METHYL ACTIVATION AND sRNA METHYLATION BY SOLUBLE FRACTIONS OF SEVERAL VERTEBRATE LIVERS

1967 ◽  
Vol 45 (10) ◽  
pp. 1513-1522 ◽  
Author(s):  
R. L. Hancock
Keyword(s):  
Mouse Liver ◽  
Specific Interaction ◽  
Adult Rabbit ◽  
X Chromosomes ◽  
E Coli ◽  
Lower Vertebrates ◽  
The Difference ◽  
Species Specific ◽  
Methylase Activity

The amount of liver ATP: L-methionine S-adenosyltransferase activity varies widely in vertebrates from trace amounts in certain lower vertebrates, e.g. shark or turtle, to relatively large quantities in mammals, e.g. mouse or rabbit. The liver from male mice of a particular stock had only two-thirds the activity of female mouse liver. This difference is not explained by the difference between the sexes in number of X-chromosomes, since female mice with only one X-chromosome (X/0) had the same levels of liver S-adenosyltransferase activity as normal females with two X-chromosomes.sRNA methylase activity from mouse, shark, and rabbit liver supernatant fractions were studied with mouse, shark, and Escherichia coli sRNA. Highspeed supernatant fractions from both rabbit embryonic liver and mouse hepatoma hypermethylated E. coli sRNA which had been previously methylated by adult rabbit or mouse liver supernatant fractions. Hepatoma methylase(s) acts in an analogous manner to that of a species-specific interaction with heterologous and homologous sRNA.A calculation is made comparing the amount of sRNA methylase activity detected in vitro with mouse liver sRNA, with one which is theoretically derived; similar values are obtained. The variation found in vertebrate liver with respect to methyl activation and sRNA methylation is discussed.

scholarly journals The Production of the Recombinant Protein of Anthrax Bacteriolysine PlyPH and In Vitro Study of the Lytic Properties of the Protein Coded for them Against Bacillus anhracis Microbial Cells

2019 ◽  
pp. 5-22
Author(s):  
Onuchina N.V., Soybanov V.D.
Keyword(s):  
In Vitro Study ◽  
Coli Strain ◽  
Vitro Study ◽  
Chromosomal Dna ◽  
Lytic Enzymes ◽  
Microbial Cells ◽  
Phage Gene ◽  
E Coli ◽  
Species Specific

The causative agent of anthrax - Bacillus anthracis, due to the prevalence of its natural foci in Russia, high virulence for humans and most mammals, the unique resistance of spore forms to environmental factors and repeated use in terrorist acts, is an extremely dangerous biological agent. Therefore, the search for new effective drugs for the diagnosis and treatment of anthrax, including diseases caused by antibiotic-resistant strains of B. anthracis is necessary. The use of lytic enzymes of species-specific bacteriophages is a new trend in the diagnosis, prevention and treatment of infectious diseases. The goal of this work is the cloning of the anthrax bacteriolysin PlyPH gene as part of the pTrcHis2C vector in Escherichia coli and the in vitro study of the lytic properties of the protein encoded by it against B. anthracis microbial cells. According to the complete sequencing of the B. anthracis genomes of the Ames, Stern 34F2 and JB17 strains, a prophage was found in their chromosomal DNA, which lost part of the structural genes necessary for its replication, but retained a gene with a high degree of homology with the bacteriolysin γ phage gene. For amplification and subsequent cloning of the PlyPH gene, we developed primers containing EcoRI and BamHI restriction enzyme recognition sites. Amplification of the PlyPH gene in a polymerase chain reaction (PCR) with a developed pair of primers was performed using the Stern 34F2 strain of the anthrax microbe as a template. Based on the obtained amplification products and the pTrcHis2C vector, we constructed a recombinant plasmid containing the bacteriolysin synthesis PlyPH gene and stably functioning in the cells of the recombinant E. coli strain. In the course of research, it has been established that microbial cells of the E. coli recombinant TOP10 strain provide for the production of the bacteriolysin of the anthrax prophage, PlyPH , which has the ability to in vitro lyse the vegetative cells of the STI-1 vaccine strain of B. anthracis

scholarly journals Domain II plays a crucial role in the function of ribosome recycling factor

