Phosphorylierung von Methylthio-β.ᴅ-galaktosid in E. coli

1968 ◽  
Vol 23 (9) ◽  
pp. 1219-1221 ◽  
Author(s):  
E. Lodemann ◽  
S. Iskrić ◽  
C. Altaner ◽  
A. Wacker
Keyword(s):  
E Coli ◽  
K 12

Methylthio-β.ᴅ-galaktosid wird in E. coli K 12, sowie in den Mutanten ML 3 und ML 308 in vivo zu einem geringen Teil in einen Phosphorsäureester, wahrscheinlich das 6-Phosphat (TMG-P) umgewandelt. TMG-P wird von E. coli K 12 aufgenommen, wirkt jedoch nicht als Induktor des Lactose-Operons. Zellfreie Extrakte aus E. coli K 12 geben die gleiche Reaktion, wobei die in vitro-Reaktion durch anorganisches Phosphat und Phosphoenolpyruvat stimuliert wird.

scholarly journals Regulatory Role of PlaR (YiaJ) for Plant Utilization in Escherichia coli K-12

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Tomohiro Shimada ◽  
Yui Yokoyama ◽  
Takumi Anzai ◽  
Kaneyoshi Yamamoto ◽  
Akira Ishihama
Keyword(s):  
Escherichia Coli ◽  
Genetic System ◽  
Regulatory Role ◽  
E Coli ◽  
Warm Blooded Animal ◽  
Plant Utilization ◽  
K 12

AbstractOutside a warm-blooded animal host, the enterobacterium Escherichia coli K-12 is also able to grow and survive in stressful nature. The major organic substance in nature is plant, but the genetic system of E. coli how to utilize plant-derived materials as nutrients is poorly understood. Here we describe the set of regulatory targets for uncharacterized IclR-family transcription factor YiaJ on the E. coli genome, using gSELEX screening system. Among a total of 18 high-affinity binding targets of YiaJ, the major regulatory target was identified to be the yiaLMNOPQRS operon for utilization of ascorbate from fruits and galacturonate from plant pectin. The targets of YiaJ also include the genes involved in the utilization for other plant-derived materials as nutrients such as fructose, sorbitol, glycerol and fructoselysine. Detailed in vitro and in vivo analyses suggest that L-ascorbate and α-D-galacturonate are the effector ligands for regulation of YiaJ function. These findings altogether indicate that YiaJ plays a major regulatory role in expression of a set of the genes for the utilization of plant-derived materials as nutrients for survival. PlaR was also suggested to play protecting roles of E. coli under stressful environments in nature, including the formation of biofilm. We then propose renaming YiaJ to PlaR (regulator of plant utilization).

scholarly journals Adaptation in a Mouse Colony Monoassociated with Escherichia coli K-12 for More than 1,000 Days

2010 ◽  
Vol 76 (14) ◽  
pp. 4655-4663 ◽  
Author(s):  
Sean M. Lee ◽  
Aaron Wyse ◽  
Aaron Lesher ◽  
Mary Lou Everett ◽  
Linda Lou ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Growth Rates ◽  
Bacterial Species ◽  
Intestinal Flora ◽  
Average Density ◽  
Host Bacterium ◽  
E Coli ◽  
K 12

ABSTRACT Although mice associated with a single bacterial species have been used to provide a simple model for analysis of host-bacteria relationships, bacteria have been shown to display adaptability when grown in a variety of novel environments. In this study, changes associated with the host-bacterium relationship in mice monoassociated with Escherichia coli K-12 over a period of 1,031 days were evaluated. After 80 days, phenotypic diversification of E. coli was observed, with the colonizing bacteria having a broader distribution of growth rates in the laboratory than the parent E. coli. After 1,031 days, which included three generations of mice and an estimated 20,000 generations of E. coli, the initially homogeneous bacteria colonizing the mice had evolved to have widely different growth rates on agar, a potential decrease in tendency for spontaneous lysis in vivo, and an increased tendency for spontaneous lysis in vitro. Importantly, mice at the end of the experiment were colonized at an average density of bacteria that was more than 3-fold greater than mice colonized on day 80. Evaluation of selected isolates on day 1,031 revealed unique restriction endonuclease patterns and differences between isolates in expression of more than 10% of the proteins identified by two-dimensional electrophoresis, suggesting complex changes underlying the evolution of diversity during the experiment. These results suggest that monoassociated mice might be used as a tool for characterizing niches occupied by the intestinal flora and potentially as a method of targeting the evolution of bacteria for applications in biotechnology.

