scholarly journals Isolation and Characterization of a Native Composite Transposon, Tn14751, Carrying 17.4 Kilobases of Corynebacterium glutamicum Chromosomal DNA

2005 ◽  
Vol 71 (1) ◽  
pp. 407-416 ◽  
Author(s):  
Masayuki Inui ◽  
Yota Tsuge ◽  
Nobuaki Suzuki ◽  
Alain A. Vert�s ◽  
Hideaki Yukawa
Keyword(s):  
Corynebacterium Glutamicum ◽  
Insertion Sequence ◽  
Kanamycin Resistance ◽  
Chromosomal Dna ◽  
Kanamycin Resistance Cassette ◽  
Isolation And Characterization ◽  
Genes Encoding ◽  
Genome Scale ◽  
Random Insertion

ABSTRACT A native composite transposon was isolated from Corynebacterium glutamicum ATCC 14751. This transposon comprises two functional copies of a corynebacterial IS31831-like insertion sequence organized as converging terminal inverted repeats. This novel 20.3-kb element, Tn14751, carries 17.4 kb of C. glutamicum chromosomal DNA containing various genes, including genes involved in purine biosynthesis but not genes related to bacterial warfare, such as genes encoding mediators of antibiotic resistance or extracellular toxins. A derivative of this element carrying a kanamycin resistance cassette, minicomposite Tn14751, transposed into the genome of C. glutamicum at an efficiency of 1.8 � 102 transformants per μg of DNA. Random insertion of the Tn14751 derivative carrying the kanamycin resistance cassette into the chromosome was verified by Southern hybridization. This work paves the way for realization of the concept of minimum genome factories in the search for metabolic engineering via genome-scale directed evolution through a combination of random and directed approaches.

Isolation and characterization of the genes encoding antimicrobial neutrophil cationic peptides, defensins.

Ensho ◽  
1995 ◽  
Vol 15 (1) ◽  
pp. 33-41
Author(s):  
Isao Nagaoka ◽  
Noriko Ishihara ◽  
Akimasa Someya ◽  
Kazuhisa Iwabuchi ◽  
Shin Yomogida ◽  
...  
Keyword(s):  
Cationic Peptides ◽  
Isolation And Characterization ◽  
Genes Encoding

scholarly journals Escherichia coli Mutants Lacking All Possible Combinations of Eight Penicillin Binding Proteins: Viability, Characteristics, and Implications for Peptidoglycan Synthesis

1999 ◽  
Vol 181 (13) ◽  
pp. 3981-3993 ◽  
Author(s):  
Sylvia A. Denome ◽  
Pamela K. Elf ◽  
Thomas A. Henderson ◽  
David E. Nelson ◽  
Kevin D. Young
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Binding Proteins ◽  
Kanamycin Resistance ◽  
Open Reading Frames ◽  
Penicillin Binding Proteins ◽  
E Coli ◽  
Peptidoglycan Biosynthesis ◽  
Kanamycin Resistance Cassette ◽  
Genes Encoding

ABSTRACT The penicillin binding proteins (PBPs) synthesize and remodel peptidoglycan, the structural component of the bacterial cell wall. Much is known about the biochemistry of these proteins, but little is known about their biological roles. To better understand the contributions these proteins make to the physiology ofEscherichia coli, we constructed 192 mutants from which eight PBP genes were deleted in every possible combination. The genes encoding PBPs 1a, 1b, 4, 5, 6, and 7, AmpC, and AmpH were cloned, and from each gene an internal coding sequence was removed and replaced with a kanamycin resistance cassette flanked by two ressites from plasmid RP4. Deletion of individual genes was accomplished by transferring each interrupted gene onto the chromosome of E. coli via λ phage transduction and selecting for kanamycin-resistant recombinants. Afterwards, the kanamycin resistance cassette was removed from each mutant strain by supplying ParA resolvase in trans, yielding a strain in which a long segment of the original PBP gene was deleted and replaced by an 8-bpres site. These kanamycin-sensitive mutants were used as recipients in further rounds of replacement mutagenesis, resulting in a set of strains lacking from one to seven PBPs. In addition, thedacD gene was deleted from two septuple mutants, creating strains lacking eight genes. The only deletion combinations not produced were those lacking both PBPs 1a and 1b because such a combination is lethal. Surprisingly, all other deletion mutants were viable even though, at the extreme, 8 of the 12 known PBPs had been eliminated. Furthermore, when both PBPs 2 and 3 were inactivated by the β-lactams mecillinam and aztreonam, respectively, several mutants did not lyse but continued to grow as enlarged spheres, so that one mutant synthesized osmotically resistant peptidoglycan when only 2 of 12 PBPs (PBPs 1b and 1c) remained active. These results have important implications for current models of peptidoglycan biosynthesis, for understanding the evolution of the bacterial sacculus, and for interpreting results derived by mutating unknown open reading frames in genome projects. In addition, members of the set of PBP mutants will provide excellent starting points for answering fundamental questions about other aspects of cell wall metabolism.

scholarly journals Cloning and Characterization of α-Methylacyl Coenzyme A Racemase from Gordonia polyisoprenivorans VH2

2008 ◽  
Vol 74 (22) ◽  
pp. 7085-7089 ◽  
Author(s):  
Quyen Arenskötter ◽  
Jens Heller ◽  
David Dietz ◽  
Matthias Arenskötter ◽  
Alexander Steinbüchel
Keyword(s):  
Cytochrome P450 ◽  
Reverse Transcription ◽  
Kanamycin Resistance ◽  
P450 Monooxygenase ◽  
Transcription Analysis ◽  
Kanamycin Resistance Cassette ◽  
Short Chain Dehydrogenase ◽  
Gordonia Polyisoprenivorans ◽  
Rubber Degradation

