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 Biosynthesis and turnover of outer-membrane proteins in Escherichia coli ML308-225

1979 ◽  
Vol 182 (2) ◽  
pp. 407-412 ◽  
Author(s):  
R J Allen ◽  
G K Scott
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Outer Membrane ◽  
Bacterial Growth ◽  
Growth Phase ◽  
Exponential Growth ◽  
Outer Membrane Proteins ◽  
Exponential Growth Phase ◽  
Outer Membranes ◽  
Dilution Experiments

Isolated outer membranes and outer-membrane extracts from Escherichia coli ML308-225 in the early-exponential growth phase contain more protein than do corresponding preparations from late-exponential- or stationary-phase bacteria. Isotope-dilution experiments show that this is due to a loss of protein from the membrane during the exponential growth phase. Inhibition of bacterial growth and protein synthesis stabilizes the outer-membrane-protein concentration. Protein synthesis in the absence of bacterial growth results in higher concentrations of protein in the outer membrane.

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 Gene Cluster for Assembly of Pilus Colonization Factor Antigen III of Enterotoxigenic Escherichia coli

2001 ◽  
Vol 69 (9) ◽  
pp. 5864-5873 ◽  
Author(s):  
Tooru Taniguchi ◽  
Yukihiro Akeda ◽  
Ayako Haba ◽  
Yoko Yasuda ◽  
Koichiro Yamamoto ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Nucleotide Sequence ◽  
Gene Cluster ◽  
Enterotoxigenic Escherichia Coli ◽  
Human Colon Carcinoma Cell ◽  
E Coli ◽  
Colonization Factor ◽  
Type Iv ◽  
K 12 ◽  
Factor Antigen

ABSTRACT The assembly of pilus colonization factor antigen III (CFA/III) of enterotoxigenic Escherichia coli (ETEC) requires the processing of CFA/III major pilin (CofA) by a prepilin peptidase (CofP), similar to other type IV pilus formation systems. CofA is produced initially as a 26.5-kDa preform pilin (prepilin) and then processed to a 20.5-kDa mature pilin by CofP which is predicted to be localized in the inner membrane. In the present experiment, we determined the nucleotide sequence of the whole region for CFA/III formation and identified a cluster of 14 genes, includingcofA and cofP. Several proteins encoded bycof genes were similar to previously described proteins, such as the toxin-coregulated pili of Vibrio cholerae and the bundle-forming pili of enteropathogenic E. coli. The G+C content of the cof gene cluster was 37%, which was significantly lower than the average for the E. coli genome (50%). The introduction of a recombinant plasmid containing thecof gene cluster into the E. coli K-12 strain conferred CFA/III biogenesis and the ability of adhesion to the human colon carcinoma cell line Caco-2. This is the first report of a complete nucleotide sequence of the type IV pili found in human ETEC, and our results provide a useful model for studying the molecular mechanism of CFA/III biogenesis and the role of CFA/III in ETEC infection.

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.

Effect of bacterial growth stage on resistance to chlorine disinfection

2011 ◽  
Vol 64 (1) ◽  
pp. 7-13 ◽  
Author(s):  
C. Cherchi ◽  
A. Z. Gu
Keyword(s):  
Escherichia Coli ◽  
Growth Phase ◽  
Growth Stage ◽  
Exponential Growth Phase ◽  
Lag Phase ◽  
Growth Phases ◽  
Bacterial Strains ◽  
E Coli ◽  
Chlorine Disinfection ◽  
The Impact

The mechanisms and factors that affect microbial resistance to chlorine disinfection have not been fully elucidated. In this study, we investigated the impact of the cell growth stage on chlorine disinfection efficiency. Specifically, we evaluated the impact of the growth stage on chlorination resistance by comparing the inactivation efficiencies of two indicator bacterial strains (Escherichia coli K12 and Escherichia coli 0157:H7) obtained from various growth phases, using Chick-Watson kinetic parameters. For both E. coli strains (K12 and 0157:H7), the inactivation rate constants are the lowest at stationary phase (0.19 and 0.32) compared to those at initial lag (0.54 and 0.76) and exponential growth phase (0.63 and 0.69), respectively. These results suggested that the abundance of resistant subpopulations increases at stressed stationary conditions and E. coli cells obtained from the stationary growth phase exhibited more resistance and lower inactivation efficiency compared to those from the lag and exponential phases. This implies that microbes in wastewater treatment process with varying solids retention times (SRTs, which indicate growth rates) may show different extents of chlorine resistance. Comparison of the coefficient of dilution (n) values in both E. coli strains for the various growth phases suggest that cells seem to be more sensitive to disinfectant concentration at the stationary-lag phase than that at the exponential stage. Comparing the two E. coli strains, higher inactivation rates were observed for the pathogenic O157:H7 than for K12 at different stages of growth. The strain-to-strain variability in survivability to chlorine exposure has to be considered when selecting indicator microorganisms for water quality monitoring.

scholarly journals The ETT2 Gene Cluster, Encoding a Second Type III Secretion System from Escherichia coli, Is Present in the Majority of Strains but Has Undergone Widespread Mutational Attrition