scholarly journals Single-Target Regulators Constitute the Minority Group of Transcription Factors in Escherichia coli K-12

2021 ◽  
Vol 12 ◽  
Author(s):  
Tomohiro Shimada ◽  
Hiroshi Ogasawara ◽  
Ikki Kobayashi ◽  
Naoki Kobayashi ◽  
Akira Ishihama
Keyword(s):  
Escherichia Coli ◽  
Transcription Factors ◽  
Binding Sites ◽  
Target Genes ◽  
Minority Group ◽  
E Coli ◽  
Single Target ◽  
K 12

The identification of regulatory targets of all transcription factors (TFs) is critical for understanding the entire network of genome regulation. A total of approximately 300 TFs exist in the model prokaryote Escherichia coli K-12, but the identification of whole sets of their direct targets is impossible with use of in vivo approaches. For this end, the most direct and quick approach is to identify the TF-binding sites in vitro on the genome. We then developed and utilized the gSELEX screening system in vitro for identification of more than 150 E. coli TF-binding sites along the E. coli genome. Based on the number of predicted regulatory targets, we classified E. coli K-12 TFs into four groups, altogether forming a hierarchy ranging from a single-target TF (ST-TF) to local TFs, global TFs, and nucleoid-associated TFs controlling as many as 1,000 targets. Using the collection of purified TFs and a library of genome DNA segments from a single and the same E. coli K-12, we identified here a total of 11 novel ST-TFs, CsqR, CusR, HprR, NorR, PepA, PutA, QseA, RspR, UvrY, ZraR, and YqhC. The regulation of single-target promoters was analyzed in details for the hitherto uncharacterized QseA and RspR. In most cases, the ST-TF gene and its regulatory target genes are adjacently located on the E. coli K-12 genome, implying their simultaneous transfer in the course of genome evolution. The newly identified 11 ST-TFs and the total of 13 hitherto identified altogether constitute the minority group of TFs in E. coli K-12.

scholarly journals Characterization of Stg Fimbriae from an Avian Pathogenic Escherichia coli O78:K80 Strain and Assessment of Their Contribution to Colonization of the Chicken Respiratory Tract

2006 ◽  
Vol 188 (18) ◽  
pp. 6449-6459 ◽  
Author(s):  
Maria H. Lymberopoulos ◽  
Sébastien Houle ◽  
France Daigle ◽  
Simon Léveillé ◽  
Annie Brée ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
Distinct Group ◽  
Immunogold Electron Microscopy ◽  
Carbohydrate Source ◽  
E Coli ◽  
Tract Infections ◽  
K 12

ABSTRACT In a previous study, ecs-3, a sequence from avian pathogenic Escherichia coli (APEC) O78:K80 strain χ7122, was found to be expressed in vivo in infected chicken tissues. The region encompassing ecs-3 carries a fimbrial gene cluster that is a putative ortholog of the stg fimbrial gene cluster of Salmonella enterica serovar Typhi. This APEC fimbrial gene cluster, which we have termed stg, is a member of a distinct group of related fimbriae that are located in the glmS-pstS intergenic region of certain E. coli and S. enterica strains. Under the control of the pBAD promoter, the production of Stg fimbriae was demonstrated by Western blotting and immunogold electron microscopy with E. coli K-12. Transcriptional fusions suggest that stg expression is influenced by the carbohydrate source and decreased by the addition of iron and that Fur plays a role in the regulation of stg expression. stg sequences were associated with APEC O78 isolates, and stg was phylogenetically distributed among E. coli reference strains and clinical isolates from human urinary tract infections. Stg fimbriae contributed to the adherence of a nonfimbriated E. coli K-12 strain to avian lung sections and human epithelial cells in vitro. Coinfection experiments with APEC strain χ7122 and an isogenic Δstg mutant demonstrated that compared to the wild-type parent, the Δstg mutant was less able to colonize air sacs, equally able to colonize lungs, and able to more effectively colonize tracheas of infected chickens. Stg fimbriae, together with other adhesins, may therefore contribute to the colonization of avian respiratory tissues by certain APEC strains.

