scholarly journals New Host-Vector System for Thermus spp. Based on the Malate Dehydrogenase Gene

2001 ◽  
Vol 183 (5) ◽  
pp. 1792-1795 ◽  
Author(s):  
Kevin J. Kayser ◽  
John J. Kilbane
Keyword(s):  
Malate Dehydrogenase ◽  
Plasmid Dna ◽  
Thermus Thermophilus ◽  
Vector System ◽  
Wild Type ◽  
New Host ◽  
Wild Type Phenotype ◽  
Dehydrogenase Gene ◽  
Small Colony ◽  
Monitoring Gene Expression

ABSTRACT A Thermus thermophilus HB27 strain was constructed in which the malate dehydrogenase (mdh) gene was deleted. The Δmdh colonies are recognized by a small-colony phenotype. Wild-type phenotype is restored by transformation with Thermus plasmids or integration vector containing an intact mdh gene. The wild-type phenotype provides a positive selection tool for the introduction of plasmid DNA into Thermus spp., and becausemdh levels can be readily quantified, this host-vector system is a convenient tool for monitoring gene expression.

scholarly journals Phenotypic Stability of Staphylococcus aureus Small Colony Variants (SCV) Isolates from Cystic Fibrosis (CF) Patients

2019 ◽  
Vol 16 (11) ◽  
pp. 1940 ◽  
Author(s):  
Clemens Kittinger ◽  
Daniela Toplitsch ◽  
Bettina Folli ◽  
Lilian Masoud Landgraf ◽  
Gernot Zarfel
Keyword(s):  
Cystic Fibrosis ◽  
Staphylococcus Aureus ◽  
Point Mutations ◽  
High Variability ◽  
Wild Type ◽  
Phenotypic Stability ◽  
Small Colony Variant ◽  
Gene Coding ◽  
Wild Type Phenotype ◽  
Small Colony

One of the most interesting features of Staphylococcus aureus is its ability to switch to a small colony variant (SCV). This switch allows the pathogen to survive periods of antibiotic treatment or pressure from the immune system of the host and further enables it to start the infection once again after the environmental stress declines. However, so far only little is known about this reversion back to the more virulent wild type phenotype. Therefore, this study aimed to analyze the frequency of reversion to the wild type phenotype of thymidine auxotroph S. aureus SCV isolates (TD-SCVs) obtained from patients with cystic fibrosis (CF). With the use of single cell starting cultures, the occurrence of the thymidine prototroph revertants was monitored. The underlying mutational cause of the SCVs and subsequent revertants were analyzed by sequencing the gene coding for thymidylate synthase (ThyA), whose mutations are known to produce thymidine auxotroph S. aureus SCV. In our study, the underlying mutational cause for the switch to the TD-SCV phenotype was primarily point mutations. Out of twelve isolates, seven isolates showed an occurrence of revertants with a frequency ranging from 90.06% to 0.16%. This high variability in the frequency of reversion to the wild type was not expected. However, this variability in the frequency of reversion may also be the key to successful re-infection of the host. Sometimes quick reversion to the wild type proves necessary for survival, whereas other times, staying hidden for a bit longer leads to success in re-colonization of the host.

scholarly journals Cloning of the .BETA.-isopropylmalate dehydrogenase gene from Acetobacter aceti and its use for construction of a new host-vector system for Acetobacter.

1988 ◽  
Vol 52 (12) ◽  
pp. 3125-3129 ◽  
Author(s):  
Hajime OKUMURA ◽  
Haruko TAGAMI ◽  
Masahiro FUKAYA ◽  
Hiroshi MASAI ◽  
Yoshiya KAWAMURA ◽  
...  
Keyword(s):  
Vector System ◽  
Acetobacter Aceti ◽  
New Host ◽  
Dehydrogenase Gene ◽  
Isopropylmalate Dehydrogenase

scholarly journals Cloning of the β-Isopropylmalate Dehydrogenase Gene fromAcetobacter acetiand Its Use for Construction of a New Host-vector System forAcetobacter

1988 ◽  
Vol 52 (12) ◽  
pp. 3125-3129
Author(s):  
Hajime Okumura ◽  
Haruko Tagami ◽  
Masahiro Fukaya ◽  
Hiroshi Masai ◽  
Yoshiya Kawamura ◽  
...  
Keyword(s):  
Vector System ◽  
New Host ◽  
Dehydrogenase Gene ◽  
Isopropylmalate Dehydrogenase

scholarly journals Plasmid DNA Supercoiling and Gyrase Activity in Escherichia coli Wild-Type and rpoS Stationary-Phase Cells