2006 ◽  
Vol 393 (3) ◽  
pp. 767-777 ◽  
Author(s):  
Peng Guo ◽  
Liqiang Zhang ◽  
Hongjie Zhang ◽  
Yanming Feng ◽  
Guozhong Jing
Keyword(s):  
Crucial Role ◽  
Specific Interaction ◽  
Elongation Factor ◽  
Wild Type ◽  
Ribosome Recycling Factor ◽  
E Coli ◽  
Thermoanaerobacter Tengcongensis ◽  
The Individual

RRF (ribosome recycling factor) consists of two domains, and in concert with EF-G (elongation factor-G), triggers dissociation of the post-termination ribosomal complex. However, the function of the individual domains of RRF remains unclear. To clarify this, two RRF chimaeras, EcoDI/TteDII and TteDI/EcoDII, were created by domain swaps between the proteins from Escherichia coli and Thermoanaerobacter tengcongensis. The ribosome recycling activity of the RRF chimaeras was compared with their wild-type RRFs by using in vivo and in vitro activity assays. Like wild-type TteRRF (T. tengcongensis RRF), the EcoDI/TteDII chimaera is non-functional in E. coli, but both wild-type TteRRF, and EcoDI/TteDII can be activated by coexpression of T. tengcongensis EF-G in E. coli. By contrast, like wild-type E. coli RRF (EcoRRF), TteDI/EcoDII is fully functional in E. coli. These findings suggest that domain II of RRF plays a crucial role in the concerted action of RRF and EF-G for the post-termination complex disassembly, and the specific interaction between RRF and EF-G on ribosomes mainly depends on the interaction between domain II of RRF and EF-G. This study provides direct genetic and biochemical evidence for the function of the individual domains of RRF.

scholarly journals In Vivo Expression of the β-Glucoside (bgl) Operon of Escherichia coli Occurs in Mouse Liver

1998 ◽  
Vol 180 (17) ◽  
pp. 4746-4749 ◽  
Author(s):  
M. Ayub Khan ◽  
Richard E. Isaacson
Keyword(s):  
Escherichia Coli ◽  
Infected Mouse ◽  
Mouse Liver ◽  
Transcription Terminator ◽  
E Coli ◽  
Reverse Transcription Pcr ◽  
Bacterial Clone ◽  
Chloramphenicol Acetyltransferase Gene

ABSTRACT An Escherichia coli DNA fragment was identified that contained part of the β-glucoside (bgl) operon. This fragment was identified because it contained a promoter that was responsible for the expression of a reporter gene, the chloramphenicol acetyltransferase gene, in a mouse liver during bacterial infection but not when a bacterial clone was grown in vitro. This fragment contained a promoter and a rho-independent transcription terminator which were flanked by the 3′ end of bglG and the 5′ end ofbglF. Reverse transcription-PCR confirmed thatcat-specific mRNA was produced in infected mouse liver but not in vitro. mRNA encoding the positive regulator of thebgl operon, bglG, also was detected in mouse liver infected with an E. coli strain. These results demonstrated that expression of the bgl operon occurs in infected mouse liver and suggests a unique role for this operon in vivo.

Use of the Direct Viable Count (D.V.C.) For the Assessment of Survival of E. Coli in Marine Environments

1993 ◽  
Vol 27 (3-4) ◽  
pp. 395-399 ◽  
Author(s):  
E. Dupray ◽  
M. Pommepuy ◽  
A. Derrien ◽  
M. P. Caprais ◽  
M. Cormier
Keyword(s):  
Sea Water ◽  
Light Stress ◽  
Viable Count ◽  
Enterotoxigenic Escherichia Coli ◽  
Culturable Bacteria ◽  
E Coli ◽  
Viable Bacteria ◽  
The Difference ◽  
Total Bacteria

