scholarly journals Characterization of rapidly labelled ribonucleic acid in Escherichia coli by deoxyribonucleic acid–ribonucleic acid hybridization

1968 ◽  
Vol 110 (2) ◽  
pp. 251-263 ◽  
Author(s):  
G. H. Pigott ◽  
J. E. M. Midgley
Keyword(s):  
Escherichia Coli ◽  
Ribosomal Rna ◽  
Ribonucleic Acid ◽  
Messenger Rna ◽  
Rna Binding ◽  
Hybridization Technique ◽  
E Coli ◽  
K 12 ◽  
Dna 2

1. Rapidly labelled RNA from Escherichia coli K 12 was characterized by hybridization to denatured E. coli DNA on cellulose nitrate membrane filters. The experiments were designed to show that, if sufficient denatured DNA is offered in a single challenge, practically all the rapidly labelled RNA will hybridize. With the technique employed, 75–80% hybridization efficiency could be obtained as a maximum. Even if an excess of DNA sites were offered, this value could not be improved upon in any single challenge of rapidly labelled RNA with denatured E. coli DNA. 2. It was confirmed that the hybridization technique can separate the rapidly labelled RNA into two fractions. One of these (30% of the total) was efficiently hybridized with the low DNA/RNA ratio (10:1, w/w) used in tests. The other fraction (70% of the total) was hybridized to DNA at low efficiencies with the DNA/RNA ratio 10:1, and was hybridized progressively more effectively as the amount of denatured DNA was increased. A practical maximum of 80% hybridization of all the rapidly labelled RNA was first achieved at a DNA/RNA ratio 210:1 (±10:1). This fraction was fully representative of the rapidly labelled RNA with regard to kind and relative amount of materials hybridized. 3. In competition experiments, where additions were made of unlabelled RNA prepared from E. coli DNA, DNA-dependent RNA polymerase (EC 2.7.7.6) and nucleoside 5′-triphosphates, the rapidly labelled RNA fraction hybridized at a low (10:1) DNA/RNA ratio was shown to be competitive with a product from genes other than those responsible for ribosomal RNA synthesis and thus was presumably messenger RNA. At higher DNA/rapidly labelled RNA ratios (200:1), competition with added unlabelled E. coli ribosomal RNA (without messenger RNA contaminants) lowered the hybridization of the rapidly labelled RNA from its 80% maximum to 23%. This proportion of rapidly labelled RNA was not competitive with E. coli ribosomal RNA even when the latter was in large excess. The ribosomal RNA would also not compete with the 23% rapidly labelled RNA bound to DNA at low DNA/RNA ratios. It was thus demonstrated that the major part of E. coli rapidly labelled RNA (70%) is ribosomal RNA, presumably a precursor to the RNA in mature ribosomes. 4. These studies have shown that, when earlier workers used low DNA/RNA ratios (about 10:1) in the assay of messenger RNA in bacterial rapidly labelled RNA, a reasonable estimate of this fraction was achieved. Criticisms that individual messenger RNA species may be synthesized from single DNA sites in E. coli at rates that lead to low efficiencies of messenger RNA binding at low DNA/RNA ratios are refuted. In accordance with earlier results, estimations of the messenger RNA content of E. coli in both rapidly labelled and randomly labelled RNA show that this fraction is 1·8–1·9% of the total RNA. This shows that, if any messenger RNA of relatively long life exists in E. coli, it does not contribute a measurable weight to that of rapidly labelled messenger RNA.

scholarly journals Identification and Characterization of a New Lipoprotein, NlpI, in Escherichia coli K-12

1999 ◽  
Vol 181 (14) ◽  
pp. 4318-4325 ◽  
Author(s):  
Masaru Ohara ◽  
Henry C. Wu ◽  
Krishnan Sankaran ◽  
Paul D. Rick
Keyword(s):  
Escherichia Coli ◽  
Direct Consequence ◽  
Insertion Mutant ◽  
Polynucleotide Phosphorylase ◽  
Wild Type ◽  
E Coli ◽  
K 12 ◽  
Identification And Characterization ◽  
Lines Of Evidence

