for complete sequencing. The four clones, provisionally named MdSOT1-4 (Malus domesitca Sorbitol Transporter), potentially encode full-length cDNAs for sorbitol transporters: Translated-BLAST searching (blastx) revealed that the open reading frames encode the complete Pfam sugar transporter domain, and the most significant alignments are with sequences encoding known- and putative polyol and sugar transporters.

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 Identification of the Initial Steps in d-Lysine Catabolism in Pseudomonas putida

2007 ◽  
Vol 189 (7) ◽  
pp. 2787-2792 ◽  
Author(s):  
Olga Revelles ◽  
Rolf-Michael Wittich ◽  
Juan L. Ramos
Keyword(s):  
Pseudomonas Putida ◽  
Nitrogen Source ◽  
Culture Medium ◽  
Point Of View ◽  
Open Reading Frames ◽  
Gene Encoding ◽  
Carbon And Nitrogen ◽  
Extension Analysis ◽  
Reading Frames ◽  
Lysine Catabolism

ABSTRACT Pseudomonas putida uses l-lysine as the sole carbon and nitrogen source which preferentially requires its metabolism through two parallel pathways. In one of the pathways δ-aminovalerate is the key metabolite, whereas in the other l-lysine is racemized to d-lysine, and l-pipecolate and α-aminoadipate are the key metabolites. All the genes and enzymes involved in the d-lysine pathway, except for those involved in the conversion of d-lysine into Δ1-piperideine-2-carboxylate, have been identified previously (30). In this study we report that the conversion of d-lysine into Δ1-piperideine-2-carboxylate can be mediated by a d-lysine aminotransferase (PP3590) and a d-lysine dehydrogenase (PP3596). From a physiological point of view PP3596 plays a major role in the catabolism of d-lysine since its inactivation leads to a marked reduction in the growth rate with l- or d-lysine as the sole carbon and nitrogen source, whereas inactivation of PP3590 leads only to slowed growth. The gene encoding PP3590, called here amaC, forms an operon with dpkA, the gene encoding the enzyme involved in conversion of Δ1-piperideine-2-carboxylate to l-pipecolate in the d-lysine catabolic pathway. The gene encoding PP3596, called here amaD, is the fifth gene in an operon made up of seven open reading frames (ORFs) encoding PP3592 through PP3597. The dpkA amaC operon was transcribed divergently from the operon ORF3592 to ORF3597. Both promoters were mapped by primer extension analysis, which showed that the divergent −35 hexamers of these operon promoters were adjacent to each other. Transcription of both operons was induced in response to l- or d-lysine in the culture medium.

scholarly journals NpdR, a Repressor Involved in 2,4,6-Trinitrophenol Degradation in Rhodococcus opacus HL PM-1

2004 ◽  
Vol 186 (1) ◽  
pp. 98-103 ◽  
Author(s):  
Dang P. Nga ◽  
Josef Altenbuchner ◽  
Gesche S. Heiss
Keyword(s):  
Wild Type Strain ◽  
Picric Acid ◽  
Open Reading Frames ◽  
Rhodococcus Opacus ◽  
Promoter Probe ◽  
Hydrogenation Reactions ◽  
Intergenic Regions ◽  
In The Wild ◽  
Promoter Probe Vector ◽  
Reading Frames

ABSTRACT Rhodococcus opacus HL PM-1 utilizes 2,4,6-trinitrophenol (picric acid) as a sole nitrogen source. The initial attack on picric acid occurs through two hydrogenation reactions. Hydride transferase II (encoded by npdI) and hydride transferase I (encoded by npdC) are responsible for the hydride transfers. Database searches with the npd genes have indicated the presence of a putative transcriptional regulator, npdR. Here, the npdR gene was expressed in Escherichia coli, and the protein was purified and shown to form a complex with intergenic regions between open reading frames A and B and between npdH and npdI within the npd gene cluster. A change in DNA-NpdR complex formation occurred in the presence of 2,4-dinitrophenol, picric acid, 2-chloro-4,6-dinitrophenol, and 2-methyl-4,6-dinitrophenol. By constructing a promoter-probe vector, we demonstrated that both intergenic regions caused the expression of reporter gene xylE. Hence, both of these regions contain promoters. A deletion mutant of R. opacus HL PM-1 was constructed in which part of npdR was deleted. The expression of npdI and npdC was induced by 2,4-dinitrophenol in the wild-type strain, while in the mutant these genes were constitutively expressed. Hence, NpdR is a repressor involved in picric acid degradation.

scholarly journals RiboNT: A Noise-Tolerant Predictor of Open Reading Frames from Ribosome-Protected Footprints

Life ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 701
Author(s):  
Bo Song ◽  
Mengyun Jiang ◽  
Lei Gao
Keyword(s):  
Ribosome Profiling ◽  
Open Reading Frames ◽  
Model Organisms ◽  
Widespread Application ◽  
Optimized Protocol ◽  
Membrane Bound ◽  
Noise Tolerant ◽  
Diverse Plant Species ◽  
Small Orfs ◽  
Reading Frames