The survival of the enterotoxigenic Escherichia coli H 10407 was observed in different marine seawaters, both in situ and in vitro by use of dialysis chambers and microcosms, exposed and not exposed to light. Survival was monitored during several days by measurement of plate counts (culturable bacteria), direct viable counts with yeast extract and nalidixic acid (DVC : viable bacteria), and acridine orange direct counts (total bacteria). Without light, culturable E. coli counts decreased slowly (1.5 log unit), whereas viable and total bacteria counts remained almost the same through 8 days. When light stress emphasized the sea-water stress, the difference between culturable and viable counts increased from the very first two days (4 log units). In all the experiments, the number of viable bacteria remained very high. On the other hand, one experiment using dialysis chambers held in a turbid and brackish estuarine water showed no marked decrease in the initial counts. This better survival was probably due to the presence of organic matter. So culturability and viability of E. coli can change according to its environment. And because of the consequences for sanitary monitoring of seawaters, it must be kept in mind that viability of E. coli can be preserved within several days, in drastic conditions.

scholarly journals Microcin J25 Uptake: His5 of the MccJ25 Lariat Ring Is Involved in Interaction with the Inner Membrane MccJ25 Transporter Protein SbmA

2006 ◽  
Vol 188 (9) ◽  
pp. 3324-3328 ◽  
Author(s):  
Ricardo E. de Cristóbal ◽  
Jose O. Solbiati ◽  
Ana M. Zenoff ◽  
Paula A. Vincent ◽  
Raul A. Salomón ◽  
...  
Keyword(s):  
Specific Interaction ◽  
Cellular Growth ◽  
Amino Acid Residues ◽  
Inner Membrane ◽  
E Coli ◽  
Transporter Protein ◽  
Intracellular Target ◽  
Dependent Transport ◽  
Microcin J25

ABSTRACT Escherichia coli microcin J25 (MccJ25) is a plasmid-encoded antibiotic peptide consisting of 21 l-amino acid residues (G1-G-A-G-H5-V-P-E-Y-F10-V-G-I-G-T15-P-I-S-F-Y20-G). E. coli RNA polymerase (RNAP) is the intracellular target of MccJ25. MccJ25 enters cells after binding to specific membrane transporters: FhuA in the outer membrane and SbmA in the inner membrane. Here, we studied MccJ25 mutants carrying a substitution of His5 by Lys, Arg, or Ala. The inhibitory effects on cellular growth and in vitro RNAP activity were determined for each mutant microcin. The results show that all mutants inhibited RNAP in vitro. However, the mutants were defective in their ability to inhibit cellular growth. Experiments in which the FhuA protein was bypassed showed that substitutions of MccJ25 His5 affected the SbmA-dependent transport. Our results thus suggest that MccJ25 His5 located in the lariat ring is involved, directly or indirectly, in specific interaction with SbmA and is not required for MccJ25 inhibition of RNAP.

scholarly journals Interaction of Bacillus subtilis CsaA with SecA and precursor proteins

2000 ◽  
Vol 348 (2) ◽  
pp. 367-373 ◽  
Author(s):  
Jörg P. MÜLLER ◽  
Jörg OZEGOWSKI ◽  
Stefan VETTERMANN ◽  
Jelto SWAVING ◽  
Karel H. M. VAN WELY ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Specific Interaction ◽  
Membrane Vesicles ◽  
Protein Export ◽  
E Coli ◽  
Bacterium Bacillus Subtilis ◽  
Bacterium Bacillus

CsaA from the Gram-positive bacterium Bacillus subtilis has been identified previously as a suppressor of the growth and protein-export defect of Escherichia coli secA(Ts) mutants. CsaA has chaperone-like activities in vivo and in vitro. To examine the role of CsaA in protein export in B. subtilis, expression of the csaA gene was repressed. While export of most proteins remained unaffected, export of at least two proteins was significantly reduced upon CsaA depletion. CsaA co-immunoprecipitates and co-purifies with the SecA proteins of E. coli and B. subtilis, and binds the B. subtilis preprotein prePhoB. Purified CsaA stimulates the translocation of prePhoB into E. coli membrane vesicles bearing the B. subtilis translocase, whereas it interferes with the SecB-mediated translocation of proOmpA into membrane vesicles of E. coli. The specific interaction with the SecA translocation ATPase and preproteins suggests that CsaA acts as a chaperone that promotes the export of a subset of preproteins in B. subtilis.

