Chromosomal Integration of Heterologous DNA in Escherichia coli with Precise Removal of Markers and Replicons Used during Construction

1999 ◽  
Vol 181 (22) ◽  
pp. 7143-7148 ◽  
Author(s):  
F. Martinez-Morales ◽  
A. C. Borges ◽  
A. Martinez ◽  
K. T. Shanmugam ◽  
L. O. Ingram
Keyword(s):  
Escherichia Coli ◽  
Helper Plasmid ◽  
Flp Recombinase ◽  
E Coli ◽  
Homologous Fragment ◽  
Genes Encoding ◽  
Dna Insertion ◽  
K 12 ◽  
Multiple Cloning Sites

ABSTRACT A set of vectors which facilitates the sequential integration of new functions into the Escherichia coli chromosome by homologous recombination has been developed. These vectors are based on plasmids described by Posfai et al. (J. Bacteriol. 179:4426–4428, 1997) which contain conditional replicons (pSC101 or R6K), a choice of three selectable markers (ampicillin, chloramphenicol, or kanamycin), and a single FRT site. The modified vectors contain twoFRT sites which bracket a modified multiple cloning region for DNA insertion. After integration, a helper plasmid expressing the flippase (FLP) recombinase allows precise in vivo excision of the replicon and the marker used for selection. Sites are also available for temporary insertion of additional functions which can be subsequently deleted with the replicon. Only the DNA inserted into the multiple cloning sites (passenger genes and homologous fragment for targeting) and a single FRT site (68 bp) remain in the chromosome after excision. The utility of these vectors was demonstrated by integrating Zymomonas mobilis genes encoding the ethanol pathway behind the native chromosomaladhE gene in strains of E. coli K-12 andE. coli B. With these vectors, a single antibiotic selection system can be used repeatedly for the successive improvement of E. coli strains with precise deletion of extraneous genes used during construction.

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 The Putrescine Importer PuuP of Escherichia coli K-12

2009 ◽  
Vol 191 (8) ◽  
pp. 2776-2782 ◽  
Author(s):  
Shin Kurihara ◽  
Yuichi Tsuboi ◽  
Shinpei Oda ◽  
Hyeon Guk Kim ◽  
Hidehiko Kumagai ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
Growth Phase ◽  
Exponential Growth ◽  
Minimal Medium ◽  
Exponential Growth Phase ◽  
E Coli ◽  
Genes Encoding ◽  
K 12 ◽  
Utilization Pathway

ABSTRACT The Puu pathway is a putrescine utilization pathway involving gamma-glutamyl intermediates. The genes encoding the enzymes of the Puu pathway form a gene cluster, the puu gene cluster, and puuP is one of the genes in this cluster. In Escherichia coli, three putrescine importers, PotFGHI, PotABCD, and PotE, were discovered in the 1990s and have been studied; however, PuuP had not been discovered previously. This paper shows that PuuP is a novel putrescine importer whose kinetic parameters are equivalent to those of the polyamine importers discovered previously. A puuP + strain absorbed up to 5 mM putrescine from the medium, but a ΔpuuP strain did not. E. coli strain MA261 has been used in previous studies of polyamine transporters, but PuuP had not been identified previously. It was revealed that the puuP gene of MA261 was inactivated by a point mutation. When E. coli was grown on minimal medium supplemented with putrescine as the sole carbon or nitrogen source, only PuuP among the polyamine importers was required. puuP was expressed strongly when putrescine was added to the medium or when the puuR gene, which encodes a putative repressor, was deleted. When E. coli was grown in M9-tryptone medium, PuuP was expressed mainly in the exponential growth phase, and PotFGHI was expressed independently of the growth phase.

scholarly journals Systematic Nomenclature for GGDEF and EAL Domain-Containing Cyclic Di-GMP Turnover Proteins of Escherichia coli: TABLE 1

2015 ◽  
Vol 198 (1) ◽  
pp. 7-11 ◽  
Author(s):  
Regine Hengge ◽  
Michael Y. Galperin ◽  
Jean-Marc Ghigo ◽  
Mark Gomelsky ◽  
Jeffrey Green ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Molecular Mechanisms ◽  
Bacterial Species ◽  
Content Type ◽  
E Coli ◽  
Diguanylate Cyclases ◽  
Genes Encoding ◽  
Systematic Nomenclature ◽  
K 12 ◽  
Encoding Genes

