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 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.

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 Catalytic Activities of the PhoQ Histidine Protein Kinase of Salmonella entericaSerovar Typhimurium

2001 ◽  
Vol 183 (5) ◽  
pp. 1787-1791 ◽  
Author(s):  
Martin Montagne ◽  
Alexandre Martel ◽  
Hervé Le Moual
Keyword(s):  
Escherichia Coli ◽  
Phosphatase Activity ◽  
Divalent Cations ◽  
Histidine Protein Kinase ◽  
Catalytic Activities ◽  
Serovar Typhimurium ◽  
High Concentrations ◽  
Kinetics Of ◽  
Autokinase Activity

ABSTRACT Studies of Escherichia coli membranes that were highly enriched in the Salmonella enterica serovar Typhimurium PhoQ protein showed that the presence of ATP and divalent cations such as Mg2+, Mn2+, Ca2+, or Ba2+ resulted in PhoQ autophosphorylation. However, when Mg2+ or Mn2+was present at concentrations higher than 0.1 mM, the kinetics of PhoQ autophosphorylation were strongly biphasic, with a rapid autophosphorylation phase followed by a slower dephosphorylation phase. A fusion protein lacking the sensory and transmembrane domains retained the autokinase activity but could not be dephosphosphorylated when Mg2+ or Mn2+ was present at high concentrations. The instability of purified [32P]phospho-PhoP in the presence of PhoQ-containing membranes indicated that PhoQ also possesses a phosphatase activity. The PhoQ phosphatase activity was stimulated by increasing the Mg2+ concentration. These data are consistent with a model in which Mg2+ binding to the sensory domain of PhoQ coordinately regulates autokinase and phosphatase activities.

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 Characterization of the Promoter Elements for the Staphylococcal Enterotoxin D Gene

2000 ◽  
Vol 182 (8) ◽  
pp. 2321-2325 ◽  
Author(s):  
Shuping Zhang ◽  
George C. Stewart
Keyword(s):  
Staphylococcus Aureus ◽  
Promoter Activity ◽  
Staphylococcal Enterotoxin ◽  
Deletion Analysis ◽  
Promoter Elements ◽  
Promoter Function ◽  
Dinucleotide Motif ◽  
Pribnow Box ◽  
Transcribed Sequences

ABSTRACT Deletion analysis of the promoter for the Staphylococcus aureus enterotoxin D determinant indicated that a 52-bp sequence, from −34 to +18, was sufficient for sed promoter function and agr regulation. A consensus −10 Pribnow box sequence, a less conserved −35 sequence, and a TG dinucleotide motif were present. Transcribed sequences (+1 to +18) are essential for promoter activity.

scholarly journals Characterization of ten species of mycobacteria by reaction-gas-liquid chromatography

1977 ◽  
Vol 6 (5) ◽  
pp. 469-473
Author(s):  
D K Ohashi ◽  
T J Wade ◽  
R J Mandle
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Liquid Chromatography ◽  
Clinical Laboratory ◽  
Tetramethylammonium Hydroxide ◽  
The Other ◽  
Gas Liquid Chromatography ◽  
Whole Cell ◽  
Chromatographic Characterization ◽  
Liquid Chromatographic

The methanolic tetramethylammonium hydroxide whole-cell lysates of nine species of mycobacteria and the "rhodochrous complex" were examined by gas-liquid chromatography. The gas chromatographic patterns produced 10 characteristic chromatographic groups that corresponded to the 10 species studied. The gas chromatograms of Mycobacterium tuberculosis were very easily distinguished from the other mycobacterial strains by high levels of a component that eluted much later than the other components. The remaining nine species could be distinguished on the basis of characteristic components and by different amounts of components common to more than one species. This study demonstrated that direct gas-liquid chromatographic characterization of M. tuberculosis and other myobacterial species was not only feasible but practical in the clinical laboratory.

scholarly journals Cloning and Characterization of Arylamine N -Acetyltransferase Genes from Mycobacterium smegmatis and Mycobacterium tuberculosis: Increased Expression Results in Isoniazid Resistance

1999 ◽  
Vol 181 (4) ◽  
pp. 1343-1347 ◽  
Author(s):  
Mark Payton ◽  
Roy Auty ◽  
Rupika Delgoda ◽  
Martin Everett ◽  
Edith Sim
Keyword(s):  
Escherichia Coli ◽  
Mycobacterium Tuberculosis ◽  
Mycobacterium Smegmatis ◽  
Antitubercular Drug ◽  
Isoniazid Resistance ◽  
E Coli ◽  
Monospecific Antiserum ◽  
Many Sources ◽  
Eukaryotic Organisms

ABSTRACT Arylamine N-acetyltransferases (NATs) are found in many eukaryotic organisms, including humans, and have previously been identified in the prokaryote Salmonella typhimurium. NATs from many sources acetylate the antitubercular drug isoniazid and so inactivate it. nat genes were cloned fromMycobacterium smegmatis and Mycobacterium tuberculosis, and expressed in Escherichia coli andM. smegmatis. The induced M. smegmatis NAT catalyzes the acetylation of isoniazid. A monospecific antiserum raised against pure NAT from S. typhimurium recognizes NAT fromM. smegmatis and cross-reacts with recombinant NAT fromM. tuberculosis. Overexpression of mycobacterialnat genes in E. coli results in predominantly insoluble recombinant protein; however, with M. smegmatisas the host using the vector pACE-1, NAT proteins from M. tuberculosis and M. smegmatis are soluble. M. smegmatis transformants induced to express the M. tuberculosis nat gene in culture demonstrated a threefold higher resistance to isoniazid. We propose that NAT in mycobacteria could have a role in acetylating, and hence inactivating, isoniazid.

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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