STUDIES ON MECHANISM OF REPAIR OF ULTRAVIOLET-IRRADIATED VIRAL AND BACTERIAL DNA IN VIVO AND IN VITRO

1965 ◽  
Vol 43 (7) ◽  
pp. 1207-1219 ◽  
Author(s):  
Emanuel Riklis
Keyword(s):  
Uv Irradiation ◽  
Soluble Fraction ◽  
Cell Extract ◽  
Host Cells ◽  
Bacterial Dna ◽  
E Coli ◽  
Thymine Dimers ◽  
K 12

The formation of thymine dimers [Formula: see text] from adjacent intrastrand thymines by ultraviolet (UV) irradiation in DNA was studied under different conditions. When thymine-2-C14 DNA was exposed in quartz tubes to 0.5 × 106 ergs/mm2 ultraviolet irradiation, two photoproducts were formed: "a" + [Formula: see text]. The concentration formed in dry DNA was only about [Formula: see text] of that formed in wet DNA.The survival of T1 phage in the dark in the resistant (uvr+) and the sensitive (uvr−) mutants of E. coli K-12 after UV irradiation of the phage in the wet and dry state is markedly dependent on the state of the phage during irradiation. Survival of T1 phage, when UV irradiated dry, was the same in the sensitive as in the resistant host cells, over a wide range of UV doses, while a marked difference in sensitivity existed when it was UV irradiated wet. Similar survival was obtained also by photoreactivation. These results correspond with the notion that thymine dimers are involved both in photoreactivation and in dark (host cell) reactivation.Thymine-requiring E. coli K-12 cells were mutated to a radioresistant strain uvr+ (AB 2416) and a radiosensitive strain uvr− (AB 2419).Irradiation of cells with a dose of 1000 ergs/mm2, followed by incubation of the cells in the dark in enriched M9 media, stopped by addition of 5% trichloroacetic acid, hydrolysis of the acid-insoluble fraction and the acid-soluble fraction in trifluoroacetic acid at 175 °C, and separation of the products by paper chromatography, showed that the two irradiation products "a" + [Formula: see text], which were formed in the bacterial DNA, are excised from the DNA in the uvr+ strain and appear in the acid-soluble fraction. No such excision occurred upon incubation of the radiosensitive strain uvr−.Incubation of the cells under light showed that photoreactivation prevails in the radiosensitive strain, i.e. disappearance of "a" + [Formula: see text] from the DNA, without their appearance in the acid-soluble fraction, while dark reactivation prevailed in the uvr+ strain, a result that indicates a stronger affinity of the dark reactivating system to UV-irradiated DNA. Cell extracts prepared by breaking the cells in a French press in Tris buffer, pH 7.5, plus 10−3 M Mg++ plus 10−3 M mercapto-ethanol showed a similar mechanism; the two irradiation products were excised from DNA by a uvr+ cell extract and not by a uvr− cell extract. Extract of uvr+ cells brought about excision of photoproducts from DNA of the uvr− cell extract.The results suggest that an enzyme, capable of excising thymine dimers, is present in the radioresistant cell as part of the system of repair of DNA from UV irradiation, and its mechanism is demonstrated, both in vivo and in vitro.

scholarly journals Dual Function of Aar, a Member of the New AraC Negative Regulator Family, in Escherichia coli Gene Expression

2020 ◽  
Vol 88 (6) ◽  
Author(s):  
Abigail S. Mickey ◽  
James P. Nataro
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Negative Regulator ◽  
Dual Function ◽  
Content Type ◽  
Virulence Gene Expression ◽  
E Coli ◽  
K 12

ABSTRACT Enteroaggregative Escherichia coli (EAEC) is an E. coli pathotype associated with diarrhea and growth faltering. EAEC virulence gene expression is controlled by the autoactivated AraC family transcriptional regulator, AggR. AggR activates transcription of a large number of virulence genes, including Aar, which in turn acts as a negative regulator of AggR itself. Aar has also been shown to affect expression of E. coli housekeeping genes, including H-NS, a global regulator that acts at multiple promoters and silences AT-rich genes (such as those in the AggR regulon). Although Aar has been shown to bind both AggR and H-NS in vitro, functional significance of these interactions has not been shown in vivo. In order to dissect this regulatory network, we removed the complex interdependence of aggR and aar by placing the genes under the control of titratable promoters. We measured phenotypic and genotypic changes on downstream genes in EAEC strain 042 and E. coli K-12 strain DH5α, which lacks the AggR regulon. In EAEC, we found that low expression of aar increases aafA fimbrial gene expression via H-NS; however, when aar is more highly expressed, it acts as a negative regulator via AggR. In DH5α, aar affected expression of E. coli genes in some cases via H-NS and in some cases independent of H-NS. Our data support the model that Aar interacts in concert with AggR, H-NS, and possibly other regulators and that these interactions are likely to be functionally significant in vivo.