In recent years,Escherichia colihas served as one of a few model bacterial species for studying cyclic di-GMP (c-di-GMP) signaling. The widely usedE. coliK-12 laboratory strains possess 29 genes encoding proteins with GGDEF and/or EAL domains, which include 12 diguanylate cyclases (DGC), 13 c-di-GMP-specific phosphodiesterases (PDE), and 4 “degenerate” enzymatically inactive proteins. In addition, six new GGDEF and EAL (GGDEF/EAL) domain-encoding genes, which encode two DGCs and four PDEs, have recently been found in genomic analyses of commensal and pathogenicE. colistrains. As a group of researchers who have been studying the molecular mechanisms and the genomic basis of c-di-GMP signaling inE. coli, we now propose a general and systematicdgcandpdenomenclature for the enzymatically active GGDEF/EAL domain-encoding genes of this model species. This nomenclature is intuitive and easy to memorize, and it can also be applied to additional genes and proteins that might be discovered in various strains ofE. coliin future studies.

scholarly journals Functional Differences between Heme Permeases: Serratia marcescens HemTUV Permease Exhibits a Narrower Substrate Specificity (Restricted to Heme) Than the Escherichia coli DppABCDF Peptide-Heme Permease

2008 ◽  
Vol 190 (6) ◽  
pp. 1866-1870 ◽  
Author(s):  
Sylvie Létoffé ◽  
Philippe Delepelaire ◽  
Cécile Wandersman
Keyword(s):  
Escherichia Coli ◽  
Serratia Marcescens ◽  
Outer Membrane Receptor ◽  
Heme Uptake ◽  
Periplasmic Binding Protein ◽  
E Coli ◽  
Iron Source ◽  
Functional Differences ◽  
Genes Encoding

ABSTRACT Serratia marcescens hemTUV genes encoding a potential heme permease were cloned in Escherichia coli recombinant mutant FB827 dppF::Km(pAM 238-hasR). This strain, which expresses HasR, a foreign heme outer membrane receptor, is potentially capable of using heme as an iron source. However, this process is invalidated due to a dppF::Km mutation which inactivates the Dpp heme/peptide permease responsible for heme, dipeptide, and δ-aminolevulinic (ALA) transport through the E. coli inner membrane. We show here that hemTUV genes complement the Dpp permease for heme utilization as an iron source and thus are functional in E. coli. However, hemTUV genes do not complement the Dpp permease for ALA uptake, indicating that the HemTUV permease does not transport ALA. Peptides do not inhibit heme uptake in vivo, indicating that, unlike Dpp permease, HemTUV permease does not transport peptides. HemT, the periplasmic binding protein, binds heme. Heme binding is saturable and not inhibited by peptides that inhibit heme uptake by the Dpp system. Thus, the S. marcescens HemTUV permease and, most likely, HemTUV orthologs present in many gram-negative pathogens form a class of heme-specific permeases different from the Dpp peptide/heme permease characterized in E. coli.

scholarly journals Iha from an Escherichia coli Urinary Tract Infection Outbreak Clonal Group A Strain Is Expressed In Vivo in the Mouse Urinary Tract and Functions as a Catecholate Siderophore Receptor

2006 ◽  
Vol 74 (6) ◽  
pp. 3427-3436 ◽  
Author(s):  
Simon Léveillé ◽  
Mélissa Caza ◽  
James R. Johnson ◽  
Connie Clabots ◽  
Mourad Sabri ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract Infection ◽  
Urinary Tract ◽  
Tract Infection ◽  
Growth Promotion ◽  
Clonal Group ◽  
E Coli ◽  
Group A ◽  
K 12

ABSTRACT Virulence factors of pathogenic Escherichia coli belonging to a recently emerged and disseminated clonal group associated with urinary tract infection (UTI), provisionally designated clonal group A (CGA), have not been experimentally investigated. We used a mouse model of ascending UTI with CGA member strain UCB34 in order to identify genes of CGA that contribute to UTI. iha was identified to be expressed by strain UCB34 in the mouse kidney using selective capture of transcribed sequences. iha from strain UCB34 demonstrated a siderophore receptor phenotype when cloned in a catecholate siderophore receptor-negative E. coli K-12 strain, as shown by growth promotion experiments and uptake of 55Fe complexed to enterobactin or its linear 2, 3-dihydroxybenzoylserine (DHBS) siderophore derivatives. Siderophore-mediated growth promotion by Iha was TonB dependent. Growth and iron uptake were more marked with linear DHBS derivatives than with purified enterobactin. The reported phenotype of adherence to epithelial cells conferred by expressing iha from a multicopy cloning vector in a poorly adherent E. coli K-12 host strain was confirmed to be specific to iha, in comparison with other siderophore receptor genes. iha expression was regulated by the ferric uptake regulator Fur and by iron availability, as shown by real-time reverse transcriptase PCR. In a competitive infection experiment using the mouse UTI model, wild-type strain UCB34 significantly outcompeted an isogenic iha null mutant. Iha thus represents a Fur-regulated catecholate siderophore receptor that, uniquely, exhibits an adherence-enhancing phenotype and is the first described urovirulence factor identified in a CGA strain.