scholarly journals Diversity and prevalence of colibactin- and yersiniabactin encoding mobile genetic elements in enterobacterial populations: insights into evolution and co-existence of two bacterial secondary metabolite determinants

2021 ◽  
Author(s):  
Haleluya Wami ◽  
Alexander Wallenstein ◽  
Daniel Sauer ◽  
Monika Stoll ◽  
Rudolf von Bünau ◽  
...  
Keyword(s):  
Structural Diversity ◽  
Eukaryotic Cell ◽  
Genomic Region ◽  
Dna Breaks ◽  
E Coli ◽  
Integrative And Conjugative Elements ◽  
Double Stranded Dna ◽  
Bacterial Genetic ◽  
Species Specific

1 AbstractThe bacterial genotoxin colibactin interferes with the eukaryotic cell cycle by causing double-stranded DNA breaks. It has been linked to bacterially induced colorectal cancer in humans. Colibactin is encoded by a 54-kb genomic region in Enterobacteriaceae. The colibactin genes commonly co-occur with the yersiniabactin biosynthetic determinant. Investigating the prevalence and sequence diversity of the colibactin determinant and its linkage to the yersiniabactin operon in prokaryotic genomes, we discovered mainly species-specific lineages of the colibactin determinant and classified three main structural settings of the colibactin-yersiniabactin genomic region in Enterobacteriaceae. The colibactin gene cluster has a similar but not identical evolutionary track to that of the yersiniabactin operon. Both determinants could have been acquired on several occasions and/or exchanged independently between enterobacteria by horizontal gene transfer. Integrative and conjugative elements play(ed) a central role in the evolution and structural diversity of the colibactin-yersiniabactin genomic region. Addition of an activating and regulating module (clbAR) to the biosynthesis and transport module (clbB-S) represents the most recent step in the evolution of the colibactin determinant. In a first attempt to correlate colibactin expression with individual lineages of colibactin determinants and different bacterial genetic backgrounds, we compared colibactin expression of selected enterobacterial isolates in vitro. Colibactin production in the tested Klebsiella spp. and Citrobacter koseri strains was more homogeneous and generally higher than that in most of the E. coli isolates studied. Our results improve the understanding of the diversity of colibactin determinants and its expression level, and may contribute to risk assessment of colibactin-producing enterobacteria.

scholarly journals Secretory IgA N-glycans contribute to the protection against E. coli O55 infection of germ-free piglets

2020 ◽  
Author(s):  
Leona Raskova Kafkova ◽  
Diana Brokesova ◽  
Michal Krupka ◽  
Zuzana Stehlikova ◽  
Jiri Dvorak ◽  
...  
Keyword(s):  
Clinical Status ◽  
Secretory Component ◽  
Secretory Iga ◽  
Intestinal Histology ◽  
E Coli ◽  
Germ Free ◽  
Mucosal Surfaces ◽  
Species Specific

Abstract Mucosal surfaces are colonized by highly diverse commensal microbiota. Coating with secretory IgA (SIgA) promotes the survival of commensal bacteria while it inhibits the invasion by pathogens. Bacterial coating could be mediated by antigen-specific SIgA recognition, polyreactivity, and/or by the SIgA-associated glycans. In contrast to many in vitro studies, only a few reported the effect of SIgA glycans in vivo. Here, we used a germ-free antibody-free newborn piglets model to compare the protective effect of SIgA, SIgA with enzymatically removed N-glycans, Fab, and Fc containing the secretory component (Fc-SC) during oral necrotoxigenic E. coli O55 challenge. SIgA, Fab, and Fc-SC were protective, whereas removal of N-glycans from SIgA reduced SIgA-mediated protection as demonstrated by piglets’ intestinal histology, clinical status, and survival. In vitro analyses indicated that deglycosylation of SIgA did not reduce agglutination of E. coli O55. These findings highlight the role of SIgA-associated N-glycans in protection. Further structural studies of SIgA-associated glycans would lead to the identification of those involved in the species-specific inhibition of attachment to corresponding epithelial cells.