scholarly journals The yefM-yoeB Toxin-Antitoxin Systems of Escherichia coli and Streptococcus pneumoniae: Functional and Structural Correlation

2006 ◽  
Vol 189 (4) ◽  
pp. 1266-1278 ◽  
Author(s):  
Concha Nieto ◽  
Izhack Cherny ◽  
Seok Kooi Khoo ◽  
Mario García de Lacoba ◽  
Wai Ting Chan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Streptococcus Pneumoniae ◽  
Structural Features ◽  
E Coli ◽  
Modeled Structure ◽  
The Family ◽  
Bona Fide ◽  
K 12

ABSTRACT Toxin-antitoxin loci belonging to the yefM-yoeB family are located in the chromosome or in some plasmids of several bacteria. We cloned the yefM-yoeB locus of Streptococcus pneumoniae, and these genes encode bona fide antitoxin (YefM Spn ) and toxin (YoeB Spn ) products. We showed that overproduction of YoeB Spn is toxic to Escherichia coli cells, leading to severe inhibition of cell growth and to a reduction in cell viability; this toxicity was more pronounced in an E. coli B strain than in two E. coli K-12 strains. The YoeB Spn -mediated toxicity could be reversed by the cognate antitoxin, YefM Spn , but not by overproduction of the E. coli YefM antitoxin. The pneumococcal proteins were purified and were shown to interact with each other both in vitro and in vivo. Far-UV circular dichroism analyses indicated that the pneumococcal antitoxin was partially, but not totally, unfolded and was different than its E. coli counterpart. Molecular modeling showed that the toxins belonging to the family were homologous, whereas the antitoxins appeared to be specifically designed for each bacterial locus; thus, the toxin-antitoxin interactions were adapted to the different bacterial environmental conditions. Both structural features, folding and the molecular modeled structure, could explain the lack of cross-complementation between the pneumococcal and E. coli antitoxins.

scholarly journals Metabolic Engineering of Escherichia coli for Enhanced Production of (R)- and (S)-3-Hydroxybutyrate

2009 ◽  
Vol 75 (10) ◽  
pp. 3137-3145 ◽  
Author(s):  
Hsien-Chung Tseng ◽  
Collin H. Martin ◽  
David R. Nielsen ◽  
Kristala L. Jones Prather
Keyword(s):  
Escherichia Coli ◽  
Shake Flask ◽  
Maximal Activity ◽  
Culture Conditions ◽  
Recombinant Enzymes ◽  
E Coli ◽  
Enhanced Production ◽  
K 12

ABSTRACT Synthetic metabolic pathways have been constructed for the production of enantiopure (R)- and (S)-3-hydroxybutyrate (3HB) from glucose in recombinant Escherichia coli strains. To promote maximal activity, we profiled three thiolase homologs (BktB, Thl, and PhaA) and two coenzyme A (CoA) removal mechanisms (Ptb-Buk and TesB). Two enantioselective 3HB-CoA dehydrogenases, PhaB, producing the (R)-enantiomer, and Hbd, producing the (S)-enantiomer, were utilized to control the 3HB chirality across two E. coli backgrounds, BL21Star(DE3) and MG1655(DE3), representing E. coli B- and K-12-derived strains, respectively. MG1655(DE3) was found to be superior for the production of each 3HB stereoisomer, although the recombinant enzymes exhibited lower in vitro specific activities than BL21Star(DE3). Hbd in vitro activity was significantly higher than PhaB activity in both strains. The engineered strains achieved titers of enantiopure (R)-3HB and (S)-3HB as high as 2.92 g liter−1 and 2.08 g liter−1, respectively, in shake flask cultures within 2 days. The NADPH/NADP+ ratio was found to be two- to three-fold higher than the NADH/NAD+ ratio under the culture conditions examined, presumably affecting in vivo activities of PhaB and Hbd and resulting in greater production of (R)-3HB than (S)-3HB. To the best of our knowledge, this study reports the highest (S)-3HB titer achieved in shake flask E. coli cultures to date.

scholarly journals Transposon-insertion sequencing screens unveil requirements for EHEC growth and intestinal colonization