ABSTRACT The mcr gene of Gordonia polyisoprenivorans VH2 is not clustered with genes required for rubber degradation. Its disruption by insertion of a kanamycin resistance cassette impaired growth on methyl-branched isoprenoids but not on linear hydrocarbons. Intact mcr from this bacterium or from Nocardia farcinica IFM 10152 complemented the mutant. Reverse transcription analysis showed similar mcr VH2 expression results during cultivation with poly(cis-1,4-isoprene) and propionate. Additional genes coding for a putative cytochrome P450 monooxygenase and a short-chain dehydrogenase/reductase involved in β-oxidation and poly(cis-1,4-isoprene) degradation were also characterized.

Analysis of the structural genes encoding M-factor in the fission yeast Schizosaccharomyces pombe: identification of a third gene, mfm3

1994 ◽  
Vol 14 (6) ◽  
pp. 3895-3905
Author(s):  
S Kjaerulff ◽  
J Davey ◽  
O Nielsen
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Mutational Analysis ◽  
M Cells ◽  
Gene Products ◽  
Structural Genes ◽  
Triple Mutant ◽  
Isolation And Characterization ◽  
Genes Encoding

We previously identified two genes, mfm1 and mfm2, with the potential to encode the M-factor mating pheromone of the fission yeast Schizosaccharomyces pombe (J. Davey, EMBO J. 11:951-960, 1992), but further analysis revealed that a mutant strain lacking both genes still produced active M-factor. Here we describe the isolation and characterization of a third M-factor gene, mfm3. A mutant lacking all three genes fails to produce M-factor, indicating that all functional M-factor genes now have been identified. The triple mutant exhibits an absolute mating defect in M cells, a defect that is not rescued by addition of exogenous M-factor. A mutational analysis reveals that all three mfm genes contribute to the production of M-factor. Their transcription is limited to M cells and requires the mat1-Mc and ste11 gene products. Each gene is induced when the cells are starved of nitrogen and further induced by a pheromone signal. Additionally, the signal transduction machinery associated with the pheromone response is required for transcription of the mfm genes in both stimulated and unstimulated cells.

scholarly journals Isolation and Characterization of a Genetically Tractable Photoautotrophic Fe(II)-Oxidizing Bacterium, Rhodopseudomonas palustris Strain TIE-1

2005 ◽  
Vol 71 (8) ◽  
pp. 4487-4496 ◽  
Author(s):  
Yongqin Jiao ◽  
Andreas Kappler ◽  
Laura R. Croal ◽  
Dianne K. Newman
Keyword(s):  
Bacterial Species ◽  
Rhodopseudomonas Palustris ◽  
Carbon Sources ◽  
Integral Membrane Protein ◽  
Transposition Frequency ◽  
Isolation And Characterization ◽  
Abc Transport ◽  
A Cell ◽  
Random Insertion

ABSTRACT We report the isolation and characterization of a phototrophic ferrous iron [Fe(II)]-oxidizing bacterium named TIE-1 that differs from other Fe(II)-oxidizing phototrophs in that it is genetically tractable. Under anaerobic conditions, TIE-1 grows photoautotrophically with Fe(II), H2, or thiosulfate as the electron donor and photoheterotrophically with a variety of organic carbon sources. TIE-1 also grows chemoheterotrophically in the dark. This isolate appears to be a new strain of the purple nonsulfur bacterial species Rhodopseudomonas palustris, based on physiological and phylogenetic analysis. Fe(II) oxidation is optimal at pH 6.5 to 6.9. The mineral products of Fe(II) oxidation are pH dependent: below pH 7.0 goethite (α-FeOOH) forms, and above pH 7.2 magnetite (Fe3O4) forms. TIE-1 forms colonies on agar plates and is sensitive to a variety of antibiotics. A hyperactive mariner transposon is capable of random insertion into the chromosome with a transposition frequency of ∼10−5. To identify components involved in phototrophic Fe(II) oxidation, mutants of TIE-1 were generated by transposon mutagenesis and screened for defects in Fe(II) oxidation in a cell suspension assay. Among approximately 12,000 mutants screened, 6 were identified that are specifically impaired in Fe(II) oxidation. Five of these mutants have independent disruptions in a gene that is predicted to encode an integral membrane protein that appears to be part of an ABC transport system; the sixth mutant has an insertion in a gene that is a homolog of CobS, an enzyme involved in cobalamin (vitamin B12) biosynthesis.

scholarly journals Isolation and Characterization of the Genes Encoding Basic and Acidic Chitinase in Arabidopsis thaliana

1990 ◽  
Vol 93 (3) ◽  
pp. 907-914 ◽  
Author(s):  
Deborah A. Samac ◽  
Cathy M. Hironaka ◽  
Peter E. Yallaly ◽  
Dilip M. Shah
Keyword(s):  
Arabidopsis Thaliana ◽  
Isolation And Characterization ◽  
Genes Encoding

Isolation and characterization of two DREB1 genes encoding dehydration-responsive element binding proteins in chicory (Cichorium intybus)

2013 ◽  
Vol 73 (1) ◽  
pp. 45-55 ◽  
Author(s):  
Mingxiang Liang ◽  
Dandan Chen ◽  
Manman Lin ◽  
Qingsong Zheng ◽  
Zengrong Huang ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Cichorium Intybus ◽  
Isolation And Characterization ◽  
Genes Encoding ◽  
Responsive Element
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