Cloning of a recA-like gene from the diazotroph Herbaspirillum seropedicae strain Z78

1993 ◽  
Vol 39 (12) ◽  
pp. 1096-1102 ◽  
Author(s):  
M. B. R. Steffens ◽  
L. U. Rigo ◽  
S. Funayama ◽  
E. M. Souza ◽  
H. B. Machado ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Genomic Library ◽  
Reca Gene ◽  
Methyl Methanesulfonate ◽  
Herbaspirillum Seropedicae ◽  
E Coli ◽  
Tn5 Mutagenesis ◽  
Ultraviolet Resistance ◽  
Restriction Sites ◽  
Reca Mutant

A recombinant plasmid, pBMR5, carrying a recA-like gene of Herbaspirillum seropedicae, was isolated from a H. seropedicae genomic library by intergeneric complementation of Escherichia coli recA mutant strain HB101. Quantitative survival experiments showed that pBMR5 restored the ultraviolet radiation and methyl methanesulfonate resistances and recombinational proficiency of this strain. Hybridization studies showed that there is DNA sequence homology between the recA gene of E. coli K12 and that of H. seropedicae. Restriction sites for EcoRI, HindIII, BamHI, and BglII were found in the DNA insert derived from H. seropedicae in pBMR5. A Tn5 insertional mutant of pBMR5, called pBMR26.2, failed to restore recombination proficiency and methyl methanesulfonate and ultraviolet resistance to recA mutants of E. coli.Key words: Herbaspirillum seropedicae, nitrogen fixation, recA-like gene, Tn5 mutagenesis.

Characterization of novel ybjG and dacC variants in Escherichia coli

2013 ◽  
Vol 62 (11) ◽  
pp. 1728-1734 ◽  
Author(s):  
Dongguo Wang ◽  
Enping Hu ◽  
Jiayu Chen ◽  
Xiulin Tao ◽  
Katelyn Gutierrez ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Multiple Drug Resistance ◽  
Multiple Drug ◽  
The Novel ◽  
Conventional Pcr ◽  
E Coli ◽  
Novel Variants ◽  
Restriction Sites ◽  
Genetic Structures

A total of 69 strains of Escherichia coli from patients in the Taizhou Municipal Hospital, China, were isolated, and 11 strains were identified that were resistant to bacitracin, chloramphenicol, tetracycline and erythromycin. These strains were PCR positive for at least two out of three genes, ybjG, dacC and mdfA, by gene mapping with conventional PCR detection. Conjugation experiments demonstrated that these genes existed in plasmids that conferred resistance. Novel ybjG and dacC variants were isolated from E. coli strains EC2163 and EC2347, which were obtained from the sputum of intensive care unit patients. Genetic mapping showed that the genes were located on 8200 kb plasmid regions flanked by EcoRI restriction sites. Three distinct genetic structures were identified among the 11 PCR-positive strains of E. coli, and two contained the novel ybjG and dacC variants. The putative amino acid differences in the ybjG and dacC gene variants were characterized. These results provide evidence for novel variants of ybjG and dacC, and suggest that multiple drug resistance in hospital strains of E. coli depends on the synergistic function of ybjG, dacC and mdfA within three distinct genetic structures in conjugative plasmids.

Identification and physical characterization of a Col E1 hybrid plasmid containing a catalase gene of Escherichia coli

1983 ◽  
Vol 61 (12) ◽  
pp. 1315-1321 ◽  
Author(s):  
Peter C. Loewen ◽  
Barbara L. Triggs ◽  
Glen R. Klassen ◽  
Joel H. Weiner
Keyword(s):  
Escherichia Coli ◽  
Catalase Activity ◽  
Physical Map ◽  
Polyacrylamide Gel Electrophoresis ◽  
Hybrid Plasmid ◽  
E Coli ◽  
Catalase Gene ◽  
Restriction Sites ◽  
Catalase Peroxidase