Ribo-seq, also known as ribosome profiling, refers to the sequencing of ribosome-protected mRNA fragments (RPFs). This technique has greatly advanced our understanding of translation and facilitated the identification of novel open reading frames (ORFs) within untranslated regions or non-coding sequences as well as the identification of non-canonical start codons. However, the widespread application of Ribo-seq has been hindered because obtaining periodic RPFs requires a highly optimized protocol, which may be difficult to achieve, particularly in non-model organisms. Furthermore, the periodic RPFs are too short (28 nt) for accurate mapping to polyploid genomes, but longer RPFs are usually produced with a compromise in periodicity. Here we present RiboNT, a noise-tolerant ORF predictor that can utilize RPFs with poor periodicity. It evaluates RPF periodicity and automatically weighs the support from RPFs and codon usage before combining their contributions to identify translated ORFs. The results demonstrate the utility of RiboNT for identifying both long and small ORFs using RPFs with either good or poor periodicity. We implemented the pipeline on a dataset of RPFs with poor periodicity derived from membrane-bound polysomes of Arabidopsis thaliana seedlings and identified several small ORFs (sORFs) evolutionarily conserved in diverse plant species. RiboNT should greatly broaden the application of Ribo-seq by minimizing the requirement of RPF quality and allowing the use of longer RPFs, which is critical for organisms with complex genomes because these RPFs can be more accurately mapped to the position from which they were derived.

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.

Characterization of the vanDGlycopeptide Resistance Gene Cluster from Enterococcus faecium BM4339

1999 ◽  
Vol 181 (12) ◽  
pp. 3644-3648 ◽  
Author(s):  
Barbara Casadewall ◽  
Patrice Courvalin
Keyword(s):  
Enterococcus Faecium ◽  
Culture Medium ◽  
Distal Part ◽  
Frameshift Mutation ◽  
Regulatory System ◽  
Open Reading Frames ◽  
Penicillin Binding Proteins ◽  
Constitutive Synthesis ◽  
Reading Frames

ABSTRACT VanD-type resistance to glycopeptides in Enterococcus faecium BM4339 is due to constitutive synthesis ofd-alanyl-d-lactate-terminating peptidoglycan precursors (B. Périchon, P. Reynolds, and P. Courvalin, Antimicrob. Agents Chemother. 41:2016–2018, 1997). The sequence of a 5,780-bp fragment was determined and revealed six open reading frames. The 3′ distal part encoded the VanHD dehydrogenase, the VanD ligase, and the VanXD dd-dipeptidase, which were highly similar to the corresponding proteins in VanA and VanB types of resistance. The deduced VanYD protein was homologous to penicillin-binding proteins that displaydd-carboxypeptidase activity. The 5′ end coded for the putative VanRD-VanSD two-component regulatory system. Due to a frameshift mutation in the chromosomal ddlgene, BM4339 produced an impairedd-alanine:d-alanine ligase. However, since expression of the resistance genes is constitutive, growth of E. faecium BM4339 was not dependent on the presence of glycopeptides in the culture medium.

scholarly journals Biosynthetic Gene Cluster of Simocyclinone, a Natural Multihybrid Antibiotic

2002 ◽  
Vol 46 (5) ◽  
pp. 1174-1182 ◽  
Author(s):  
A. Trefzer ◽  
S. Pelzer ◽  
J. Schimana ◽  
S. Stockert ◽  
C. Bihlmaier ◽  
...  
Keyword(s):  
Gene Cluster ◽  
High Performance ◽  
Biosynthetic Gene Cluster ◽  
Open Reading Frames ◽  
Primary Metabolites ◽  
Streptomyces Antibioticus ◽  
Mutant Strains ◽  
In The Wild ◽  
Simocyclinone D8 ◽  
Reading Frames

ABSTRACT The entire simocyclinone biosynthetic cluster (sim gene cluster) from the producer Streptomyces antibioticus Tü6040 was identified on six overlapping cosmids (1N1, 5J10, 2L16, 2P6, 4G22, and 1K3). In total, 80.7 kb of DNA from these cosmids was sequenced, and the analysis revealed 49 complete open reading frames (ORFs). These ORFs include genes responsible for the formation and attachment of four different moieties originating from at least three different pools of primary metabolites. Also in the sim gene cluster, four ORFs were detected that resemble putative regulatory and export functions. Based on the putative function of the gene products, a model for simocyclinone D8 biosynthesis was proposed. Biosynthetic mutants were generated by insertional gene inactivation experiments, and culture extracts of these mutants were analyzed by high-performance liquid chromatography. Production of simocyclinone D8 was clearly detectable in the wild-type strain but was not detectable in the mutant strains. This indicated that indeed the sim gene cluster had been cloned.

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