2003 ◽  
Vol 185 (3) ◽  
pp. 1097-1100 ◽  
Author(s):  
Yazmid Reyes-Domínguez ◽  
Gabriel Contreras-Ferrat ◽  
Jesús Ramírez-Santos ◽  
Jorge Membrillo-Hernández ◽  
M. Carmen Gómez-Eichelmann
Keyword(s):  
Escherichia Coli ◽  
Stationary Phase ◽  
Plasmid Dna ◽  
Bimodal Distribution ◽  
Dna Supercoiling ◽  
Wild Type

ABSTRACT Stationary-phase cells displayed a distribution of relaxed plasmids and had the ability to recover plasmid supercoiling as soon as nutrients became available. Preexisting gyrase molecules in these cells were responsible for this recovery. Stationary-phase rpoS cells showed a bimodal distribution of plasmids and failed to supercoil plasmids after the addition of nutrients, suggesting that rpoS plays a role in the regulation of plasmid topology during the stationary phase.

scholarly journals The UDP N-Acetylgalactosamine 4-Epimerase Gene Is Essential for Mesophilic Aeromonas hydrophila Serotype O34 Virulence

2006 ◽  
Vol 74 (1) ◽  
pp. 537-548 ◽  
Author(s):  
Rocío Canals ◽  
Natalia Jiménez ◽  
Silvia Vilches ◽  
Miguel Regué ◽  
Susana Merino ◽  
...  
Keyword(s):  
Aeromonas Hydrophila ◽  
Type Strain ◽  
Wild Type Strain ◽  
Sugar Residue ◽  
Serum Resistance ◽  
Single Mutation ◽  
Wild Type ◽  
O Antigen ◽  
Wild Type Phenotype

ABSTRACT Mesophilic Aeromonas hydrophila strains of serotype O34 typically express smooth lipopolysaccharide (LPS) on their surface. A single mutation in the gene that codes for UDP N-acetylgalactosamine 4-epimerase (gne) confers the O− phenotype (LPS without O-antigen molecules) on a strain in serotypes O18 and O34, but not in serotypes O1 and O2. The gne gene is present in all the mesophilic Aeromonas strains tested. No changes were observed for the LPS core in a gne mutant from A. hydrophila strain AH-3 (serotype O34). O34 antigen LPS contains N-acetylgalactosamine, while no such sugar residue forms part of the LPS core from A. hydrophila AH-3. Some of the pathogenic features of A. hydrophila AH-3 gne mutants are drastically reduced (serum resistance or adhesion to Hep-2 cells), and the gne mutants are less virulent for fish and mice compared to the wild-type strain. Strain AH-3, like other mesophilic Aeromonas strains, possess two kinds of flagella, and the absence of O34 antigen molecules by gne mutation in this strain reduced motility without any effect on the biogenesis of both polar and lateral flagella. The reintroduction of the single wild-type gne gene in the corresponding mutants completely restored the wild-type phenotype (presence of smooth LPS) independently of the O wild-type serotype, restored the virulence of the wild-type strain, and restored motility (either swimming or swarming).

Rescue of the mutant phenotype by reexpression of full-length vinculin in null F9 cells; effects on cell locomotion by domain deleted vinculin

1998 ◽  
Vol 111 (11) ◽  
pp. 1535-1544 ◽  
Author(s):  
W. Xu ◽  
J.L. Coll ◽  
E.D. Adamson
Keyword(s):  
Marked Effect ◽  
Control Cell ◽  
Covalent Attachment ◽  
Tail Domain ◽  
Wild Type ◽  
F9 Cells ◽  
Wild Type Phenotype ◽  
Correct Location ◽  
Low Levels ◽  
Lamellipodia Formation

Vinculin plays a role in signaling between integrins and the actin cytoskeleton. We reported earlier that F9-derived cells lacking vinculin are less spread, less adhesive, and move two times faster than wild-type F9 cells. Expression of intact vinculin in null cells restored all wild-type characteristics. In contrast, expression of the head (90 kDa) fragment exaggerated mutant characteristics, especially locomotion, which was double that of vinculin null cells. Expression of the tail domain also had a marked effect on locomotion in the opposite direction, reducing it to very low levels. The expression of the head plus tail domains together (no covalent attachment) effected a partial rescue towards wild-type phenotype, thus indicating that reexpressed polypeptides may be in their correct location and are interacting normally. Therefore, we conclude that: (1) the head domain is part of the locomotory force of the cell, modulated by the tail, and driven by the integrin/matrix connection; (2) intact vinculin is required for normal regulation of cell behavior, suggesting that vinculin head-tail interactions control cell adhesion, spreading, lamellipodia formation and locomotion.