A hybrid Escherichia coli: Col E1 plasmid, pLC36-19, containing a catalase gene has been identified in the Clarke and Carbon colony bank. Catalase activity was amplified two- to three-fold in the pLC36-19-containing strain relative to other hybrid-plasmid-containing strains and this activity could be induced three- or four-fold by hydrogen peroxide or ascorbic acid. The plasmid was transferred to a strain chromosomally deficient in catalase synthesis, resulting in a strain with high and inducible levels of catalase. The plasmid was also transferred to a minicell-producing strain and minicells harbouring the plasmid were found to synthesize a labelled protein with a molecular weight of 84 000 characteristic of catalase from E. coli. A catalase activity was also synthesized by the plasmid-containing minicells. Two catalase activities with associated peroxidase activities coded for by the plasmid were separable by polyacrylamide gel electrophoresis and migrated coincident with chromosomally encoded catalase–peroxidase activities. A third catalase activity which did not have an associated peroxidase activity was not coded for by the plasmid. A physical map of the 25.5-kilobase pair plasmid was constructed by restriction nuclease analysis and the relative positions of 38 restriction sites were defined.

Selection of In Vivo Expressed Genes of Escherichia coli O157:H7 Strain EDL933 in Ground Meat under Elevated Temperature Conditions

2012 ◽  
Vol 75 (10) ◽  
pp. 1743-1750 ◽  
Author(s):  
ANDREA KROJ ◽  
HERBERT SCHMIDT
Keyword(s):  
Escherichia Coli ◽  
Elevated Temperatures ◽  
Genomic Library ◽  
Transport Processes ◽  
Enterohemorrhagic Escherichia Coli ◽  
Escherichia Coli O157 ◽  
Ground Meat ◽  
E Coli ◽  
Selection Of

Enterohemorrhagic Escherichia coli O157:H7 strains are important foodborne pathogens that are often transmitted to humans by the ingestion of raw or undercooked meat of bovine origin. To investigate adaptation of this pathogen during persistence and growth in ground meat, we established an in vivo expression technology model to identify genes that are expressed during growth in this food matrix under elevated temperatures (42°C). To improve on the antibiotic-based selection method, we constructed the promoter trap vector pAK-1, containing a promoterless kanamycin resistance gene. A genomic library of E. coli O157:H7 strain EDL933 was constructed in pAK-1 and used for promoter selection in ground meat. The 20 in vivo expressed genes identified were associated with transport processes, metabolism, macromolecule synthesis, and stress response. For most of the identified genes, only hypothetical functions could be assigned. The results of our study provide the first insights into the complex response of E. coli O157:H7 to a ground meat environment under elevated temperatures and establish a suitable vector for promoter studies or selection of in vivo induced promoters in foods such as ground meat.

scholarly journals TleA, a Tsh-Like Autotransporter Identified in a Human Enterotoxigenic Escherichia coli Strain

2015 ◽  
Vol 83 (5) ◽  
pp. 1893-1903 ◽  
Author(s):  
Daniela Gutiérrez ◽  
Mirka Pardo ◽  
David Montero ◽  
Angel Oñate ◽  
Mauricio J. Farfán ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Genomic Library ◽  
Coli Strain ◽  
Enterotoxigenic Escherichia Coli ◽  
Watery Diarrhea ◽  
Temperature Sensitive ◽  
Content Type ◽  
E Coli ◽  
Colonization Factor ◽  
Adhesion Level

EnterotoxigenicEscherichia coli(ETEC), a leading cause of acute diarrhea, colonizes the intestine by means of adhesins. However, 15 to 50% of clinical isolates are negative for known adhesins, making it difficult to identify antigens for broad-coverage vaccines. The ETEC strain 1766a, obtained from a child with watery diarrhea in Chile, harbors the colonization factor CS23 but is negative for other known adhesins. One clone, derived from an ETEC 1766a genomic library (clone G10), did not produce CS23 yet was capable of adhering to Caco-2 cells. The goal of this study was to identify the gene responsible for this capacity. Random transposon-based mutagenesis allowed the identification of a 4,110-bp gene that codes for a homologue of the temperature-sensitive hemagglutinin (Tsh) autotransporter described in avianE. colistrains (97% identity, 90% coverage) and that is called TleA (Tsh-like ETEC autotransporter) herein. An isogenic ETEC 1766a strain with atleAmutation showed an adhesion level similar to that of the wild-type strain, suggesting that the gene does not direct attachment to Caco-2 cells. However, expression oftleAconferred the capacity for adherence to nonadherentE. coliHB101. This effect coincided with the detection of TleA on the surface of nonpermeabilized bacteria, while, conversely, ETEC 1766a seems to secrete most of the produced autotransporter to the medium. On the other hand, TleA was capable of degrading bovine submaxillary mucin and leukocyte surface glycoproteins CD45 and P-selectin glycoprotein ligand 1 (PSGL-1). These results suggest that TleA promotes colonization of the intestinal epithelium and that it may modulate the host immune response.

