scholarly journals Characterization of ssfR andssgA, Two Genes Involved in Sporulation ofStreptomyces griseus

2000 ◽  
Vol 182 (19) ◽  
pp. 5521-5529 ◽  
Author(s):  
Hao Jiang ◽  
Kathleen E. Kendrick
Keyword(s):  
Stop Codon ◽  
Streptomyces Griseus ◽  
Large Family ◽  
Resistant Mutant ◽  
Open Reading Frames ◽  
Dna Fragment ◽  
Morphological Differences ◽  
White Mutant ◽  
Reading Frames

ABSTRACT In the presence of cefoxitin, which inhibits septum formation during sporulation, Streptomyces griseus is unable to sporulate, retaining the sonication sensitivity of nonsporulating hyphae. Cefoxitin- and sonication-resistant mutant SKK2600 was isolated and showed many morphological differences from its parental strain. A 3.6-kb DNA fragment that complemented the mutations of SKK2600 contained two open reading frames (ORFs), either of which could complement SKK2600. One ORF, designated ssfR, encoded a protein containing a potential DNA-binding helix-turn-helix motif close to its N terminus. SsfR is similar to members of a large family of transcriptional regulators, particularly IclR of Escherichia coli. The second ORF was identified as ssgA, a previously described sporulation gene from S. griseus (S. Kawamoto and J. C. Ensign, Actinomycetology 9:136–151, 1995). A point mutation of C to T seven nucleotides upstream of the UGA stop codon of ssfR was responsible for the phenotype of isolated mutant strain SKK2600. Surprisingly, this mutation should not change the primary structure of SsfR. The ssfR andssgA disruption mutants were constructed and showed the “white” mutant phenotype, with some growth medium dependence. In addition, the ssfR null mutant sporulated ectopically in phosphate starvation medium.

scholarly journals Genetic Localization and Molecular Characterization of the nonS Gene Required for Macrotetrolide Biosynthesis inStreptomyces griseus DSM40695

2000 ◽  
Vol 44 (7) ◽  
pp. 1809-1817 ◽  
Author(s):  
Wyatt C. Smith ◽  
Longkuan Xiang ◽  
Ben Shen
Keyword(s):  
Polyketide Synthase ◽  
Isotope Labeling ◽  
Streptomyces Griseus ◽  
Open Reading Frames ◽  
A Cell ◽  
Cyclic Polyethers ◽  
The Difference ◽  
Reading Frames

ABSTRACT The macrotetrolides are a family of cyclic polyethers derived from tetramerization, in a stereospecific fashion, of the enantiomeric nonactic acid (NA) and its homologs. Isotope labeling experiments established that NA is of polyketide origin, and biochemical investigations demonstrated that 2-methyl-6,8-dihydroxynon-2E-enoic acid can be converted into NA by a cell-free preparation from Streptomyces lividans that expresses nonS. These results lead to the hypothesis that macrotetrolide biosynthesis involves a pair of enantiospecific polyketide pathways. In this work, a 55-kb contiguous DNA region was cloned from Streptomyces griseus DSM40695, a 6.3-kb fragment of which was sequenced to reveal five open reading frames, including the previously reported nonR andnonS genes. Inactivation of nonS in vivo completely abolished macrotetrolide production. Complementation of thenonS mutant by the expression of nonS intrans fully restored its macrotetrolide production ability, with a distribution of individual macrotetrolides similar to that for the wild-type producer. In contrast, fermentation of thenonS mutant in the presence of exogenous (±)-NA resulted in the production of nonactin, monactin, and dinactin but not in the production of trinactin and tetranactin. These results prove the direct involvement of nonS in macrotetrolide biosynthesis. The difference in macrotetrolide production between in vivo complementation of the nonS mutant by the plasmid-borne nonSgene and fermentation of the nonS mutant in the presence of exogenously added (±)-NA suggests that NonS catalyzes the formation of (−)-NA and its homologs, supporting the existence of a pair of enantiospecific polyketide pathways for macrotetrolide biosynthesis inS. griseus. The latter should provide a model that can be used to study the mechanism by which polyketide synthase controls stereochemistry during polyketide biosynthesis.

scholarly journals Cloning and Characterization of a New Site-Specific Methyl-Directed ElmI Endonuclease Recognizing and Cleaving C5-methylated DNA Sequence 5’-G(5mC)^NG(5mC)-3’

Acta Naturae ◽  
2016 ◽  
Vol 8 (1) ◽  
pp. 117-125 ◽  
Author(s):  
V. A. Chernukhin ◽  
D. A. Gonchar ◽  
M. A. Abdurashitov ◽  
O. A. Belichenko ◽  
V. S. Dedkov ◽  
...  
Keyword(s):  
Dna Sequence ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Site Specific ◽  
Pcr Screening ◽  
Methylated Dna ◽  
Chromatographic Techniques ◽  
Dna Fragment ◽  
Reading Frames

