scholarly journals Characterization of the protein gp1 from bacteriophage φIN93

2011 ◽  
Vol 48 (1) ◽  
pp. 47-56 ◽  
Author(s):  
Isao Matsushita ◽  
Hideshi Yanase
Keyword(s):  
Escherichia Coli ◽  
Recombinant Plasmid ◽  
Shuttle Vector ◽  
Thermus Thermophilus ◽  
The Other ◽  
Replication Protein

Characterization of the protein gp1 from bacteriophage φIN93The protein gp1, encoded by theORF1gene in the genome of theThermusphage φIN93, is similar to the replication protein encoded byrepAin theThermus sp.plasmid. To confirm that gp1 functions as a replication protein, we constructed the recombinant plasmid pIA1, by insertingORF1and the kanamycin acetyltransferase (kat) gene into pBluescriptII SK(+), which enabled replication of pIA1 inThermus thermophilusHB27. By contrast, plasmid pIA1-del, which contained only a part ofORF1(the other part deleted usingKpnI), was not replicated. These results show that gp1 functions as a replication protein and that pIA1 can be used as a shuttle vector betweenThermus thermophilusHB27 andEscherichia coli.

scholarly journals Characterization of the Two Mycobacterium tuberculosis recA Promoters

2003 ◽  
Vol 185 (20) ◽  
pp. 6005-6015 ◽  
Author(s):  
Krishna K. Gopaul ◽  
Patricia C. Brooks ◽  
Jean-François Prost ◽  
Elaine O. Davis
Keyword(s):  
Escherichia Coli ◽  
Dna Damage ◽  
Mycobacterium Tuberculosis ◽  
Promoter Activity ◽  
The Other ◽  
Expression Level ◽  
Promoter Elements ◽  
Kinetics Of

ABSTRACT The recA gene of Mycobacterium tuberculosis is unusual in that it is expressed from two promoters, one of which, P1, is DNA damage inducible independently of LexA and RecA, while the other, P2, is regulated by LexA in the classical way (E. O. Davis, B. Springer, K. K. Gopaul, K. G. Papavinasasundaram, P. Sander, and E. C. Böttger, Mol. Microbiol. 46:791-800, 2002). In this study we characterized these two promoters in more detail. Firstly, we localized the promoter elements for each of the promoters, and in so doing we identified a mutation in each promoter which eliminates promoter activity. Interestingly, a motif with similarity to Escherichia coli σ70 −35 elements but located much closer to the −10 element is important for optimal expression of P1, whereas the sequence at the −35 location is not. Secondly, we found that the sequences flanking the promoters can have a profound effect on the expression level directed by each of the promoters. Finally, we examined the contribution of each of the promoters to recA expression and compared their kinetics of induction following DNA damage.

scholarly journals Characterization of the Bacillus subtilis ywtD Gene, Whose Product Is Involved in γ-Polyglutamic Acid Degradation

2003 ◽  
Vol 185 (7) ◽  
pp. 2379-2382 ◽  
Author(s):  
Takao Suzuki ◽  
Yasutaka Tahara
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Glutamic Acid ◽  
Recombinant Plasmid ◽  
Signal Sequence ◽  
High Molecular Mass ◽  
Enzymatic Analysis ◽  
Polyglutamic Acid ◽  
Partial Similarity

ABSTRACT The ywtD gene, which codes for an enzyme that degrades γ-polyglutamic acid (PGA), was cloned from Bacillus subtilis IFO16449. The gene is located immediately downstream of ywsC and ywtABC, a PGA operon involved in PGA biosynthesis, and it showed partial similarity to genes coding for dl-endopeptidase, a peptidoglycan-degrading enzyme. The ywtD gene, from which signal sequence is excised, was inserted into pET15b, and the recombinant plasmid was then transformed into Escherichia coli. Histidine-tagged YwtD was purified from sonicated cells of the transformant. The purified YwtD degraded PGA to yield two hydrolyzed products, a high-molecular-mass product (490 kDa with nearly 100% l-glutamic acid) and an 11-kDa product (with d-glutamic acid and l-glutamic acid in an 80:20 ratio). This finding and results of enzymatic analysis of the two products with carboxypeptidase G suggest that YwtD is a novel enzyme cleaving the γ-glutamyl bond only between d- and l-glutamic acids of PGA, and it may be designated γ-dl-glutamyl hydrolase.