scholarly journals Integrated Genomic Map from Uropathogenic Escherichia coli J96

2000 ◽  
Vol 68 (10) ◽  
pp. 5933-5942 ◽  
Author(s):  
Lyla J. Melkerson-Watson ◽  
Christopher K. Rode ◽  
Lixin Zhang ◽  
Betsy Foxman ◽  
Craig A. Bloch
Keyword(s):  
Escherichia Coli ◽  
Virulence Genes ◽  
Housekeeping Genes ◽  
E Coli ◽  
Restriction Sites ◽  
Physical And Genetic Map ◽  
K 12 ◽  
Genomic Map ◽  
Insertion Mutants

ABSTRACT Escherichia coli J96 is a uropathogen having both broad similarities to and striking differences from nonpathogenic, laboratoryE. coli K-12. Strain J96 contains three large (>100-kb) unique genomic segments integrated on the chromosome; two are recognized as pathogenicity islands containing urovirulence genes. Additionally, the strain possesses a fourth smaller accessory segment of 28 kb and two deletions relative to strain K-12. We report an integrated physical and genetic map of the 5,120-kb J96 genome. The chromosome contains 26 NotI, 13 BlnI, and 7 I-CeuI macrorestriction sites. Macrorestriction mapping was rapidly accomplished by a novel transposon-based procedure: analysis of modified minitransposon insertions served to align the overlapping macrorestriction fragments generated by three different enzymes (each sharing a common cleavage site within the insert), thus integrating the three different digestion patterns and ordering the fragments. The resulting map, generated from a total of 54 mini-Tn10insertions, was supplemented with auxanography and Southern analysis to indicate the positions of insertionally disrupted aminosynthetic genes and cloned virulence genes, respectively. Thus, it contains not only physical, macrorestriction landmarks but also the loci for eight housekeeping genes shared with strain K-12 and eight acknowledged urovirulence genes; the latter confirmed clustering of virulence genes at the large unique accessory chromosomal segments. The 115-kb J96 plasmid was resolved by pulsed-field gel electrophoresis inNotI digests. However, because the plasmid lacks restriction sites for the enzymes BlnI and I-CeuI, it was visualized in BlnI and I-CeuI digests only of derivatives carrying plasmid inserts artificially introducing these sites. Owing to an I-SceI site on the transposon, the plasmid could also be visualized and sized from plasmid insertion mutants after digestion with this enzyme. The insertional strains generated in construction of the integrated genomic map provide useful physical and genetic markers for further characterization of the J96 genome.

scholarly journals Expression of an exogenous isopentenyl diphosphate isomerase gene enhances isoprenoid biosynthesis in Escherichia coli

1997 ◽  
Vol 324 (2) ◽  
pp. 421-426 ◽  
Author(s):  
Susumu KAJIWARA ◽  
Paul D. FRASER ◽  
Keiji KONDO ◽  
Norihiko MISAWA
Keyword(s):  
Escherichia Coli ◽  
Nucleotide Sequence ◽  
Genomic Library ◽  
Haematococcus Pluvialis ◽  
Isoprenoid Biosynthesis ◽  
Nucleotide Sequence Analysis ◽  
Efficient Production ◽  
Isopentenyl Diphosphate ◽  
E Coli ◽  
Β Carotene

