scholarly journals KLONING GEN carB Salmonella typhi MENGGUNAKAN VEKTOR EKSPRESI pET-16b dan SEL INANG Escherichia coli JM109

2019 ◽  
Vol 3 (3) ◽  
pp. 57
Author(s):  
Elisawati Wonohadi ◽  
Mariana Wahyudi
Keyword(s):  
Escherichia Coli ◽  
Host Cell ◽  
Recombinant Plasmid ◽  
Expression Vector ◽  
Restriction Enzymes ◽  
Salmonella Typhi ◽  
Luria Bertani ◽  
E Coli ◽  
Escherichia Coli Jm109 ◽  
Large Plasmids

The effort of cloning the Lister strain of Salmonella typhi (NCTC 786, BCC 712) carB gene using pET-16b expression vector and E. coli JM109 as host cell has been done. The carB gene and the pET-16b expression vector were both prepared from their recombinant plasmid digested using BamHI and NdeI as restriction enzymes. The pG-carB-11-ST recombinant plasmid was isolated from Escherichia coli XL10(pG-carB-11-ST) and the pET-carA-ST recombinant plasmid was from Escherichia coli DH5α(pET-carA-ST). After being ligated (in a ratio of vector:gene  of 1:3, 1:5 and 1:6) in the presence of T4 Bacteriofage DNA Ligase, the ligation mixture was used to transform Escherichia coli JM109 cells and plated out onto Luria Bertani medium containing ampicillin. An amount of 369 produced colonies were screened for the presence of the appropriate recombinant plasmid using combination of plasmid miniprep and agarose gel electrophoresis. None of the recombinant plasmids being suspected to carry the Salmonella typhi carB gene. It is suggested  to repeat cloning process using E. coli JM109 or using E. coli XL10 as host cell which were known to have large cloning efficiency and can be used for large plasmids up to 25 kb.

scholarly journals degS Is Necessary for Virulence and Is among Extraintestinal Escherichia coli Genes Induced in Murine Peritonitis

2003 ◽  
Vol 71 (6) ◽  
pp. 3088-3096 ◽  
Author(s):  
Peter Redford ◽  
Paula L. Roesch ◽  
Rodney A. Welch
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract Infection ◽  
Urinary Tract ◽  
Tract Infection ◽  
Luria Bertani ◽  
Broth Culture ◽  
Wild Type ◽  
E Coli ◽  
Virulence Attenuation

ABSTRACT Extraintestinal Escherichia coli strains cause meningitis, sepsis, urinary tract infection, and other infections outside the bowel. We examined here extraintestinal E. coli strain CFT073 by differential fluorescence induction. Pools of CFT073 clones carrying a CFT073 genomic fragment library in a promoterless gfp vector were inoculated intraperitoneally into mice; bacteria were recovered by lavage 6 h later and then subjected to fluorescence-activated cell sorting. Eleven promoters were found to be active in the mouse but not in Luria-Bertani (LB) broth culture. Three are linked to genes for enterobactin, aerobactin, and yersiniabactin. Three others are linked to the metabolic genes metA, gltB, and sucA, and another was linked to iha, a possible adhesin. Three lie before open reading frames of unknown function. One promoter is associated with degS, an inner membrane protease. Mutants of the in vivo-induced loci were tested in competition with the wild type in mouse peritonitis. Of the mutants tested, only CFT073 degS was found to be attenuated in peritoneal and in urinary tract infection, with virulence restored by complementation. CFT073 degS shows growth similar to that of the wild type at 37°C but is impaired at 43°C or in 3% ethanol LB broth at 37°C. Compared to the wild type, the mutant shows similar serum survival, motility, hemolysis, erythrocyte agglutination, and tolerance to oxidative stress. It also has the same lipopolysaccharide appearance on a silver-stained gel. The basis for the virulence attenuation is unclear, but because DegS is needed for σE activity, our findings implicate σE and its regulon in E. coli extraintestinal pathogenesis.

scholarly journals Cloning of Salmonella enterica Serovar Enteritidis Fimbrial Protein SefA as a Surface Protein in Escherichia coli Confers the Ability To Attach to Eukaryotic Cell Lines

