scholarly journals Uptake of 2-Oxoglutarate inSynechococcus Strains Transformed with the Escherichia coli kgtP Gene

2000 ◽  
Vol 182 (1) ◽  
pp. 211-215 ◽  
Author(s):  
María Félix Vázquez-Bermúdez ◽  
Antonia Herrero ◽  
Enrique Flores
Keyword(s):  
Escherichia Coli ◽  
Gene Cassette ◽  
Kanamycin Resistance ◽  
Kanamycin Resistance Gene ◽  
Gene Encoding ◽  
Resistance Gene Cassette ◽  
Low Levels ◽  
Active Transport Process ◽  
Taxonomic Groups ◽  
Pcc 7942

ABSTRACT A number of cyanobacteria from different taxonomic groups exhibited very low levels of uptake of 2-[U-14C]oxoglutarate.Synechococcus sp. strain PCC 7942 was transformed with DNA constructs carrying the Escherichia coli kgtP gene encoding a 2-oxoglutarate permease and a kanamycin resistance gene cassette. The Synechococcus sp. strains bearing thekgtP gene incorporated 2-oxoglutarate into the cells through an active transport process. About 75% of the radioactivity from the 2-[U-14C]oxoglutarate taken up that was recovered in soluble metabolites was found as glutamate and glutamine. 2-Oxoglutarate was, however, detrimental to the growth of aSynechococcus sp. strain bearing the kgtP gene.

scholarly journals Insertion Mutagenesis of wca Reduces Acid and Heat Tolerance of Enterohemorrhagic Escherichia coliO157:H7

2001 ◽  
Vol 183 (12) ◽  
pp. 3811-3815 ◽  
Author(s):  
Ying Mao ◽  
Michael P. Doyle ◽  
Jinru Chen
Keyword(s):  
Uronic Acid ◽  
Gene Cassette ◽  
Kanamycin Resistance ◽  
Insertion Mutagenesis ◽  
Kanamycin Resistance Gene ◽  
Macconkey Agar ◽  
E Coli ◽  
Resistance Gene Cassette ◽  
Glucose Agar ◽  
Adverse Environmental Conditions

ABSTRACT Strains of enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 produce under stress copious amounts of exopolysaccharide (EPS) composed of colanic acid (CA). Studies were performed to evaluate the association of production of CA with survival of EHEC under adverse environmental conditions. A CA-deficient mutant, M4020, was obtained from a CA-proficient parental strain, E. coli O157:H7 W6-13, by inserting a kanamycin resistance gene cassette (kan) into wcaD and wcaE, 2 of the 21 genes required for CA biosynthesis. M4020 was defective in CA production as determined from the ratio of uronic acid to protein (UA/P) of cells grown from 1 to 4 days at 25°C on minimal glucose agar (MGA), MacConkey agar, and sorbitol-MacConkey agar, and by colony morphology on MGA. The results of stress treatment revealed that M4020 was substantially less tolerant to acid (pH 4.5 and 5.5) and heat (55 and 60°C) in comparison to W6-13, indicating that CA confers onE. coli O157:H7 a protective effect from the environmental stresses of acid and heat.

Formaldehyde effects on kanamycin resistance gene of inactivated recombinant Escherichia coli vaccines

2020 ◽  
Vol 42 (11) ◽  
pp. 2223-2230
Author(s):  
Rafael A. Donassolo ◽  
Marcos Roberto A. Ferreira ◽  
Clóvis Moreira Jr ◽  
Lucas M. dos Santos ◽  
Emili Griep ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Resistance Gene ◽  
Kanamycin Resistance ◽  
Recombinant Escherichia Coli ◽  
Kanamycin Resistance Gene

scholarly journals Melanin-Based High-Throughput Screen for l-Tyrosine Production in Escherichia coli