Putative open reading frames of MD-endonucleases have been identified in Enterobacteria genomes as a result of the search for amino acid sequences homologous to MD-endonuclease BisI. A highly conserved DNA primary structure of these open reading frames in different genera of Enterobacteria (Escherichia, Klebsiella and Cronobacter) has allowed researchers to create primers for PCR screening, which was carried out on Enterobacteria DNA collected from natural sources. The DNA fragment, about 440 bp in length, was amplified by use of the genomic DNA of a wild E.coli LM N17 strain as a template and was inserted into the pMTL22 vector. Endonuclease activity was detected in an E.coli ER 2267 strain transformed with the obtained construction. A new enzyme named ElmI was purified by chromatographic techniques from the recombinant strain biomass. It was discovered that similarly to BisI this enzyme specifically cleaves the methylated DNA sequence 5-GCNGC- 3 before the central nucleotide N if this sequence contains two 5-methylcytosines. However, unlike BisI, ElmI more efficiently cleaves this sequence if more than two cytosine residues are methylated.

scholarly journals Characterization of the Bacillus subtilis ywsC Gene, Involved in γ-Polyglutamic Acid Production

2002 ◽  
Vol 184 (2) ◽  
pp. 337-343 ◽  
Author(s):  
Yuji Urushibata ◽  
Shinji Tokuyama ◽  
Yasutaka Tahara
Keyword(s):  
Bacillus Subtilis ◽  
Western Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Nitrilotriacetic Acid ◽  
Open Reading Frames ◽  
Polyglutamic Acid ◽  
Dna Fragment ◽  
Reading Frames

ABSTRACT The genes required for γ-polyglutamic acid (PGA) production were cloned from Bacillus subtilis IFO16449, a strain isolated from fermented soybeans. There were four open reading frames in the cloned 4.2-kb DNA fragment, and they were almost identical to those in the ywsC and ywtABC genes of B. subtlis 168. Northern blot analysis showed that the four genes constitute an operon. Three genes, ywsC, ywtA, and ywtB, were disrupted to determine which gene plays a central role in PGA biosynthesis. No PGA was produced in ΔywsC and ΔywtA strains, indicating that both of these genes are essential for PGA production. To clarify the function of the YwsC protein, histidine-tagged YwsC (YwsC-His) was produced in the ΔywsC strain and purified from the lysozyme-treated lysate of the transformant by Ni-nitrilotriacetic acid affinity chromatography. Western blot analysis revealed that the YwsC-His protein consists of two subunits, the 44-kDa and 33-kDa proteins, which are encoded by in-phase overlapping in the ywsC gene. 14C-labeled PGA was synthesized by the purified proteins from l-[14C]-glutamate in the presence of ATP and MnCl2, through an acylphosphate intermediate, indicating that the ywsC gene encodes PGA synthetase (EC 6.3.2), a crucial enzyme in PGA biosynthesis.

scholarly journals Characterization of the Type 8 Capsular Gene Cluster of Streptococcus pneumoniae

1999 ◽  
Vol 181 (19) ◽  
pp. 6214-6219 ◽  
Author(s):  
Rosario Muñoz ◽  
Marta Mollerach ◽  
Rubens López ◽  
Ernesto García
Keyword(s):  
Streptococcus Pneumoniae ◽  
Nucleotide Sequence ◽  
Gene Cluster ◽  
Complete Nucleotide Sequence ◽  
Capsular Polysaccharide ◽  
Open Reading Frames ◽  
Dna Fragment ◽  
Type 3 ◽  
Reading Frames

ABSTRACT The complete nucleotide sequence of the capsular gene cluster (cap8) responsible for the biosynthesis of the capsular polysaccharide of Streptococcus pneumoniae type 8 has been determined. The cap8 gene cluster, located between the genes dexB and aliA, is composed of 12 open reading frames. A 14.7-kb DNA fragment embracing the cap8genes was sufficient to transform an unencapsulated type 3 S. pneumoniae strain to a strain with the type 8 capsule. A possible scenario for the evolution of pneumococcal types 2 and 8 is outlined.

scholarly journals Characterization of the Serogroup O11 O-Antigen Locus of Pseudomonas aeruginosa PA103

1999 ◽  
Vol 181 (14) ◽  
pp. 4275-4284 ◽  
Author(s):  
C. R. Dean ◽  
C. V. Franklund ◽  
J. D. Retief ◽  
M. J. Coyne ◽  
K. Hatano ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Open Reading Frames ◽  
Striking Similarity ◽  
Insertion Mutation ◽  
Significant Similarity ◽  
O Antigen ◽  
Dna Fragment ◽  
Similarity Searches ◽  
Reading Frames