2009 ◽  
Vol 75 (20) ◽  
pp. 6622-6625 ◽  
Author(s):  
Douglas L. Rank ◽  
Mahdi A. Saeed ◽  
Peter M. Muriana
Keyword(s):  
Escherichia Coli ◽  
Salmonella Enterica ◽  
Expression Vector ◽  
Surface Protein ◽  
Surface Expression ◽  
Eukaryotic Cell ◽  
Salmonella Enterica Serovar Enteritidis ◽  
Western Blot Assay ◽  
E Coli ◽  
Fimbrial Protein

ABSTRACT The gene for the Salmonella enterica serovar Enteritidis fimbrial protein SefA was cloned into an Escherichia coli surface expression vector and confirmed by Western blot assay. E. coli clones expressing SefA attached to avian ovary granulosa cells and HEp-2 cells, providing evidence for the involvement of SefA in the ability of Salmonella to attach to eukaryotic cells.

A Comparison of Hand Washing Techniques To Remove Escherichia coli and Caliciviruses under Natural or Artificial Fingernails

2003 ◽  
Vol 66 (12) ◽  
pp. 2296-2301 ◽  
Author(s):  
CHIA-MIN LIN ◽  
FONE-MAO WU ◽  
HOI-KYUNG KIM ◽  
MICHAEL P. DOYLE ◽  
BARRY S. MICHAELS ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Tap Water ◽  
Hand Washing ◽  
Microbial Populations ◽  
Liquid Soap ◽  
E Coli ◽  
Hand Sanitizer ◽  
Before And After ◽  
Escherichia Coli Jm109 ◽  
Viral Indicators

Compared with other parts of the hand, the area beneath fingernails harbors the most microorganisms and is most difficult to clean. Artificial fingernails, which are usually long and polished, reportedly harbor higher microbial populations than natural nails. Hence, the efficacy of different hand washing methods for removing microbes from natural and artificial fingernails was evaluated. Strains of nonpathogenic Escherichia coli JM109 and feline calicivirus (FCV) strain F9 were used as bacterial and viral indicators, respectively. Volunteers with artificial or natural nails were artificially contaminated with ground beef containing E. coli JM109 or artificial feces containing FCV. Volunteers washed their hands with tap water, regular liquid soap, antibacterial liquid soap, alcohol-based hand sanitizer gel, regular liquid soap followed by alcohol gel, or regular liquid soap plus a nailbrush. The greatest reduction of inoculated microbial populations was obtained by washing with liquid soap plus a nailbrush, and the least reduction was obtained by rubbing hands with alcohol gel. Lower but not significantly different (P > 0.05) reductions of E. coli and FCV counts were obtained from beneath artificial than from natural fingernails. However, significantly (P ≤ 0.05) higher E. coli and FCV counts were recovered from hands with artificial nails than from natural nails before and after hand washing. In addition, microbial cell numbers were correlated with fingernail length, with greater numbers beneath fingernails with longer nails. These results indicate that best practices for fingernail sanitation of food handlers are to maintain short fingernails and scrub fingernails with soap and a nailbrush when washing hands.

scholarly journals Níveis de sensibilidade de enterobactérias, em particular Salmonella typhi, a rifamicina S.V.

1969 ◽  
Vol 3 (2) ◽  
pp. 87-93
Author(s):  
Ivone R. Suassuna ◽  
I. Suassuna ◽  
C. E. de V. Serpa
Keyword(s):  
Escherichia Coli ◽  
Proteus Mirabilis ◽  
Salmonella Typhi ◽  
E Coli

