scholarly journals BacS: An Abundant Bacteroid Protein in Rhizobium etli Whose Expression Ex Planta Requires nifA

2003 ◽  
Vol 16 (1) ◽  
pp. 65-73 ◽  
Author(s):  
Olivia J. Jahn ◽  
Guillermo Davila ◽  
David Romero ◽  
K. Dale Noel
Keyword(s):  
Dna Hybridization ◽  
Open Reading Frames ◽  
Nucleotide Sequencing ◽  
Rhizobium Etli ◽  
Low Oxygen ◽  
Sigma 54 ◽  
Rhizobial Species ◽  
Dna Fragment ◽  
Low Oxygen Concentration ◽  
Reading Frames

Rhizobium etli CFN42 bacteroids from bean nodules possessed an abundant 16-kDa protein (BacS) that was found in the membrane pellet after cell disruption. This protein was not detected in bacteria cultured in tryptone-yeast extract. In minimal media, it was produced at low oxygen concentration but not in a mutant whose nifA was disrupted. N-terminal sequencing of the protein led to isolation of a bacS DNA fragment. DNA hybridization and nucleotide sequencing revealed three copies of the bacS gene, all residing on the main symbiotic plasmid of strain CFN42. A stretch of 304 nucleotides, exactly conserved upstream of all three bacS open reading frames, had very close matches with the NifA and sigma 54 consensus binding sequences. The only bacS homology in the genetic sequence databases was to three hypothetical proteins of unknown function, all from rhizobial species. Mutation and genetic complementation indicated that each of the bacS genes gives rise to a BacS polypeptide. Mutants disrupted or deleted in all three genes did not produce the BacS polypeptide but were Nod+ and Fix+ on Phaseolus vulgaris.

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.

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 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 An endogenous retrovirus presumed to have been endogenized or relocated recently in a marsupial, the red-necked wallaby

Genome ◽  
2022 ◽  
Author(s):  
Sakura Hayashi ◽  
Konami Shimizu ◽  
Yusuke Honda ◽  
Yukako Katsura ◽  
Akihiko Koga
Keyword(s):  
Endogenous Retrovirus ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Length Variation ◽  
Wild Type ◽  
Long Terminal Repeats ◽  
Recent Event ◽  
Melanin Biosynthesis ◽  
Dna Fragment ◽  
Reading Frames

An albino infant wallaby was born to a mother with the wild-type body color. PCR and sequencing analyses of <i>TYR</i> (encoding tyrosinase, which is essential for melanin biosynthesis) of this albino wallaby revealed a 7.1-kb-long DNA fragment inserted in the first exon. Because the fragment carried long terminal repeats, we assumed it to be a copy of an endogenous retrovirus, which we named <i>walb</i>. We cloned other <i>walb</i> copies residing in the genomes of this species and another wallaby species. The copies exhibited length variation, and the longest copy (>8.0 kb) contained open reading frames whose deduced amino acid sequences were well aligned with those of <i>gag</i>, <i>pol</i>, and <i>env</i> of retroviruses. It is not known through which of the following likely processes the walb copy was inserted into <i>TYR</i>: endogenization (infection of a germline cell by an exogenous virus), reinfection (infection by a virus produced from a previously endogenized provirus), or retrotransposition (intracellular relocation of a provirus). In any case, the insertion into <i>TYR</i> is considered to have been a recent event on an evolutionary timescale because albino mutant alleles generally do not persist for long because of their deleterious effects in wild circumstances. 

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.

RHD gene deletion occurred in the Rhesus box

Blood ◽  
2000 ◽  
Vol 95 (12) ◽  
pp. 3662-3668 ◽  
Author(s):  
Franz F. Wagner ◽  
Willy A. Flegel
Keyword(s):  
Open Reading Frames ◽  
Chromosome 1 ◽  
Nucleotide Sequencing ◽  
Blood Group Antigens ◽  
Specific Detection ◽  
Pcr Rflp ◽  
Polymerase Chain ◽  
Specific Priming ◽  
Rh Blood Group ◽  
Reading Frames