scholarly journals RNA Polymerases from Bacillus subtilisand Escherichia coli Differ in Recognition of Regulatory Signals In Vitro

2000 ◽  
Vol 182 (21) ◽  
pp. 6027-6035 ◽  
Author(s):  
Irina Artsimovitch ◽  
Vladimir Svetlov ◽  
Larry Anthony ◽  
Richard R. Burgess ◽  
Robert Landick
Keyword(s):  
Escherichia Coli ◽  
Bacterial Species ◽  
In Vitro Transcription ◽  
Rna Polymerases ◽  
Bacterial Cells ◽  
Signal Recognition ◽  
E Coli ◽  
Promoter Recognition ◽  
Species Specific

ABSTRACT Adaptation of bacterial cells to diverse habitats relies on the ability of RNA polymerase to respond to various regulatory signals. Some of these signals are conserved throughout evolution, whereas others are species specific. In this study we present a comprehensive comparative analysis of RNA polymerases from two distantly related bacterial species, Escherichia coli and Bacillus subtilis, using a panel of in vitro transcription assays. We found substantial species-specific differences in the ability of these enzymes to escape from the promoter and to recognize certain types of elongation signals. Both enzymes responded similarly to other pause and termination signals and to the general E. coli elongation factors NusA and GreA. We also demonstrate that, although promoter recognition depends largely on the ς subunit, promoter discrimination exhibited in species-specific fashion by both RNA polymerases resides in the core enzyme. We hypothesize that differences in signal recognition are due to the changes in contacts made between the β and β′ subunits and the downstream DNA duplex.

scholarly journals Characterization of FimY as a Coactivator of Type 1 Fimbrial Expression in Salmonella enterica Serovar Typhimurium

2000 ◽  
Vol 68 (6) ◽  
pp. 3305-3313 ◽  
Author(s):  
Juliette K. Tinker ◽  
Steven Clegg
Keyword(s):  
Dna Binding ◽  
Salmonella Enterica ◽  
Specific Interaction ◽  
Binding Assays ◽  
Reporter Construct ◽  
E Coli ◽  
Fimbrial Subunit ◽  
Serovar Typhimurium

ABSTRACT Type 1 fimbriae of Salmonella enterica serovar Typhimurium are surface appendages that carry adhesins specific for mannosylated host glycoconjugates. Regulation of the major fimbrial subunit is thought to be controlled by a number of ancillaryfim genes, including fimZ, fimY,fimW, and fimU. Previous studies using a FimZ mutant have indicated that this protein is necessary forfimA expression, and in vitro DNA binding assays determined that FimZ is a transcriptional activator that binds directly to thefimA promoter. To determine the role of FimY as a potential regulator of fimbrial expression, a fimY mutant of serovar Typhimurium was generated by allelic exchange. This mutant was found to be phenotypically nonfimbriate. No transcription from thefimA promoter was detected in a fimY mutant containing a fimA-lacZ reporter construct located on the chromosome. In addition, transcription from the cloned fimYpromoter was not detected in Escherichia coli unless both FimZ and FimY were present, indicating that these proteins also act as coactivators of fimY expression. Consistent with these results, there is no transcription from a fimY-lacZreporter construct within a serovar Typhimurium fimY orfimZ mutant. Studies using the fimY-lacZconstruct reveal that expression of this gene varies with environmental conditions in a manner similar to fimA expression. Extensive in vitro DNA binding assays using extracts from E. coli that overexpress FimY, as well as partially purified FimY, were unable to identify a specific interaction between FimY and thefimA or fimY promoter. The results indicate that FimY is a positive regulator of fimbrial expression and that this protein acts in cooperation with FimZ to regulate the expression ofSalmonella type 1 fimbrial appendages.

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