Em face à predominante eliminação biliar da rifamicina S.V. atingindo concentrações muitas vêzes superiores aos níveis séricos obtidos com as doses terapêuticas, e pelo possível interêsse dessa verificação para o tratamento dos portadores biliares crônicos de Salmonella typhi determinou-se a concentração mínima inibitória de 165 estirpes de enterobactérias, incluindo 77 amostras de S. typhi. Foi verificado que a maioria das cepas de Escherichia coli, Shigella e Proteus mirabilis correspondiam a uma concentração inibitória mínima entre 33 a 65 μg/ml. Entre 65 e 128 μg/ml foram determinadas as concetrações inibitórias mínimas da maioria das outras espécies de Proteus, de Providencia e de Klebsiella. Para Salmonella e Enterobacter o limite mínino de sensibilidade foi, em regra, igual ou superior a 128 μg/ml. Diferenças mais acentuadas de comportamento entre as enterobactérias foram observadas quanto à ação bactericida da rifamicidas S.V. De uma maneira geral, para E. coli e Shigella, as concentrações inibitórias mínimas já referidas. Para as espécies de Proteus e Providencia houve variação maior de comportamento, mas tendência a que o efeito bactericidas fôsse encontrado em concentrações que correspondiam a 4 vêzes as bacteriostáticas para as mesmas espécies. Finalmente, de modo pouco feliz para os propósitos visados, em Salmonella, com a inclusão de S. typhi, não foi atingido um efeito bactericida, com as mais altas concentrações usadas as quais corresponderam em média a 6 vêzes as concentrações bacteriostáticas para esse gênero.

scholarly journals MazF-Mediated Cell Death in Escherichia coli: a Point of No Return

2004 ◽  
Vol 186 (24) ◽  
pp. 8295-8300 ◽  
Author(s):  
Shahar Amitai ◽  
Yussuf Yassin ◽  
Hanna Engelberg-Kulka
Keyword(s):  
Escherichia Coli ◽  
Cell Death ◽  
Programmed Cell Death ◽  
Inhibitory Effect ◽  
Luria Bertani ◽  
E Coli ◽  
Ectopic Overexpression ◽  
Point Of No Return ◽  
The Rich ◽  
Overexpression System

ABSTRACT mazEF is a stress-induced toxin-antitoxin module, located on the chromosome of Escherichia coli, that we have previously described to be responsible for programmed cell death in E. coli. mazF specifies a stable toxin, and mazE specifies a labile antitoxin. Recently, it was reported that inhibition of translation and cell growth by ectopic overexpression of the toxin MazF can be reversed by the action of the antitoxin MazE ectopically overexpressed at a later time. Based on these results, it was suggested that rather than inducing cell death, mazF induces a state of reversible bacteriostasis (K. Pederson, S. K. Christensen, and K. Gerdes, Mol. Microbiol. 45:501-510, 2002). Using a similar ectopic overexpression system, we show here that overexpression of MazE could reverse MazF lethality only over a short window of time. The size of that window depended on the nature of the medium in which MazF was overexpressed. Thus, we found “a point of no return,” which occurred sooner in minimal M9 medium than it did in the rich Luria-Bertani medium. We also describe a state in which the effect of MazF on translation could be separated from its effect on cell death: MazE overproduction could completely reverse the inhibitory effect of MazF on translation, while not affecting the bacteriocidic effect of MazF at all. Our results reported here support our view that the mazEF module mediates cell death and is part of a programmed cell death network.

scholarly journals Cell Death in Escherichia coli dnaE(Ts) Mutants Incubated at a Nonpermissive Temperature Is Prevented by Mutation in the cydA Gene

2004 ◽  
Vol 186 (7) ◽  
pp. 2147-2155 ◽  
Author(s):  
Bernard Strauss ◽  
Kemba Kelly ◽  
Toros Dincman ◽  
Damian Ekiert ◽  
Theresa Biesieda ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Death ◽  
Dna Synthesis ◽  
Dna Content ◽  
Electrophoretic Pattern ◽  
Luria Bertani ◽  
E Coli ◽  
Viable Cells ◽  
Rna And Protein Synthesis ◽  
Transformation Activity