2004 ◽  
Vol 186 (11) ◽  
pp. 3547-3560 ◽  
Author(s):  
Chuan-Peng Ren ◽  
Roy R. Chaudhuri ◽  
Amanda Fivian ◽  
Christopher M. Bailey ◽  
Martin Antonio ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Genome Sequences ◽  
Phylogenetic Structure ◽  
E Coli ◽  
Type Iii ◽  
K 12

ABSTRACT ETT2 is a second cryptic type III secretion system in Escherichia coli which was first discovered through the analysis of genome sequences of enterohemorrhagic E. coli O157:H7. Comparative analyses of Escherichia and Shigella genome sequences revealed that the ETT2 gene cluster is larger than was previously thought, encompassing homologues of genes from the Spi-1, Spi-2, and Spi-3 Salmonella pathogenicity islands. ETT2-associated genes, including regulators and chaperones, were found at the same chromosomal location in the majority of genome-sequenced strains, including the laboratory strain K-12. Using a PCR-based approach, we constructed a complete tiling path through the ETT2 gene cluster for 79 strains, including the well-characterized E. coli reference collection supplemented with additional pathotypes. The ETT2 gene cluster was found to be present in whole or in part in the majority of E. coli strains, whether pathogenic or commensal, with patterns of distribution and deletion mirroring the known phylogenetic structure of the species. In almost all strains, including enterohemorrhagic E. coli O157:H7, ETT2 has been subjected to varying degrees of mutational attrition that render it unable to encode a functioning secretion system. A second type III secretion system-associated locus that likely encodes the ETT2 translocation apparatus was found in some E. coli strains. Intact versions of both ETT2-related clusters are apparently present in enteroaggregative E. coli strain O42.

scholarly journals Fr-Spezifische RNA in Escherichia coli während der Phagenvermehrung

1967 ◽  
Vol 22 (11) ◽  
pp. 1118-1129 ◽  
Author(s):  
Peter Knolle
Keyword(s):  
Escherichia Coli ◽  
Latent Period ◽  
Minimal Medium ◽  
E Coli ◽  
Replicative Intermediates ◽  
K 12

The appearance of fr-specific RNA components was followed in two strains of E. coli K 12 growing in supplemented minimal medium. Under these conditions the eclipse period was 25 min. and the latent period 50 minutes. Using pulses with 14C-uracil for 5 min followed by chases for 10 min, labelled RNase-resistant components appeared simultaneously with labelled material corresponding to phage RNA. The peak of the RNase-resistant components was found to sediment initially at 6 s. As replication progressed, it sedimented with increasingly higher values, reaching a maximum of 11.5 s 80 min after infection. Properties of this RNase-resistant fraction resemble those expected of partially double-stranded material as it is assumed to be present in “replicative intermediates I and II”.

scholarly journals Identification and Characterization of a Novel Uropathogenic Escherichia coli-Associated Fimbrial Gene Cluster

2004 ◽  
Vol 72 (7) ◽  
pp. 3890-3901 ◽  
Author(s):  
Eric L. Buckles ◽  
Farah K. Bahrani-Mougeot ◽  
Anita Molina ◽  
C. Virgina Lockatell ◽  
David E. Johnson ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Gene Cluster ◽  
Amorphous Material ◽  
Exponential Growth Phase ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
T7 Promoter ◽  
E Coli ◽  
Tract Infections

ABSTRACT Recently, we identified a fimbrial usher gene in uropathogenic Escherichia coli strain CFT073 that is absent from an E. coli laboratory strain. Analysis of the CFT073 genome indicates that this fimbrial usher gene is part of a novel fimbrial gene cluster, aufABCDEFG. Analysis of a collection of pathogenic and commensal strains of E. coli and related species revealed that the auf gene cluster was significantly associated with uropathogenic E. coli isolates. For in vitro expression analysis of the auf gene cluster, RNA was isolated from CFT073 bacteria grown to the exponential or stationary phase in Luria-Bertani broth and reverse transcriptase PCR (RT-PCR) with oligonucleotide primers specific to the major subunit, aufA, was performed. We found that aufA is expressed in CFT073 only during the exponential growth phase; however, no expression of AufA protein was observed by Western blotting, indicating that under these conditions, the expression of the auf gene cluster is low. To determine if the auf gene cluster is expressed in vivo, RT-PCR was performed on bacteria from urine samples of mice infected with CFT073. Out of three independent experiments, we were able to detect expression of aufA at least once at 4, 24, and 48 h of infection, indicating that the auf gene cluster is expressed in the murine urinary tract. Furthermore, antisera from mice infected with CFT073 reacted with recombinant AufA in an enzyme-linked immunosorbent assay. To identify the structure encoded by the auf gene cluster, a recombinant plasmid containing the auf gene cluster under the T7 promoter was introduced into the E. coli BL-21 (AI) strain. Immunogold labeling using AufA antiserum revealed the presence of amorphous material extending from the surface of BL-21 cells. No hemagglutination or cellular adherence properties were detected in association with expression of AufA. Deletion of the entire auf gene cluster had no effect on the ability of CFT073 to colonize the kidney, bladder, or urine of mice. In addition, no significant histological differences between the parent and aufC mutant strain were observed. Therefore, Auf is a uropathogenic E. coli-associated structure that plays an uncertain role in the pathogenesis of urinary tract infections.