ABSTRACT We report here the identification of a new lipoprotein, NlpI, inEscherichia coli K-12. The NlpI structural gene (nlpI) is located between the genes pnp(polynucleotide phosphorylase) and deaD (RNA helicase) at 71 min on the E. coli chromosome. The nlpI gene encodes a putative polypeptide of approximately 34 kDa, and multiple lines of evidence clearly demonstrate that NlpI is indeed a lipoprotein. An nlpI::cm mutation rendered growth of the cells osmotically sensitive, and incubation of the insertion mutant at an elevated temperature resulted in the formation of filaments. The altered phenotype of the mutant was a direct consequence of the mutation in nlpI, since it was complemented by the wild-type nlpI gene alone. Overexpression of the unaltered nlpI gene in wild-type cells resulted in the loss of the rod morphology and the formation of single prolate ellipsoids and pairs of prolate ellipsoids joined by partial constrictions. NlpI may be important for an as-yet-undefined step in the overall process of cell division.

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 Molecular Characterization of UpaB and UpaC, Two New Autotransporter Proteins of Uropathogenic Escherichia coli CFT073

2011 ◽  
Vol 80 (1) ◽  
pp. 321-332 ◽  
Author(s):  
Luke P. Allsopp ◽  
Christophe Beloin ◽  
Glen C. Ulett ◽  
Jaione Valle ◽  
Makrina Totsika ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Molecular Characterization ◽  
Host Responses ◽  
Upec Strain ◽  
Content Type ◽  
Pcr Screening ◽  
E Coli ◽  
K 12 ◽  
Encoding Genes

ABSTRACTUropathogenicEscherichia coli(UPEC) is the primary cause of urinary tract infection (UTI) in the developed world. The major factors associated with virulence of UPEC are fimbrial adhesins, which mediate specific attachment to host receptors and trigger innate host responses. Another group of adhesins is represented by the autotransporter (AT) subgroup of proteins. The genome-sequenced prototype UPEC strain CFT073 contains 11 putative AT-encoding genes. In this study, we have performed a detailed molecular characterization of two closely related AT adhesins from CFT073: UpaB (c0426) and UpaC (c0478). PCR screening revealed that theupaBandupaCAT-encoding genes are common inE. coli. TheupaBandupaCgenes were cloned and characterized in a recombinantE. coliK-12 strain background. This revealed that they encode proteins located at the cell surface but possess different functional properties: UpaB mediates adherence to several ECM proteins, while UpaC expression is associated with increased biofilm formation. In CFT073,upaBis expressed whileupaCis transcriptionally repressed by the global regulator H-NS. In competitive colonization experiments employing the mouse UTI model, CFT073 significantly outcompeted itsupaB(but notupaC) isogenic mutant strain in the bladder. This attenuated phenotype was also observed in single-challenge experiments, where deletion of theupaBgene in CFT073 significantly reduced early colonization of the bladder.

scholarly journals Isolation of Generalized Transducing Bacteriophages for Uropathogenic Strains of Escherichia coli

2011 ◽  
Vol 77 (18) ◽  
pp. 6630-6635 ◽  
Author(s):  
E. J. Battaglioli ◽  
G. A. Baisa ◽  
A. E. Weeks ◽  
R. A. Schroll ◽  
A. J. Hryckowian ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance Gene ◽  
Treatment Plant ◽  
Content Type ◽  
E Coli ◽  
Multiple Loci ◽  
Isolation And Characterization ◽  
Strain Cft073 ◽  
K 12

ABSTRACTThe traditional genetic procedure for random or site-specific mutagenesis inEscherichia coliK-12 involves mutagenesis, isolation of mutants, and transduction of the mutation into a clean genetic background. The transduction step reduces the likelihood of complications due to secondary mutations. Though well established, this protocol is not tenable for many pathogenicE. colistrains, such as uropathogenic strain CFT073, because it is resistant to known K-12 transducing bacteriophages, such as P1. CFT073 mutants generated via a technique such as lambda Red mutagenesis may contain unknown secondary mutations. Here we describe the isolation and characterization of transducing bacteriophages for CFT073. Seventy-seven phage isolates were acquired from effluent water samples collected from a wastewater treatment plant in Madison, WI. The phages were differentiated by a host sensitivity-typing scheme with a panel ofE. colistrains from the ECOR collection and clinical uropathogenic isolates. We found 49 unique phage isolates. These were then examined for their ability to transduce antibiotic resistance gene insertions at multiple loci between different mutant strains of CFT073. We identified 4 different phages capable of CFT073 generalized transduction. These phages also plaque on the model uropathogenicE. colistrains 536, UTI89, and NU14. The highest-efficiency transducing phage, ΦEB49, was further characterized by DNA sequence analysis, revealing a double-stranded genome 47,180 bp in length and showing similarity to other sequenced phages. When combined with a technique like lambda Red mutagenesis, the newly characterized transducing phages provide a significant development in the genetic tools available for the study of uropathogenicE. coli.