scholarly journals Feedback Inhibition of Chorismate Mutase/Prephenate Dehydrogenase (TyrA) of Escherichia coli: Generation and Characterization of Tyrosine-Insensitive Mutants

2005 ◽  
Vol 71 (11) ◽  
pp. 7224-7228 ◽  
Author(s):  
Tina Lütke-Eversloh ◽  
Gregory Stephanopoulos
Keyword(s):  
Escherichia Coli ◽  
Dna Sequences ◽  
Molecular Level ◽  
Feedback Inhibition ◽  
Feedback Regulation ◽  
The Other ◽  
Chorismate Mutase ◽  
Amino Acid Substitutions ◽  
Prephenate Dehydrogenase

ABSTRACT In order to get insights into the feedback regulation by tyrosine of the Escherichia coli chorismate mutase/prephenate dehydrogenase (CM/PDH), which is encoded by the tyrA gene, feedback-inhibition-resistant (fbr) mutants were generated by error-prone PCR. The tyrA fbr mutants were selected by virtue of their resistance toward m-fluoro-d,l-tyrosine, and seven representatives were characterized on the biochemical as well as on the molecular level. The PDH activities of the purified His6-tagged TyrA proteins exhibited up to 35% of the enzyme activity of TyrAWT, but tyrosine did not inhibit the mutant PDH activities. On the other hand, CM activities of the TyrAfbr mutants were similar to those of the TyrAWT protein. Analyses of the DNA sequences of the tyrA genes revealed that tyrA fbr contained amino acid substitutions either at Tyr263 or at residues 354 to 357, indicating that these two sites are involved in the feedback inhibition by tyrosine.

Construction and characterization of a new shuttle vector, pLES2, capable of functioning in Escherichia coli and Neisseria gonorrhoeae

Gene ◽  
1983 ◽  
Vol 25 (2-3) ◽  
pp. 241-247 ◽  
Author(s):  
Daniel C. Stein ◽  
Lin E. Silver ◽  
Virginia L. Clark ◽  
Frank E. Young
Keyword(s):  
Escherichia Coli ◽  
Neisseria Gonorrhoeae ◽  
Shuttle Vector

scholarly journals Characterization of four plasmids harboured in a Lactobacillus brevis strain encoding a novel bacteriocin, brevicin 925A, and construction of a shuttle vector for lactic acid bacteria and Escherichia coli

Microbiology ◽  
2009 ◽  
Vol 155 (5) ◽  
pp. 1726-1737 ◽  
Author(s):  
Takaomi Wada ◽  
Masafumi Noda ◽  
Fumi Kashiwabara ◽  
Hyung Joon Jeon ◽  
Ayano Shirakawa ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Shuttle Vector ◽  
Lactobacillus Brevis ◽  
Fermented Food ◽  
Lactobacillus Helveticus ◽  
Significant Similarity ◽  
Molecular Sizes

In this study we isolated over 250 lactic acid bacteria (LAB) candidates from fruit, flowers, vegetables and a fermented food to generate an LAB library. One strain, designated 925A, isolated from kimchi (a traditional Korean fermented dish made from Chinese cabbage) produced a novel type of bacteriocin, brevicin 925A, which is effective against certain LAB, including strains of Lactobacillus, Enterococcus, Streptococcus, Bacillus and Listeria. Strain 925A, identified as Lactobacillus brevis, harboured at least four plasmids and we determined the entire nucleotide sequence of each one. The four plasmids were designated pLB925A01–04, and have molecular sizes of 1815, 3524, 8881 and 65 037 bp, respectively. We obtained bacteriocin non-producing derivatives by treatment of strain 925A with novobiocin. All of these derivatives, which were susceptible to their own antibacterial product, lost the largest plasmid, pLB925A04, suggesting that the genes for bacteriocin biosynthesis (breB and breC) and immunity (breE) are located on pLB925A04. The partial amino acid sequence of purified brevicin 925A and sequence analysis of pLB925A04 showed that breB is the structural gene for brevicin 925A. We constructed a shuttle vector (pLES003, 6134 bp) that can replicate in both Escherichia coli and LAB such as Lactobacillus plantarum, Lb. brevis, Lactobacillus helveticus, Lactobacillus hilgardii and Enterococcus hirae. To determine the function of gene breE, which displays no significant similarity to any other sequences in the blast search database, the gene was inserted into pLES003. A pLB925A04-cured derivative transformed with pLES003 carrying breE acquired immunity to brevicin 925A, suggesting that breE encodes an immunity protein.