ABSTRACT Cells of the Escherichia coli dnaE(Ts) dnaE74 and dnaE486 mutants die after 4 h of incubation at 40°C in Luria-Bertani medium. Cell death is preceded by elongation, is inhibited by chloramphenicol, tetracycline, or rifampin, and is dependent on cell density. Cells survive at 40°C when they are incubated at a high population density or at a low density in conditioned medium, but they die when the medium is supplemented with glucose and amino acids. Deletion of recA or sulA has no effect. We isolated suppressors which survived for long periods at 40°C but did not form colonies. The suppressors protected against hydroxyurea-induced killing. Sequence and complementation analysis indicated that suppression was due to mutation in the cydA gene. The DNA content of dnaE mutants increased about eightfold in 4 h at 40°C, as did the DNA content of the suppressed strains. The amount of plasmid pBR322 in a dnaE74 strain increased about fourfold, as measured on gels, and the electrophoretic pattern appeared to be normal even though the viability of the parent cells decreased 2 logs. Transformation activity also increased. 4′,6′-Diamidino-2-phenylindole staining demonstrated that there were nucleoids distributed throughout the dnaE filaments formed at 40°C, indicating that there was segregation of the newly formed DNA. We concluded that the DNA synthesized was physiologically competent, particularly since the number of viable cells of the suppressed strain increased during the first few hours of incubation. These observations support the view that E. coli senses the rate of DNA synthesis and inhibits septation when the rate of DNA synthesis falls below a critical level relative to the level of RNA and protein synthesis.

Changes in the permeability of Escherichia coli during parasitization by Bdellovibrio bacteriovorus

1971 ◽  
Vol 17 (5) ◽  
pp. 689-697 ◽  
Author(s):  
S. F. Crothers ◽  
J. Robinson
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Host Cell ◽  
Culture Fluid ◽  
Infection Cycle ◽  
Bdellovibrio Bacteriovorus ◽  
E Coli ◽  
Hepes Buffer ◽  
Per Se ◽  
Ph Range

Bdellovibrio bacteriovorus strain 6-5-S completed a typical infection cycle when incubated with E. coli ML 35 (lac i−z+y−) in 0.025 M Hepes buffer, pH 7.8, supplemented with 0.002 M CaCl2∙2H2O. Growth of this strain of B. bacteriovorus was optimal over the range of pH 7.5–8.5. No growth occurred at pH 6.5. The broad pH range may occur because a buffer per se is not required for growth and multiplication. The parasite failed to multiply in a two-membered culture unsupplemented with Ca2+ or Mg2+.Growth and multiplication of B. bacteriovorus in a two-membered culture were assessed by various parameters, including decrease in absorbance at 520 mμ, and release of materials absorbing at 260 mμ, and at 280 mμ. The onset of lysis of the host cell was accompanied by an increase in the release of materials absorbing at 260 mμ and at 280 mμ. The ratio of the absorbance of these materials at 280 and 260 mμ increased at the same time, from which it may be inferred that probably amino acids or proteins were being released.No β-galactosidase could be detected in the culture fluid of the two-membered culture. The infected E. coli cells were more permeable than uninfected cells to o-nitrophenyl-β-D-galactoside, and to the fluorescent dye 8-aniIino-1-naphthalenesulfonic acid.

scholarly journals Plasmid DNA Supercoiling and Survival in Long-Term Cultures of Escherichia coli: Role of NaCl

2003 ◽  
Vol 185 (17) ◽  
pp. 5324-5327 ◽  
Author(s):  
Annie Conter
Keyword(s):  
Escherichia Coli ◽  
Dna Supercoiling ◽  
Luria Bertani ◽  
Plasmid Pbr322 ◽  
E Coli ◽  
Topological State ◽  
Negative Supercoiling ◽  
The Relationship

ABSTRACT The relationship between the survival of Escherichia coli during long-term starvation in rich medium and the supercoiling of a reporter plasmid (pBR322) has been studied. In aerated continuously shaken cultures, E. coli lost the ability to form colonies earlier in rich NaCl-free Luria-Bertani medium than in NaCl-containing medium, and the negative supercoiling of plasmid pBR322 declined more rapidly in the absence of NaCl. Addition of NaCl at the 24th hour restored both viability and negative supercoiling in proportion to the concentration of added NaCl. Addition of ofloxacin, a quinolone inhibitor of gyrase, abolished rescue by added NaCl in proportion to the ofloxacin added. This observation raises the possibility that cells had the ability to recover plasmid supercoiling even if nutrients were not available and could survive during long-term starvation in a manner linked, at least in part, to the topological state of DNA and gyrase activity.

scholarly journals Plasmid-Encoded asp Operon Confers a Proton Motive Metabolic Cycle Catalyzed by an Aspartate-Alanine Exchange Reaction