scholarly journals Plasmid-Encoded Phthalate Catabolic Pathway in Arthrobacter keyseri 12B

2001 ◽  
Vol 183 (12) ◽  
pp. 3689-3703 ◽  
Author(s):  
Richard W. Eaton
Keyword(s):  
Escherichia Coli ◽  
Minimal Medium ◽  
Restriction Endonucleases ◽  
Phthalate Ester ◽  
Carboxyl Group ◽  
Catabolic Pathway ◽  
E Coli ◽  
Cell Extracts ◽  
Atp Binding Cassette Transporter ◽  
Genes Encoding

ABSTRACT Several 2-substituted benzoates (including 2-trifluoromethyl-, 2-chloro-, 2-bromo-, 2-iodo-, 2-nitro-, 2-methoxy-, and 2-acetyl-benzoates) were converted by phthalate-grown Arthrobacter keyseri (formerlyMicrococcus sp.) 12B to the corresponding 2-substituted 3,4-dihydroxybenzoates (protocatechuates). Because these products lack a carboxyl group at the 2 position, they were not substrates for the next enzyme of the phthalate catabolic pathway, 3,4-dihydroxyphthalate 2-decarboxylase, and accumulated. When these incubations were carried out in iron-containing minimal medium, the products formed colored chelates. This chromogenic response was subsequently used to identify recombinant Escherichia colistrains carrying genes encoding the responsible enzymes, phthalate 3,4-dioxygenase and 3,4-dihydroxy-3,4-dihydrophthalate dehydrogenase, from the 130-kbp plasmid pRE1 of strain 12B. Beginning with the initially cloned 8.14-kbp PstI fragment of pRE824 as a probe to identify recombinant plasmids carrying overlapping fragments, a DNA segment of 33.5 kbp was cloned from pRE1 on several plasmids and mapped using restriction endonucleases. From these plasmids, the sequence of 26,274 contiguous bp was determined. Sequenced DNA included several genetic units: tnpR, pcmoperon, ptr genes, pehA, norA fragment, and pht operon, encoding a transposon resolvase, catabolism of protocatechuate (3,4-dihydroxybenzoate), a putative ATP-binding cassette transporter, a possible phthalate ester hydrolase, a fragment of a norfloxacin resistance-like transporter, and the conversion of phthalate to protocatechuate, respectively. Activities of the eight enzymes involved in the catabolism of phthalate through protocatechuate to pyruvate and oxaloacetate were demonstrated in cells or cell extracts of recombinant E. coli strains.

scholarly journals The Flag-2 Locus, an Ancestral Gene Cluster, Is Potentially Associated with a Novel Flagellar System from Escherichia coli

2005 ◽  
Vol 187 (4) ◽  
pp. 1430-1440 ◽  
Author(s):  
Chuan-Peng Ren ◽  
Scott A. Beatson ◽  
Julian Parkhill ◽  
Mark J. Pallen
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
Type Iii Secretion ◽  
Yersinia Pseudotuberculosis ◽  
Close Relative ◽  
Secretion Systems ◽  
Ancestral Gene ◽  
E Coli ◽  
Type Iii ◽  
K 12

ABSTRACT Escherichia coli K-12 possesses two adjacent, divergent, promoterless flagellar genes, fhiA-mbhA, that are absent from Salmonella enterica. Through bioinformatics analysis, we found that these genes are remnants of an ancestral 44-gene cluster and are capable of encoding a novel flagellar system, Flag-2. In enteroaggregative E. coli strain 042, there is a frameshift in lfgC that is likely to have inactivated the system in this strain. Tiling path PCR studies showed that the Flag-2 cluster is present in 15 of 72 of the well-characterized ECOR strains. The Flag-2 system resembles the lateral flagellar systems of Aeromonas and Vibrio, particularly in its apparent dependence on RpoN. Unlike the conventional Flag-1 flagellin, the Flag-2 flagellin shows a remarkable lack of sequence polymorphism. The Flag-2 gene cluster encodes a flagellar type III secretion system (including a dedicated flagellar sigma-antisigma combination), thus raising the number of distinct type III secretion systems in Escherichia/Shigella to five. The presence of the Flag-2 cluster at identical sites in E. coli and its close relative Citrobacter rodentium, combined with its absence from S. enterica, suggests that it was acquired by horizontal gene transfer after the former two species diverged from Salmonella. The presence of Flag-2-like gene clusters in Yersinia pestis, Yersinia pseudotuberculosis, and Chromobacterium violaceum suggests that coexistence of two flagellar systems within the same species is more common than previously suspected. The fact that the Flag-2 gene cluster was not discovered in the first 10 Escherichia/Shigella genome sequences studied emphasizes the importance of maintaining an energetic program of genome sequencing for this important taxonomic group.

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