2007 ◽  
Vol 74 (4) ◽  
pp. 1190-1197 ◽  
Author(s):  
Christine Nicole S. Santos ◽  
Gregory Stephanopoulos
Keyword(s):  
Escherichia Coli ◽  
High Throughput ◽  
Combinatorial Libraries ◽  
Selection Strategy ◽  
High Throughput Screen ◽  
Bacterial Strains ◽  
Gene Encoding ◽  
Strong Linear Correlation ◽  
Low Levels ◽  
Tyrosine Content

ABSTRACT We present the development of a simple, high-throughput screen for identifying bacterial strains capable of l-tyrosine production. Through the introduction of a heterologous gene encoding a tyrosinase, we were able to link l-tyrosine production in Escherichia coli with the synthesis of the black and diffusible pigment melanin. Although melanin was initially produced only at low levels in morpholinepropanesulfonic acid (MOPS) minimal medium, phosphate supplementation was found to be sufficient for increasing both the rates of synthesis and the final titers of melanin. Furthermore, a strong linear correlation between extracellular l-tyrosine content and melanin formation was observed by use of this new medium formulation. A selection strategy that utilizes these findings has been developed and has been shown to be effective in screening large combinatorial libraries in the search for l-tyrosine-overproducing strains.

scholarly journals Identification of a Gene Essential for Sheathed Structure Formation in Sphaerotilus natans, a Filamentous Sheathed Bacterium

2002 ◽  
Vol 68 (1) ◽  
pp. 365-371 ◽  
Author(s):  
Toshihiko Suzuki ◽  
Takahiro Kanagawa ◽  
Yoichi Kamagata
Keyword(s):  
Gene Cassette ◽  
Kanamycin Resistance ◽  
Reading Frame ◽  
Sphaerotilus Natans ◽  
Crucial Component ◽  
Rich Media ◽  
Resistance Gene Cassette ◽  
Sheath Formation ◽  
Bacterial Exopolysaccharide ◽  
Activated Sludge Processes

ABSTRACT Sphaerotilus natans, a filamentous bacterium that causes bulking in activated sludge processes, can assume two distinct morphologies, depending on the substrate concentration for growth; in substrate-rich media it grows as single rod-shaped cells, whereas in substrate-limited media it grows as filaments. To identify genes responsible for sheath formation, we carried out transposon Tn5 mutagenesis. Of the approximately 20,000 mutants obtained, 7 did not form sheathed structures. Sequencing of the Tn5-flanking regions showed that five of the seven Tn5 insertions converged at the same open reading frame, designated sthA. The deduced amino acids encoded by sthA were found to be homologous to glycosyltransferase, which is known to be involved in linking sugars to lipid carriers during bacterial exopolysaccharide biosynthesis. Disruption of the gene of the wild-type strain by inserting a kanamycin resistance gene cassette also resulted in sheathless growth under either type of nutrient condition. These findings indicate that sthA is a crucial component responsible for sheath formation.

scholarly journals Construction and Complementation of In-Frame Deletions of the Essential Escherichia coli Thymidylate Kinase Gene

2006 ◽  
Vol 72 (2) ◽  
pp. 1288-1294 ◽  
Author(s):  
David-Nicolas Chaperon
Keyword(s):  
Escherichia Coli ◽  
Resistance Gene ◽  
Homo Sapiens ◽  
Bacteriophage T4 ◽  
Kanamycin Resistance ◽  
Kanamycin Resistance Gene ◽  
Kinase Gene ◽  
Thymidylate Kinase ◽  
E Coli ◽  
Putative Operon