Characterization of pC7 from Lactobacillus paraplantarum C7 derived from Kimchi and development of lactic acid bacteria–Escherichia coli shuttle vector

Plasmid ◽  
2004 ◽  
Vol 52 (2) ◽  
pp. 84-88 ◽  
Author(s):  
Woo Jung Park ◽  
Kwan Hoon Lee ◽  
Jung Min Lee ◽  
Hyong Joo Lee ◽  
Jeong Hwan Kim ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Shuttle Vector ◽  
Lactobacillus Paraplantarum

Colicinogeny among Escherichia coli Serotypes, Including O157:H7, Representing Four Closely Related Diarrheagenic Clones

1998 ◽  
Vol 61 (11) ◽  
pp. 1431-1438 ◽  
Author(s):  
SHELTON E. MURINDA ◽  
SHU-MIN LIU ◽  
ROBERT F. ROBERTS ◽  
RICHARD A. WILSON
Keyword(s):  
Escherichia Coli ◽  
Toxin Production ◽  
Coli Strain ◽  
The Other ◽  
E Coli ◽  
Diarrheagenic Escherichia Coli ◽  
Reference Set ◽  
Almost All ◽  
Luria Agar

Twenty-seven diarrheagenic Escherichia coli (DEC) strains from five closely related, genetically distinct clones (DEC 3, 4, 8, 9, and 10), representing serotypes commonly associated with Shiga-like toxin production, i.e., 015:H−, 026:(H11, H−), 0111:(H8, H11, H−), and O157:H7, were evaluated for colicinogeny on Luria agar or Luria agar containing 0.25 μg/ml mitomycin C to induce colicin production. Ten (37%) of the DEC strains tested were colicinogenic. One of 11 serotype O157:H7 strains, DEC strain 4E, produced a colicin identified as Col D. DEC strains 8B, 9D, and 10B produced Col E1, whereas DEC strain 10A produced Col E2. DEC strains 8A, 8E, 10C, 10E, and 10F produced “untypable” colicins that killed almost all Pugsley Colicin Reference Set strains and the other DEC strains tested. To aid with further characterization of the colicins, plasmids extracted from each colicin-producing (Col+) DEC strain were used to transform E. coli strain DH5α. All Col+ DH5α transformants contained one plasmid ranging in size from 1.3 to 10 kb. Some transformants were stable colicin producers whereas others were unstable. The inhibitory activity and colicin sensitivity and insensitivity profiles of the Col+ transformants were similar to those of the corresponding Col+ donor DEC strains. It appears that the untypable colicins are novel and, thus, warrant further study. Colicin production by some of the DEC strains evaluated partly explains why they were insensitive to standard colicins in a previous study.

scholarly journals Cloning, Expression, and Characterization of the Superantigen Streptococcal Pyrogenic Exotoxin G from Streptococcus dysgalactiae

2007 ◽  
Vol 75 (4) ◽  
pp. 1721-1729 ◽  
Author(s):  
Jizi Zhao ◽  
Tomohito Hayashi ◽  
Susanna Saarinen ◽  
Anastassios C. Papageorgiou ◽  
Hidehito Kato ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Mononuclear Cells ◽  
The Other ◽  
Human Pbmcs ◽  
Streptococcus Dysgalactiae ◽  
Pathogenic Role ◽  
Peripheral Blood Mononuclear ◽  
Novel Variants ◽  
Blood Mononuclear Cells

ABSTRACT We identified seven novel variants of streptococcal pyrogenic exotoxin G (SPEGG), a superantigen, in Streptococcus dysgalactiae subsp. dysgalactiae or equisimilis isolates from clinical cases of infection in humans and animals. Phylogenetic analysis of the SPEGG variants indicated two clades in the dendrogram: one composed of variants derived from the bacteria isolated from the humans and the other composed of variants from the bacteria isolated from the animals. Bovine peripheral blood mononuclear cells (PBMCs) were stimulated effectively by recombinant SPEGGs (rSPEGGs) expressed in Escherichia coli, while human PBMCs were not stimulated well by any of the rSPEGGs tested. SPEGGs selectively stimulated bovine T cells bearing Vβ1,10 and Vβ4. Bovine serum showed reactivity to the rSPEGG proteins. These results indicated that SPEGGs have properties as superantigens, and it is likely that SPEGGs play a pathogenic role in animals.

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