ABSTRACT We previously cloned a genomic DNA fragment from the serogroup O11Pseudomonas aeruginosa strain PA103 that contained all genes necessary for O-antigen synthesis and directed the expression of serogroup O11 antigen on recombinant Escherichia coli andSalmonella. To elucidate the pathway of serogroup O11 antigen synthesis, the nucleotide sequence of the biosynthetic genes was determined. Eleven open reading frames likely to be involved in serogroup O11 O-antigen biosynthesis were identified and are designated in order as wzz PaO111 (wzz fromP. aeruginosa serogroup O11),wzx PaO11, wbjA,wzy PaO11, wbjB to wbjF,wbpL O11 and wbpM O11(wbpL and wbpM from serogroup O11). Consistent with previous descriptions of O-antigen biosynthetic gene loci, the entire region with the exception of wbpM O11 has a markedly reduced G+C content relative to the chromosomal average. WzyPaO11 shows no significant similarity at the protein or DNA sequence level to any database sequence and is very hydrophobic, with 10 to 12 putative transmembrane domains, both typical characteristics of O-antigen polymerases. A nonpolar chromosomal insertion mutation in wzy PaO11 in P. aeruginosa PA103 confirmed the identity of this gene. There is striking similarity between WbjBCDE and Cap(5/8)EFGL, involved in type 5 and type 8 capsule biosynthesis in Staphylococcus aureus. There is nearly total identity between wbpM O11and wbpM O5, previously shown by others to be present in all 20 P. aeruginosa serogroups. Using similarity searches, we have assigned functions to the proteins encoded by the PA103 O-antigen locus and present the potential steps in the pathway for the biosynthesis of P. aeruginosa serogroup O11 O antigen.

scholarly journals Genetic and Functional Analysis of the tbc Operons for Catabolism of Alkyl- and Chloroaromatic Compounds inBurkholderia sp. Strain JS150

2001 ◽  
Vol 67 (10) ◽  
pp. 4805-4816 ◽  
Author(s):  
Hyung-Yeel Kahng ◽  
Juliana C. Malinverni ◽  
Michelle M. Majko ◽  
Jerome J. Kukor
Keyword(s):  
Functional Characterization ◽  
Open Reading Frames ◽  
Gene Products ◽  
Product Analysis ◽  
Initial Substrate ◽  
Wide Range ◽  
Dna Fragment ◽  
Similarity Searches ◽  
Reading Frames

ABSTRACT Burkholderia sp. strain JS150 is able to metabolize a wide range of alkyl-and chloroaromatic hydrocarbons through multiple, apparently redundant catabolic pathways. Previous research has shown that strain JS150 is able to synthesize enzymes for multiple upper pathways as well as multiple lower pathways to accommodate variously substituted catechols that result from degradation of complex mixtures of monoaromatic compounds. We report here the genetic organization and functional characterization of a gene cluster, designatedtbc (for toluene, benzene, and chlorobenzene utilization), which has been cloned as a 14.3-kb DNA fragment from strain JS150 into vector pRO1727. The cloned DNA fragment expressed in Pseudomonas aeruginosa PAO1c allowed the recombinant to grow on toluene or benzene and to transform chlorobenzene, trichloroethylene, phenol, and cresols. The tbc genes are organized into two divergently transcribed operons, tbc1 and tbc2, each comprised of six open reading frames. Similarity searches of databases revealed that the tbc1 and tbc2 genes showed significant homology to multicomponent cresol and phenol hydroxylases and to toluene and benzene monooxygenases, respectively. Deletion mutagenesis and product analysis were used to demonstrate thattbc2 plays a role in the initial catabolism of the unactivated alkyl- or chloroaromatic substrate and that thetbc1 gene products play a role in the catabolism of the first metabolite that results from transformation of the initial substrate. Phylogenetic analysis was used to compare individual components of these tbc monooxygenases with similar sequences in the databases. These results provide further evidence for the existence of multiple, functionally redundant alkyl- and chloroaromatic monooxygenases in strain JS150.

scholarly journals Isolation and Characterization of Salmonella Jumbo-Phage pSal-SNUABM-04

Viruses ◽  
2020 ◽  
Vol 13 (1) ◽  
pp. 27
Author(s):  
Jun Kwon ◽  
Sang Guen Kim ◽  
Hyoun Joong Kim ◽  
Sib Sankar Giri ◽  
Sang Wha Kim ◽  
...  
Keyword(s):  
Morphological Traits ◽  
Open Reading Frames ◽  
Genome Comparison ◽  
The Novel ◽  
Promising Alternative ◽  
Isolation And Characterization ◽  
Global Issue ◽  
Reading Frames ◽  
Predicted Function