Escherichia coli expressing the Erwinia carotenoid biosynthesis genes, crtE, crtB, crtI and crtY, form yellow-coloured colonies due to the presence of β-carotene. This host was used as a visible marker for evaluating regulatory systems operating in isoprenoid biosynthesis of E. coli. cDNAs enhancing carotenoid levels were isolated from the yeast Phaffia rhodozyma and the green alga Haematococcus pluvialis. Nucleotide sequence analysis indicated that they coded for proteins similar to isopentenyl diphosphate (IPP) isomerase of the yeast Saccharomyces cerevisiae. Determination of enzymic activity confirmed the identity of the gene products as IPP isomerases. The corresponding gene was isolated from the genomic library of S. cerevisiae based on its nucleotide sequence, and was confirmed to have the same effect as the above two IPP isomerase genes when introduced into the E. coli transformant accumulating β-carotene. In the three E. coli strains carrying the individual exogenous IPP isomerase genes, the increases in carotenoid levels are comparable to the increases in IPP isomerase enzyme activity with reference to control strains possessing the endogenous gene alone. These results imply that IPP isomerase forms an influential step in isoprenoid biosynthesis of the prokaryote E. coli, with potential for the efficient production of industrially useful isoprenoids by metabolic engineering.

Cloning and characterization of the N-acetylglucosamine operon of Escherichia coli

1990 ◽  
Vol 68 (1) ◽  
pp. 123-137 ◽  
Author(s):  
Krishna G. Peri ◽  
Hughes Goldie ◽  
E. Bruce Waygood
Keyword(s):  
Escherichia Coli ◽  
Genomic Library ◽  
Sodium Dodecyl ◽  
Open Reading Frames ◽  
Open Reading Frame ◽  
Gene Products ◽  
Reading Frame ◽  
E Coli ◽  
Palindromic Sequences ◽  
Repetitive Extragenic Palindromic

Three enzymes are required for N-acetylglucosamine (NAG) utilization in Escherichia coli: enzyme IInag (gene nagE), N-acetylglucosamine-6-phosphate deacetylase (gene nagA), and glucosamine-6-phosphate isomerase (gene nagB). The three genes are located near 16 min on the E. coli chromosome. A strain of E. coli, KPN9, incapable of utilizing N-acetylglucosamine, was used to screen a genomic library of E. coli for a complementing recombinant colicin E1 plasmid that allowed for growth on N-acetylglucosamine. Plasmid pLC5-21 was found to contain all three known nag genes on a 5.7-kilobase (5.7-kb) fragment of DNA. The products of these nag genes were identified by complementation of E. coli strains with mutations in nagA, nagB, and nagE. The gene products from the 5.7-kb fragment were identified by [35S]methionine-labelled maxicells and autoradiography of sodium dodecyl sulphate – polyacrylamide electrophoresis gels. The gene products had the following relative masses (Mrs: nagE, 62 000; nagA, 45 000; nagB, 29 000. In addition, another product of Mr 44 000 was detected. The genes have been sequenced to reveal an additional open reading frame (nagC), a putative catabolite activator protein binding site that may control nagB and nagE, putative rho-independent terminator sites for nagB and nagE, and sequence homologies for RNA polymerase binding sites preceding each of the open reading frames, except for nagA. The calculated molecular weights (MWs) of the gene products derived from the sequence are as follows: nagA, 40 954; nagB, 29 657; nagC, 44 664; nagE, 68 356. No role is known for nagC, although a number of regulatory roles appear to be plausible. No obvious transcriptional termination site distal to nagC was found and another open reading frame begins after nagC. This gene, nagD, was isolated separately from pLC5-21, and the sequence revealed a protein with a calculated MW of 27 181. The nagD gene is followed by repetitive extragenic palindromic sequences. The nag genes appear to be organized in an operon: [Formula: see text]Key words: N-acetylglucosamine, N-acetylglucosamine-6-P deacetylase, glucosamine-6-P isomerase, repetitive extragenic palindromic sequences, catabolite repression.

scholarly journals Molecular Cloning and Characterization of the Salmonella enterica Serovar Paratyphi B rma Gene, Which Confers Multiple Drug Resistance in Escherichia coli

2002 ◽  
Vol 46 (2) ◽  
pp. 360-366 ◽  
Author(s):  
Mahmoud. A. Yassien ◽  
Hosam E. Ewis ◽  
Chung-Dar Lu ◽  
Ahmed T. Abdelal
Keyword(s):  
Escherichia Coli ◽  
Drug Resistance ◽  
Salmonella Enterica ◽  
Genomic Library ◽  
Multiple Drug Resistance ◽  
Protein Profile ◽  
Multiple Drug ◽  
E Coli ◽  
Paratyphi B ◽  
High Degree