scholarly journals Evolved Cobalamin-Independent Methionine Synthase (MetE) Improves the Acetate and Thermal Tolerance of Escherichia coli

2013 ◽  
Vol 79 (24) ◽  
pp. 7905-7915 ◽  
Author(s):  
Elena A. Mordukhova ◽  
Jae-Gu Pan
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Elevated Temperatures ◽  
Defined Medium ◽  
Methionine Synthase ◽  
Mutant Enzyme ◽  
Content Type ◽  
E Coli ◽  
K 12

ABSTRACTAcetate-mediated growth inhibition ofEscherichia colihas been found to be a consequence of the accumulation of homocysteine, the substrate of the cobalamin-independent methionine synthase (MetE) that catalyzes the final step of methionine biosynthesis. To improve the acetate resistance ofE. coli, we randomly mutated the MetE enzyme and isolated a mutant enzyme, designated MetE-214 (V39A, R46C, T106I, and K713E), that conferred accelerated growth in theE. coliK-12 WE strain in the presence of acetate. Additionally, replacement of cysteine 645, which is a unique site of oxidation in the MetE protein, with alanine improved acetate tolerance, and introduction of the C645A mutation into the MetE-214 mutant enzyme resulted in the highest growth rate in acetate-treatedE. colicells among three mutant MetE proteins.E. coliWE strains harboring acetate-tolerant MetE mutants were less inhibited by homocysteine inl-isoleucine-enriched medium. Furthermore, the acetate-tolerant MetE mutants stimulated the growth of the host strain at elevated temperatures (44 and 45°C). Unexpectedly, the mutant MetE enzymes displayed a reduced melting temperature (Tm) but an enhancedin vivostability. Thus, we demonstrate improvedE. coligrowth in the presence of acetate or at elevated temperatures solely due to mutations in the MetE enzyme. Furthermore, when anE. coliWE strain carrying the MetE mutant was combined with a previously found MetA (homoserineo-succinyltransferase) mutant enzyme, the MetA/MetE strain was found to grow at 45°C, a nonpermissive growth temperature forE. coliin defined medium, with a similar growth rate as if it were supplemented byl-methionine.

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.

Biosynthetic ornithine and arginine decarboxylases: correlation of rates of synthesis with activities in Escherichia coli during exponential growth and following nutritional shift-up

1982 ◽  
Vol 28 (8) ◽  
pp. 945-950
Author(s):  
Stephen M. Boyle ◽  
Kazuo Adachi
Keyword(s):  
Escherichia Coli ◽  
Steady State ◽  
Ornithine Decarboxylase ◽  
Biological Activities ◽  
Decarboxylase Activity ◽  
Biosynthetic Enzyme ◽  
Guanosine Tetraphosphate ◽  
E Coli ◽  
K 12

Whether guanosine tetraphosphate (ppGpp) has a role in the regulation of the putrescine biosynthetic enzyme, ornithine decarboxylase, in Escherichia coli is controversial. Different laboratories have reported either direct or indirect correlations between ppGpp levels and ornithine decarboxylase activity using different in vivo conditions. In this report, using conditions in vivo to modulate ppGpp levels, experiments are described which bear on the controversy. The rates of synthesis and biological activities of the biosynthetic ornithine and arginine decarboxylases (ODC and ADC) were measured in E. coli K-12 during experimental growth and during nutritional shift-up. There were good correlations between changes in their respective activities and the rates of synthesis of these enzymes during steady state or shift-up. ODC activity or rate of synthesis changed directly in concert with ppGpp levels, while ADC activity or rate of synthesis changed inversely with ppGpp levels. These observations support the contention that ppGpp does not inhibit ODC activity.

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