scholarly journals The control of ribonucleic acid synthesis in bacteria. Steady-state content of messenger ribonucleic acid in Escherichia coli M.R.E. 600

1970 ◽  
Vol 120 (2) ◽  
pp. 279-288 ◽  
Author(s):  
W. J. H. Gray ◽  
J. E. M. Midgley
Keyword(s):  
Escherichia Coli ◽  
Ribosomal Rna ◽  
Ribonucleic Acid ◽  
Messenger Rna ◽  
Acid Synthesis ◽  
Growth Conditions ◽  
Transfer Rna ◽  
Average Lifetime ◽  
Messenger Ribonucleic Acid ◽  
Wide Range

1. The technique of DNA–RNA hybridization was used to follow changes in the amount and average lifetime of unstable messenger RNA in Escherichia coli M.R.E. 600 over a wide range of different growth conditions. The method of analysis was based on the kinetics of incorporation of exogenous labelled nucleic acid bases into the RNA of steadily growing cultures, as described by Bolton & McCarthy (1962). 2. The ratio of the average lifetime of messenger RNA to the mean generation time of E. coli cultures was constant over the temperature range 25–45°C in a given medium, but the constant varied with the nature of the growth medium. For cultures growing in sodium lactate–salts or glucose–salts media the ratio was 0.046±0.005 and in enriched broth it was 0.087±0.009. Measurements of the amounts of transfer RNA, ribosomal RNA and messenger RNA were also made. The results confirmed earlier reports that the ratio of the amount of messenger RNA to the amount of ribosomes in the cells is virtually constant. On the other hand, the ratio of the amount of transfer RNA to the amount of ribosomal RNA decreased with increasing growth rate at a given temperature. 3. In cultures at temperatures higher than necessary for optimum rates of growth the average lifetime of messenger RNA lengthened in harmony with the increased time required for cell division. It seems that suboptimum growth rates at higher temperatures cannot be explained simply as a combination of increased rates of synthesis and breakdown of messenger RNA with a grossly decreased efficiency of translation. The absolute rate of messenger RNA synthesis was lowered, and its amount in the cells was typical of all other cultures grown at lower temperatures in the same medium. 4. The rate of entry of exogenous labelled uracil into unstable messenger RNA and stable ribosomal RNA was constant in all media at all temperatures in the approximate ratio 1:2. In media supporting a lower rate of growth, e.g. lactate–salts or glucose–salts media, the messenger RNA fraction constituted 2.2±0.3% of the total cellular RNA. In enriched broth 3.6±0.3% of the total RNA was messenger.

scholarly journals Biofilm Formation and Dispersal under the Influence of the Global Regulator CsrA of Escherichia coli

2002 ◽  
Vol 184 (1) ◽  
pp. 290-301 ◽  
Author(s):  
Debra W. Jackson ◽  
Kazushi Suzuki ◽  
Lawrence Oakford ◽  
Jerry W. Simecka ◽  
Mark E. Hart ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Rna Binding ◽  
Glycogen Synthase ◽  
Regulatory Protein ◽  
Ectopic Expression ◽  
Knockout Mutant ◽  
E Coli ◽  
K 12 ◽  
Biofilm Development