2019 ◽  
Author(s):  
Alyson R. Warr ◽  
Troy P. Hubbard ◽  
Diana Munera ◽  
Carlos J. Blondel ◽  
Pia Abel zur Wiesch ◽  
...  
Keyword(s):  
Type Iii Secretion ◽  
Intestinal Colonization ◽  
E Coli ◽  
Rabbit Colon ◽  
Transposon Insertion ◽  
Food Borne ◽  
K 12 ◽  
Transposon Insertion Sequencing

AbstractEnterohemorrhagicEscherichia coliO157:H7 (EHEC) is an important food-borne pathogen that colonizes the colon. Transposon-insertion sequencing (TIS) was used to identify genes required for EHEC and commensalE. coliK-12 growth in vitro and for EHEC growth in vivo in the infant rabbit colon. Surprisingly, many conserved loci contribute to EHEC’s but not to K-12’s growth in vitro, suggesting that gene acquisition during EHEC evolution has heightened the pathogen’s reliance on certain metabolic processes that are dispensable for K-12. There was a restrictive bottleneck for EHEC colonization of the rabbit colon, which complicated identification of EHEC genes facilitating growth in vivo. Both a refined version of an existing analytic framework as well as PCA-based analysis were used to compensate for the effects of the infection bottleneck. These analyses confirmed that the EHEC LEE-encoded type III secretion apparatus is required for growth in vivo and revealed that only a few effectors are critical for in vivo fitness. Numerous mutants not previously associated with EHEC survival/growth in vivo also appeared attenuated in vivo, and a subset of these putative in vivo fitness factors were validated. Some were found to contribute to efficient type-three secretion while others, includingtatABC, oxyR, envC, acrAB, andcvpA, promote EHEC resistance to host-derived stresses encountered in vivo.cvpA, which is also required for intestinal growth of several other enteric pathogens, proved to be required for EHEC,Vibrio choleraeandVibrio parahaemolyticusresistance to the bile salt deoxycholate. Collectively, our findings provide a comprehensive framework for understanding EHEC growth in the intestine.Author SummaryEnterohemorrhagicE. coli(EHEC) are important food-borne pathogens that infect the colon. We created a highly saturated EHEC transposon library and used transposon insertion sequencing to identify the genes required for EHEC growth in vitro and in vivo in the infant rabbit colon. We found that there is a large infection bottleneck in the rabbit model of intestinal colonization, and refined two analytic approaches to facilitate rigorous identification of new EHEC genes that promote fitness in vivo. Besides the known type III secretion system, more than 200 additional genes were found to contribute to EHEC survival and/or growth within the intestine. The requirement for some of these new in vivo fitness factors was confirmed, and their contributions to infection were investigated. This set of genes should be of considerable value for future studies elucidating the processes that enable the pathogen to proliferate in vivo and for design of new therapeutics.

scholarly journals Transduction of Porcine Enteropathogenic Escherichia coli with a Derivative of a Shiga Toxin 2-Encoding Bacteriophage in a Porcine Ligated Ileal Loop System

2003 ◽  
Vol 69 (12) ◽  
pp. 7242-7247 ◽  
Author(s):  
István Tóth ◽  
Herbert Schmidt ◽  
Mohamed Dow ◽  
Anna Malik ◽  
Eric Oswald ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Prototype Strain ◽  
Epec Strain ◽  
Ileal Loop ◽  
E Coli ◽  
Shiga Toxin 2 ◽  
K 12

ABSTRACT In this study, we have investigated the ability of detoxified Shiga toxin (Stx)-converting bacteriophages Φ3538 (Δstx 2::cat) (H. Schmidt et al., Appl. Environ. Microbiol. 65:3855-3861, 1999) and H-19B::Tn10d-bla (D. W. Acheson et al., Infect. Immun. 66:4496-4498, 1998) to lysogenize enteropathogenic Escherichia coli (EPEC) strains in vivo. We were able to transduce the porcine EPEC strain 1390 (O45) withΦ 3538 (Δstx 2::cat) in porcine ligated ileal loops but not the human EPEC prototype strain E2348/69 (O127). Neither strain 1390 nor strain E2348/69 was lysogenized under these in vivo conditions when E. coli K-12 containing H-19B::Tn10d-bla was used as the stx1 phage donor. The repeated success in the in vivo transduction of an Stx2-encoding phage to a porcine EPEC strain in pig loops was in contrast to failures in the in vitro trials with these and other EPEC strains. These results indicate that in vivo conditions are more effective for transduction of Stx2-encoding phages than in vitro conditions.

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