ABSTRACT A genomic library from a strain of Salmonella enterica serovar Paratyphi B that exhibits multiple drug resistance (MDR) was constructed in Escherichia coli. Two of the recombinant plasmids, pNOR5 and pNOR5, conferred resistance only to fluoroquinolones in E. coli, whereas the third, pNCTR4, conferred the MDR phenotype. Sequence and subcloning analysis showed that it is the presence of RecA on the first two plasmids which confers resistance to fluoroquinolones in E. coli. A similar analysis established that the MDR phenotype conferred by pNCTR4 is due to a gene, rma (resistance to multiple antibiotics), which encodes a 13.5-kDa polypeptide. The derived sequence for Rma exhibits a high degree of similarity to those of a group of MarA-like activators that confer MDR in E. coli. A MalE-Rma fusion protein was purified to near homogeneity and was shown to interact with a DNA fragment carrying a MarA operator sequence. Furthermore, overexpression of rma in E. coli caused changes in the outer membrane protein profile that were similar to those reported for MarA. These results suggest that Rma might act as a transcriptional activator of the marA regulon.

scholarly journals Cloning of the groE operon of the marine bacterium Vibrio harveyi using a lambda vector.

1998 ◽  
Vol 45 (1) ◽  
pp. 261-270 ◽  
Author(s):  
D Kuchanny ◽  
G Klein ◽  
J Krzewska ◽  
A Czyz ◽  
B Lipińska
Keyword(s):  
Escherichia Coli ◽  
Nucleotide Sequence ◽  
Marine Bacterium ◽  
Southern Hybridization ◽  
Genomic Library ◽  
Vibrio Harveyi ◽  
Bacteriophage Lambda ◽  
E Coli ◽  
Escherichia Coli Cells ◽  
Dna Fragment

groES and groEL genes encode two co-operating proteins GroES and GroEL, belonging to a class of chaperone proteins highly conserved during evolution. The GroE chaperones are indispensable for the growth of bacteriophage lambda in Escherichia coli cells. In order to clone the groEL and groES genes of the marine bacterium Vibrio harveyi, we constructed the V. harveyi genomic library in the lambdaEMBL1 vector, and selected clones which were able to complement mutations in both groE genes of E. coli for bacteriophage lambda growth. Using Southern hybridization, in one of these clones we identified a DNA fragment homologous to the E. coli groE region. Analysis of the nucleotide sequence of this fragment showed that the cloned region contained a sequence in 71.7% homologous to the 3' end of the groEL gene of E. coli. This confirmed that the lambda clone indeed carries the groE region of V. harveyi. The positive result of our strategy of cloning with the use of the genomic library in lambda vector suggests that the same method might be useful in the isolation of the groE homologues from other bacteria. The V. harveyi cloned groE genes did not suppress thermosensitivity of the E. coli groE mutants.

scholarly journals Chromosomal Changes during Experimental Evolution in Laboratory Populations of Escherichia coli

1999 ◽  
Vol 181 (4) ◽  
pp. 1360-1363 ◽  
Author(s):  
Ulfar Bergthorsson ◽  
Howard Ochman
Keyword(s):  
Escherichia Coli ◽  
Experimental Evolution ◽  
Restriction Enzymes ◽  
Short Term ◽  
E Coli ◽  
Restriction Sites ◽  
Laboratory Populations ◽  
Chromosome Alteration ◽  
Experimental Populations ◽  
Chromosomal Changes

ABSTRACT Short-term rates of chromosome evolution were analyzed in experimental populations of Escherichia coli B that had been propagated for 2,000 generations under four thermal regimens. Chromosome alterations were monitored in 24 independent populations by pulsed-field gel electrophoresis of DNA treated with five rare-cutting restriction enzymes. A total of 11 changes, 8 affecting chromosome size and 3 altering restriction sites, were observed in these populations, with none occurring in strains cultured at 37°C. Considering the changes detected in these experimental populations, the rate of chromosome alteration of E. coli is estimated to be half of that observed in experimental populations of yeast.

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