ABSTRACT The predominant mode of growth of bacteria in the environment is within sessile, matrix-enclosed communities known as biofilms. Biofilms often complicate chronic and difficult-to-treat infections by protecting bacteria from the immune system, decreasing antibiotic efficacy, and dispersing planktonic cells to distant body sites. While the biology of bacterial biofilms has become a major focus of microbial research, the regulatory mechanisms of biofilm development remain poorly defined and those of dispersal are unknown. Here we establish that the RNA binding global regulatory protein CsrA (carbon storage regulator) of Escherichia coli K-12 serves as both a repressor of biofilm formation and an activator of biofilm dispersal under a variety of culture conditions. Ectopic expression of the E. coli K-12 csrA gene repressed biofilm formation by related bacterial pathogens. A csrA knockout mutation enhanced biofilm formation in E. coli strains that were defective for extracellular, surface, or regulatory factors previously implicated in biofilm formation. In contrast, this csrA mutation did not affect biofilm formation by a glgA (glycogen synthase) knockout mutant. Complementation studies with glg genes provided further genetic evidence that the effects of CsrA on biofilm formation are mediated largely through the regulation of intracellular glycogen biosynthesis and catabolism. Finally, the expression of a chromosomally encoded csrA′-′lacZ translational fusion was dynamically regulated during biofilm formation in a pattern consistent with its role as a repressor. We propose that global regulation of central carbon flux by CsrA is an extremely important feature of E. coli biofilm development.

scholarly journals Phenotypic and multi-omics characterization of Escherichia coli K-12 adapted to quaternary ammonium compounds identifies lipid A and cell envelope alterations regulated by mar-sox-rob and stress inducible pathways

2020 ◽  
Author(s):  
Kari A.C. Green ◽  
Branden S.J. Gregorchuk ◽  
Shelby L. Reimer ◽  
Nicola H. Cartwright ◽  
Daniel R. Beniac ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Quaternary Ammonium ◽  
Lipid A ◽  
Quaternary Ammonium Compounds ◽  
Cross Resistance ◽  
E Coli ◽  
Lipid A Biosynthesis ◽  
K 12 ◽  
Ammonium Compounds

AbstractQuaternary ammonium compounds (QACs) benzalkonium (BZK) and cetrimide (CET) are common disinfectants used to inhibit or eradicate Gram-negative bacteria in clinical and agricultural products. QAC tolerance in Escherichia coli and other Enterobacterales species can confer cross-resistance to various clinically used antibiotics, making it important to understand mechanisms of QAC tolerance in greater depth. QAC adaptation by E. coli is hypothesized to alter MarRAB regulated genes that converge on the outer membrane, specifically, lipid A biosynthesis and transport genes, porins, and efflux pump systems. To test this, we performed a ‘multi’-omics and phenotypic characterization of E. coli K-12 adapted to BZK and CET, to assess how QACs alter cell growth, genomics, and proteomics. E. coli adapted to either BZK and CET resulted in strains with stable QAC tolerance when either drug was omitted, elongated and narrower cell morphologies by scanning electron microscopy, and reduced growth fitness when compared to un-adapted E. coli. Antimicrobial susceptibility testing revealed that QAC adaptation increased E. coli tolerance by ≥4-fold to BZK, CET, and other QACs but no antibiotic cross-resistance. Single nucleotide variants identified by whole genome sequencing and differentially accumulated proteins by liquid chromatography-mass spectrometry identified alterations to various QAC-adapted E. coli genes and proteins belonging to: lipid A biosynthesis and transport (lpxLM, msbA, mla), the mar-sox-rob regulatory pathway (marR, rob), DNA/protein translation (gyrA, rpsA, rpoB, rapA). These alterations validate the hypothesis that mar-sox-rob network plays a role in QAC tolerance and identifies additional stress inducible genetic and protein QAC tolerant biomarkers.ImportanceBacterial tolerance mechanisms associated with disinfectant QAC adaptation is hypothesized to overlap with the mar-sox-rob multiple antimicrobial resistance pathway but has not been directly shown. Here, we generate QAC tolerant E. coli strains and identify phenotypic changes associated with protein and genetic alterations caused by prolonged QAC exposure. We identified genes that overlap with known antibiotic resistance mechanisms as well as distinct genes and proteins specific to QAC adaptation that are useful for future bacterial disinfectant tolerance mechanism studies. However, these altered genes and proteins implicate MarR and Rob pathways specifically in QAC tolerance but, surprisingly, the involvement of mar-sox-rob pathways did not increase antibiotic cross-resistance. Many altered genes we identified were essential genes in lipid A biosynthesis/transport, DNA and RNA transcription, and protein regulation systems potentially explaining why only QAC cross-tolerance was observed and why we observed greater cell fitness costs despite MarR and Rob pathway involvement.

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