ABSTRACT This work reports the construction of Escherichia coli in-frame deletion strains of tmk, which encodes thymidylate kinase, Tmk. The tmk gene is located at the third position of a putative five-gene operon at 24.9 min on the E. coli chromosome, which comprises the genes pabC, yceG, tmk, holB, and ycfH. To avoid potential polar effects on downstream genes of the operon, as well as recombination with plasmid-encoded tmk, the tmk gene was replaced by the kanamycin resistance gene kka1, encoding amino glycoside 3′-phosphotransferase kanamycin kinase. The kanamycin resistance gene is expressed under the control of the natural promoter(s) of the putative operon. The E. coli tmk gene is essential under any conditions tested. To show functional complementation in bacteria, the E. coli tmk gene was replaced by thymidylate kinases of bacteriophage T4 gp1, E. coli tmk, Saccharomyces cerevisiae cdc8, or the Homo sapiens homologue, dTYMK. Growth of these transgenic E. coli strains is completely dependent on thymidylate kinase activities of various origin expressed from plasmids. The substitution constructs show no polar effects on the downstream genes holB and ycfH with respect to cell viability. The presented transgenic bacteria could be of interest for testing of thymidylate kinase-specific phosphorylation of nucleoside analogues that are used in therapies against cancer and infectious diseases.

scholarly journals Osmoregulated periplasmic glucans of Salmonella enterica serovar Typhimurium are required for optimal virulence in mice

Microbiology ◽  
2009 ◽  
Vol 155 (1) ◽  
pp. 229-237 ◽  
Author(s):  
Arvind A. Bhagwat ◽  
Won Jun ◽  
Liu Liu ◽  
Porteen Kannan ◽  
Mahesh Dharne ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Type Strain ◽  
Wild Type Strain ◽  
Gene Cassette ◽  
Growth Conditions ◽  
Kanamycin Resistance ◽  
Wild Type ◽  
Serovar Typhimurium ◽  
Resistance Gene Cassette ◽  
Reduced Rate

We purified osmoregulated periplasmic glucans (OPGs) from Salmonella enterica serovar Typhimurium and found them to be composed of 100 % glucose with 2-linked glucose as the most abundant residue, with terminal glucose, 2,3-linked and 2,6-linked glucose also present in high quantities. The two structural genes for OPG biosynthesis, opgG and opgH, form a bicistronic operon, and insertion of a kanamycin resistance gene cassette into this operon resulted in a strain devoid of OPGs. The opgGH mutant strain was impaired in motility and growth under low osmolarity conditions. The opgGH mutation also resulted in a 2 log increase in the LD50 in mice compared to the wild-type strain SL1344. Inability to synthesize OPGs had no significant impact on the organism's lipopolysaccharide pattern or its ability to survive antimicrobial peptides-, detergent-, pH- and nutrient-stress conditions. We observed that the opgGH-defective strain respired at a reduced rate under acidic growth conditions (pH 5.0) and had lower ATP levels compared to the wild-type strain. These data indicate that OPGs of S. Typhimurium contribute towards mouse virulence as well as growth and motility under low osmolarity growth conditions.

scholarly journals Lytic and Lysogenic Infection of DiverseEscherichia coli and Shigella Strains with a Verocytotoxigenic Bacteriophage

2001 ◽  
Vol 67 (9) ◽  
pp. 4335-4337 ◽  
Author(s):  
Chloe E. James ◽  
Karen N. Stanley ◽  
Heather E. Allison ◽  
Harry J. Flint ◽  
Colin S. Stewart ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Resistance Gene ◽  
Plaque Assay ◽  
Kanamycin Resistance ◽  
Lytic Infection ◽  
Lytic Cycle ◽  
Animal Origin ◽  
Kanamycin Resistance Gene ◽  
E Coli ◽  
Infected Cells

ABSTRACT A verocytotoxigenic bacteriophage isolated from a strain of enterohemorrhagic Escherichia coli O157, into which a kanamycin resistance gene (aph3) had been inserted to inactivate the verocytotoxin gene (vt2 ), was used to infect Enterobacteriaceae strains. A number ofShigella and E. coli strains were susceptible to lysogenic infection, and a smooth E. coli isolate (O107) was also susceptible to lytic infection. The lysogenized strains included different smooth E. coli serotypes of both human and animal origin, indicating that this bacteriophage has a substantial capacity to disseminate verocytotoxin genes. A novel indirect plaque assay utilizing an E. coli recA441 mutant in which phage-infected cells can enter only the lytic cycle, enabling detection of all infective phage, was developed.

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