scholarly journals Mutation of a single residue in the ba3 oxidase specifically impairs protonation of the pump site

2015 ◽  
Vol 112 (11) ◽  
pp. 3397-3402 ◽  
Author(s):  
Christoph von Ballmoos ◽  
Nathalie Gonska ◽  
Peter Lachmann ◽  
Robert B. Gennis ◽  
Pia Ädelroth ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Time Constant ◽  
Thermus Thermophilus ◽  
Proton Translocation ◽  
Proton Pumping ◽  
Wild Type ◽  
Structural Variant ◽  
In The Wild ◽  
Membrane Bound ◽  
Proton Uptake

The ba3-type cytochrome c oxidase from Thermus thermophilus is a membrane-bound protein complex that couples electron transfer to O2 to proton translocation across the membrane. To elucidate the mechanism of the redox-driven proton pumping, we investigated the kinetics of electron and proton transfer in a structural variant of the ba3 oxidase where a putative “pump site” was modified by replacement of Asp372 by Ile. In this structural variant, proton pumping was uncoupled from internal electron transfer and O2 reduction. The results from our studies show that proton uptake to the pump site (time constant ∼65 μs in the wild-type cytochrome c oxidase) was impaired in the Asp372Ile variant. Furthermore, a reaction step that in the wild-type cytochrome c oxidase is linked to simultaneous proton uptake and release with a time constant of ∼1.2 ms was slowed to ∼8.4 ms, and in Asp372Ile was only associated with proton uptake to the catalytic site. These data identify reaction steps that are associated with protonation and deprotonation of the pump site, and point to the area around Asp372 as the location of this site in the ba3 cytochrome c oxidase.

scholarly journals Analysis of junction sequences resulting from integration at nonhomologous loci in Neurospora crassa.

Genetics ◽  
1992 ◽  
Vol 130 (4) ◽  
pp. 737-748
Author(s):  
D K Asch ◽  
G Frederick ◽  
J A Kinsey ◽  
D D Perkins
Keyword(s):  
Neurospora Crassa ◽  
Plasmid Dna ◽  
Repetitive Sequence ◽  
Single Copy ◽  
The Other ◽  
Chromosomal Dna ◽  
End Joining ◽  
Dehydrogenase Gene ◽  
The Right ◽  
Complete Deletion

Abstract We have analyzed the junctions involved in two examples of ectopic integration of plasmids containing the am+ (glutamate dehydrogenase) gene into a strain of Neurospora crassa bearing a complete deletion of the am locus. In one transformed strain a single copy of plasmid DNA had been integrated into linkage group (LG) III DNA without the loss of chromosomal DNA. In contrast, 450 bp had been lost from plasmid sequences at the site of integration. The transforming DNA used was circular, so we postulate that the plasmid was linearized and truncated prior to its integration by end joining into a break in LG III DNA. There was no significant homology between the incoming DNA and DNA at the site of integration. The second transformed strain resulted from transformation with a linearized plasmid. It contained multiple integrated copies of plasmid DNA, one of which was recloned, together with adjacent chromosomal DNA, by plasmid rescue in Escherichia coli. Prior to integration into chromosomal DNA, the linear plasmid had been truncated by 64 bp on one end and 3.2 kbp on the other end. One end of the integrated DNA was adjacent to DNA from the right arm of LG I, while the other end was integrated into a copy of a repetitive sequence. Restriction fragment length polymerism mapping showed that integration was in a copy of the repetitive sequence that is linked to the previously unassigned telomere M11 and is distantly linked to the LG VI marker con-11. Genetic analysis revealed that a long segment of LG I containing all markers from un-1 to the right tip has been translocated to the right end of LG VI. Tetrad analysis showed that the integrated DNA was closely linked to the translocation. We conclude that the transforming DNA was truncated and joined to DNA from two different chromosomes by end joining during the formation of a quasiterminal translocation, T(IR----VIR) UK-T12. We also conclude that the previously unassigned telomere, M11, is the right end of LG VI.

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