2002 ◽  
Vol 184 (11) ◽  
pp. 2906-2913 ◽  
Author(s):  
Keietsu Abe ◽  
Fumito Ohnishi ◽  
Kyoko Yagi ◽  
Tasuku Nakajima ◽  
Takeshi Higuchi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Sequence Analysis ◽  
Amino Acid Sequence ◽  
Expression Vector ◽  
Alcaligenes Faecalis ◽  
E Coli ◽  
Cell Extracts ◽  
Membrane Spanning ◽  
Metabolic Cycle

ABSTRACT Tetragenococcus halophila D10 catalyzes the decarboxylation of l-aspartate with nearly stoichiometric release of l-alanine and CO2. This trait is encoded on a 25-kb plasmid, pD1. We found in this plasmid a putative asp operon consisting of two genes, which we designated aspD and aspT, encoding an l-aspartate-β-decarboxylase (AspD) and an aspartate-alanine antiporter (AspT), respectively, and determined the nucleotide sequences. The sequence analysis revealed that the genes of the asp operon in pD1 were in the following order: promoter → aspD → aspT. The deduced amino acid sequence of AspD showed similarity to the sequences of two known l-aspartate-β-decarboxylases from Pseudomonas dacunhae and Alcaligenes faecalis. Hydropathy analyses suggested that the aspT gene product encodes a hydrophobic protein with multiple membrane-spanning regions. The operon was subcloned into the Escherichia coli expression vector pTrc99A, and the two genes were cotranscribed in the resulting plasmid, pTrcAsp. Expression of the asp operon in E. coli coincided with appearance of the capacity to catalyze the decarboxylation of aspartate to alanine. Histidine-tagged AspD (AspDHis) was also expressed in E. coli and purified from cell extracts. The purified AspDHis clearly exhibited activity of l-aspartate-β-decarboxylase. Recombinant AspT was solubilized from E. coli membranes and reconstituted in proteoliposomes. The reconstituted AspT catalyzed self-exchange of aspartate and electrogenic heterologous exchange of aspartate with alanine. Thus, the asp operon confers a proton motive metabolic cycle consisting of the electrogenic aspartate-alanine antiporter and the aspartate decarboxylase, which keeps intracellular levels of alanine, the countersubstrate for aspartate, high.

scholarly journals A Predicted ABC Transporter, FtsEX, Is Needed for Cell Division in Escherichia coli

2004 ◽  
Vol 186 (3) ◽  
pp. 785-793 ◽  
Author(s):  
Kari L. Schmidt ◽  
Nicholas D. Peterson ◽  
Ryan J. Kustusch ◽  
Mark C. Wissel ◽  
Becky Graham ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Abc Transporter ◽  
Luria Bertani ◽  
Wild Type ◽  
E Coli ◽  
Division Site ◽  
Site Localization ◽  
Free Media ◽  
Mass Increase

ABSTRACT FtsE and FtsX have homology to the ABC transporter superfamily of proteins and appear to be widely conserved among bacteria. Early work implicated FtsEX in cell division in Escherichia coli, but this was subsequently challenged, in part because the division defects in ftsEX mutants are often salt remedial. Strain RG60 has an ftsE::kan null mutation that is polar onto ftsX. RG60 is mildly filamentous when grown in standard Luria-Bertani medium (LB), which contains 1% NaCl, but upon shift to LB with no NaCl growth and division stop. We found that FtsN localizes to potential division sites, albeit poorly, in RG60 grown in LB with 1% NaCl. We also found that in wild-type E. coli both FtsE and FtsX localize to the division site. Localization of FtsX was studied in detail and appeared to require FtsZ, FtsA, and ZipA, but not the downstream division proteins FtsK, FtsQ, FtsL, and FtsI. Consistent with this, in media lacking salt, FtsA and ZipA localized independently of FtsEX, but the downstream proteins did not. Finally, in the absence of salt, cells depleted of FtsEX stopped dividing before any change in growth rate (mass increase) was apparent. We conclude that FtsEX participates directly in the process of cell division and is important for assembly or stability of the septal ring, especially in salt-free media.

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