The increasing emergence of antimicrobial resistance has become a global issue. Therefore, many researchers have attempted to develop alternative antibiotics. One promising alternative is bacteriophage. In this study, we focused on a jumbo-phage infecting Salmonella isolated from exotic pet markets. Using a Salmonella strain isolated from reptiles as a host, we isolated and characterized the novel jumbo-bacteriophage pSal-SNUABM-04. This phage was investigated in terms of its morphology, host infectivity, growth and lysis kinetics, and genome. The phage was classified as Myoviridae based on its morphological traits and showed a comparatively wide host range. The lysis efficacy test showed that the phage can inhibit bacterial growth in the planktonic state. Genetic analysis revealed that the phage possesses a 239,626-base pair genome with 280 putative open reading frames, 76 of which have a predicted function and 195 of which have none. By genome comparison with other jumbo phages, the phage was designated as a novel member of Machinavirus composed of Erwnina phages.

scholarly journals Pan-cancer analysis of transcripts encoding novel open-reading frames (nORFs) and their potential biological functions

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Chaitanya Erady ◽  
Adam Boxall ◽  
Shraddha Puntambekar ◽  
N. Suhas Jagannathan ◽  
Ruchi Chauhan ◽  
...  
Keyword(s):  
Transcript Level ◽  
Open Reading Frames ◽  
The Cancer Genome Atlas ◽  
Small Subset ◽  
Cancer Tissue ◽  
Post Translational Modifications ◽  
Cancer Genome Atlas ◽  
Systematic Identification ◽  
Reading Frames

AbstractUncharacterized and unannotated open-reading frames, which we refer to as novel open reading frames (nORFs), may sometimes encode peptides that remain unexplored for novel therapeutic opportunities. To our knowledge, no systematic identification and characterization of transcripts encoding nORFs or their translation products in cancer, or in any other physiological process has been performed. We use our curated nORFs database (nORFs.org), together with RNA-Seq data from The Cancer Genome Atlas (TCGA) and Genotype-Expression (GTEx) consortiums, to identify transcripts containing nORFs that are expressed frequently in cancer or matched normal tissue across 22 cancer types. We show nORFs are subject to extensive dysregulation at the transcript level in cancer tissue and that a small subset of nORFs are associated with overall patient survival, suggesting that nORFs may have prognostic value. We also show that nORF products can form protein-like structures with post-translational modifications. Finally, we perform in silico screening for inhibitors against nORF-encoded proteins that are disrupted in stomach and esophageal cancer, showing that they can potentially be targeted by inhibitors. We hope this work will guide and motivate future studies that perform in-depth characterization of nORF functions in cancer and other diseases.

scholarly journals Genetic Investigation of the Catabolic Pathway for Degradation of Abietane Diterpenoids by Pseudomonas abietaniphilaBKME-9

2000 ◽  
Vol 182 (13) ◽  
pp. 3784-3793 ◽  
Author(s):  
Vincent J. J. Martin ◽  
William W. Mohn
Keyword(s):  
Gene Cluster ◽  
Sequence Similarity ◽  
Open Reading Frames ◽  
Ring Cleavage ◽  
Catabolic Pathway ◽  
Transcriptional Fusion ◽  
Abietane Diterpenoids ◽  
Genes Encoding ◽  
Dna Fragment ◽  
Reading Frames

ABSTRACT We have cloned and sequenced the dit gene cluster encoding enzymes of the catabolic pathway for abietane diterpenoid degradation by Pseudomonas abietaniphila BKME-9. Thedit gene cluster is located on a 16.7-kb DNA fragment containing 13 complete open reading frames (ORFs) and 1 partial ORF. The genes ditA1A2A3 encode the α and β subunits and the ferredoxin of the dioxygenase which hydroxylates 7-oxodehydroabietic acid to 7-oxo-11,12-dihydroxy-8,13-abietadien acid. The dioxygenase mutant strain BKME-941 (ditA1::Tn5) did not grow on nonaromatic abietanes, and transformed palustric and abietic acids to 7-oxodehydroabietic acid in cell suspension assays. Thus, nonaromatic abietanes are aromatized prior to further degradation. Catechol 2,3-dioxygenase activity of xylEtranscriptional fusion strains showed induction of ditA1and ditA3 by abietic, dehydroabietic, and 7-oxodehydroabietic acids, which support the growth of strain BKME-9, as well as by isopimaric and 12,14-dichlorodehydroabietic acids, which are diterpenoids that do not support the growth of strain BKME-9. In addition to the aromatic-ring-hydroxylating dioxygenase genes, thedit cluster includes ditC, encoding an extradiol ring cleavage dioxygenase, and ditR, encoding an IclR-type transcriptional regulator. Although ditR is not strictly required for the growth of strain BKME-9 on abietanes, aditR::Kmr mutation in aditA3::xylE reporter strain demonstrated that it encodes an inducer-dependent transcriptional activator of ditA3. An ORF with sequence similarity to genes encoding permeases (ditE) is linked with genes involved in abietane degradation.

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