Abstract The Rh blood group antigens derive from 2 genes,RHD and RHCE, that are located at chromosomal position 1p34.1-1p36 (chromosome 1, short arm, region 3, band 4, subband 1, through band 6). In whites, a cde haplotype with a deletion of the whole RHD gene occurs with a frequency of approximately 40%. The relative position of the 2 RH genes and the location of the RHD deletion was previously unknown. A model has been developed for the RH locus using RHD- and RHCE-related nucleotide sequences deposited in nucleotide sequence databases along with polymerase chain reaction (PCR) and nucleotide sequencing. The open reading frames of bothRH genes had opposite orientations. The 3′ ends of the genes faced each other and were separated by about 30 000 base pair (bp) that contained the SMP1 gene. The RHD gene was flanked by 2 DNA segments, dubbed Rhesus boxes, with a length of approximately 9000 bp, 98.6% homology, and identical orientation. The Rhesus box contained the RHD deletion occurring within a stretch of 1463 bp of identity. PCR with sequence-specific priming (PCR-SSP) and PCR with restriction fragment length polymorphism (PCR-RFLP) were used for specific detection of the RHDdeletion. The molecular structure of the RH gene locus explains the mechanisms for generating RHD/RHCE hybrid alleles and the RHD deletion. Specific detection of theRHD− genotype is now possible.

scholarly journals Characterization and Genomic Analysis of Phage asccφ28, a Phage of the Family Podoviridae Infecting Lactococcus lactis

2008 ◽  
Vol 74 (11) ◽  
pp. 3453-3460 ◽  
Author(s):  
Steven E. Kotsonis ◽  
Ian B. Powell ◽  
Christopher J. Pillidge ◽  
Gaëtan K. Y. Limsowtin ◽  
Alan J. Hillier ◽  
...  
Keyword(s):  
Lactococcus Lactis ◽  
Dna Hybridization ◽  
Burst Size ◽  
Genomic Analysis ◽  
Open Reading Frames ◽  
Full Genome Sequence ◽  
The Family ◽  
Reading Frames ◽  
Dairy Fermentation

ABSTRACT Bacteriophage asccφ28 infects dairy fermentation strains of Lactococcus lactis. This report describes characterization of asccφ28 and its full genome sequence. Phage asccφ28 has a prolate head, whiskers, and a short tail (C2 morphotype). This morphology and DNA hybridization to L. lactis phage P369 DNA showed that asccφ28 belongs to the P034 phage species, a group rarely encountered in the dairy industry. The burst size of asccφ28 was found to be 121 ± 18 PFU per infected bacterial cell after a latent period of 44 min. The linear genome (18,762 bp) contains 28 possible open reading frames (ORFs) comprising 90% of the total genome. The ORFs are arranged bidirectionally in recognizable functional modules. The genome contains 577 bp inverted terminal repeats (ITRs) and putatively eight promoters and four terminators. The presence of ITRs, a phage-encoded DNA polymerase, and a terminal protein that binds to the DNA, along with BLAST and morphology data, show that asccφ28 more closely resembles streptococcal phage Cp-1 and the φ29-like phages that infect Bacillus subtilis than it resembles common lactococcal phages. The sequence of this phage is the first published sequence of a P034 species phage genome.

scholarly journals The Legionella pneumophila icmGCDJBFGenes Are Required for Killing of Human Macrophages

1998 ◽  
Vol 66 (5) ◽  
pp. 2245-2255 ◽  
Author(s):  
Mary Purcell ◽  
Howard A. Shuman
Keyword(s):  
Legionella Pneumophila ◽  
Dna Hybridization ◽  
Open Reading Frames ◽  
Nucleotide Sequencing ◽  
Human Macrophages ◽  
Cytopathic Effects ◽  
Significant Homology ◽  
Genomic Insert ◽  
Transcriptional Units ◽  
Macrophage Killing

ABSTRACT Previously, a collection of mutants of Legionella pneumophila that had lost the ability to multiply within and kill human macrophages was generated by Tn903dIIlacZ transposon mutagenesis and classified into DNA hybridization groups. A subset of these mutants was complemented by a plasmid, pMW100, containing a 13.5-kb genomic DNA insert. This plasmid restored the ability to multiply within and produce cytopathic effects on human macrophages to members of DNA hybridization groups II, IV, VI, and XVII. A region of the genomic insert of pMW100 was sequenced, and eight potential genes were identified and named icmE,icmG, icmC, icmD, icmJ,icmB, icmF, and tphA. None of the genes encode potential protein products with significant homology to previously characterized proteins, except for tphA, whose product has significant homology to a family of metabolite/H+ symport proteins from gram-negative bacteria. The positions of the Tn903dIIlacZ insertions within the genes were determined by nucleotide sequencing. No Tn903dIIlacZ insertions mapped toicmG, icmJ, or tphA; therefore, these loci were mutated to test whether they were required for macrophage killing. Complementation analysis was used to evaluate the roles of the potential gene products and provide information on the organization of transcriptional units within the region. The results indicate that all identified open reading frames excepttphA are required for killing of human macrophages.

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