Construction and Initial Characterization of Escherichia coli Strains with Few or No Intact Chromosomal rRNA Operons

ABSTRACT The Escherichia coli genome carries seven rRNA (rrn) operons, each containing three rRNA genes. The presence of multiple operons has been an obstacle to many studies of rRNA because the effect of mutations in one operon is diluted by the six remaining wild-type copies. To create a tool useful for manipulating rRNA, we sequentially inactivated from one to all seven of these operons with deletions spanning the 16S and 23S rRNA genes. In the final strain, carrying no intact rRNA operon on the chromosome, rRNA molecules were expressed from a multicopy plasmid containing a single rRNA operon (prrn). Characterization of these rrndeletion strains revealed that deletion of two operons was required to observe a reduction in the growth rate and rRNA/protein ratio. When the number of deletions was extended from three to six, the decrease in the growth rate was slightly more than the decrease in the rRNA/protein ratio, suggesting that ribosome efficiency was reduced. This reduction was most pronounced in the Δ7 prrn strain, in which the growth rate, unlike the rRNA/protein ratio, was not completely restored to wild-type levels by a cloned rRNA operon. The decreases in growth rate and rRNA/protein ratio were surprisingly moderate in the rrndeletion strains; the presence of even a single operon on the chromosome was able to produce as much as 56% of wild-type levels of rRNA. We discuss possible applications of these strains in rRNA studies.

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Identification and characterization of intervening sequences within 23S rRNA genes from more than 200 Campylobacter isolates from seven species including atypical campylobacters

BMC Microbiology ◽

10.1186/1471-2180-9-256 ◽

2009 ◽

Vol 9 (1) ◽

pp. 256 ◽

Cited By ~ 6

Author(s):

Akihiro Tazumi ◽

Yuki Kakinuma ◽

Naoaki Misawa ◽

John E Moore ◽

Beverley C Millar ◽

...

Keyword(s):

Rrna Genes ◽

23S Rrna ◽

Intervening Sequences ◽

23S Rrna Genes ◽

Identification And Characterization

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Characterization of Agrobacterium vitis Strains Isolated from Feral Vitis riparia

Plant Disease ◽

10.1094/pdis.1999.83.2.102 ◽

1999 ◽

Vol 83 (2) ◽

pp. 102-107 ◽

Cited By ~ 10

Author(s):

T. J. Burr ◽

C. L. Reid ◽

C. E. Adams ◽

E. A. Momol

Keyword(s):

New York ◽

New York State ◽

Rrna Genes ◽

23S Rrna ◽

Agrobacterium Vitis ◽

Control Activity ◽

Vitis Riparia ◽

23S Rrna Genes ◽

Physiological Tests

Agrobacterium vitis was isolated from roots of 41 of 66 feral Vitis riparia vines collected in three different regions of New York State. Two of the regions were more than 150 km from commercial vineyards. The strains were highly diverse as determined by DNA fingerprinting of the chromosomal region lying between the 16S and 23S rRNA genes. Of 24 strains examined, 15 different fingerprints were generated, and none was identical to fingerprints generated by previously identified groups of tumorigenic A. vitis strains. Results of physiological tests that were done to characterize strains from V. riparia conformed closely to those expected for A. vitis, except that 23 of 26 strains did not utilize tartrate. All strains were nontumorigenic, did not hybridize with a probe consisting of T-DNA genes, did not utilize octopine or nopaline, and carried zero to three plasmids. Of 26 strains, 7 inhibited A. vitis strain K306 from causing galls at wound sites on grape as well as or better than a previously studied nontumorigenic A. vitis strain, F2/5, that is known to have biological control activity.

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Ribosomal Mutations in Arcanobacterium pyogenes Confer a Unique Spectrum of Macrolide Resistance

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.48.3.1021-1023.2004 ◽

2004 ◽

Vol 48 (3) ◽

pp. 1021-1023 ◽

Cited By ~ 8

Author(s):

B. Helen Jost ◽

Hien T. Trinh ◽

J. Glenn Songer ◽

Stephen J. Billington

Keyword(s):

Escherichia Coli ◽

Macrolide Resistance ◽

Rrna Genes ◽

23S Rrna ◽

23S Rrna Genes ◽

Arcanobacterium Pyogenes

ABSTRACT Four macrolide-resistant Arcanobacterium pyogenes isolates contained A2058T, A2058G, or C2611G (Escherichia coli numbering) mutations in their 23S rRNA genes. While these mutations conferred resistance to erythromycin, oleandomycin, and spiramycin, they did not confer resistance to tylosin.

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Molecular characterization of intervening sequences in 23S rRNA genes and 23S rRNA fragmentation in Taylorella equigenitalis

Folia Microbiologica ◽

10.1007/s12223-008-0076-0 ◽

2008 ◽

Vol 53 (6) ◽

pp. 486-492 ◽

Cited By ~ 4

Author(s):

A. Tazumi ◽

T. Sekizuka ◽

J. E. Moore ◽

B. C. Millar ◽

I. Taneike ◽

...

Keyword(s):

Molecular Characterization ◽

Rrna Genes ◽

23S Rrna ◽

Taylorella Equigenitalis ◽

Intervening Sequences ◽

23S Rrna Genes

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Identification of Cold Shock Gene Loci inSinorhizobium meliloti by Using a luxABReporter Transposon

Applied and Environmental Microbiology ◽

10.1128/aem.66.1.401-405.2000 ◽

2000 ◽

Vol 66 (1) ◽

pp. 401-405 ◽

Cited By ~ 12

Author(s):

Kevin P. O'Connell ◽

Ann M. Gustafson ◽

M. Deane Lehmann ◽

Michael F. Thomashow

Keyword(s):

Escherichia Coli ◽

Sinorhizobium Meliloti ◽

Cold Shock ◽

Insertion Site ◽

Temperature Shift ◽

Rrna Genes ◽

23S Rrna ◽

Shock Gene ◽

23S Rrna Genes ◽

Transposon Insertions

ABSTRACT Using a luxAB reporter transposon, seven mutants ofSinorhizobium meliloti were identified as containing insertions in four cold shock loci. LuxAB activity was strongly induced (25- to 160-fold) after a temperature shift from 30 to 15°C. The transposon and flanking host DNA from each mutant was cloned, and the nucleic acid sequence of the insertion site was determined. Unexpectedly, five of the seven luxAB mutants contained transposon insertions in the 16S and 23S rRNA genes of two of the threerrn operons of S. meliloti. Directed insertion of luxAB genes into each of the three rrnoperons revealed that all three operons were similarly affected by cold shock. Two other insertions were found to be located downstream of a homolog of the major Escherichia coli cold shock gene,cspA. Although the cold shock loci were highly induced in response to a shift to low temperature, none of the insertions resulted in a statistically significant decrease in growth rate at 15°C.

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Simultaneous detection and genotyping of three genomic groups of Borrelia burgdorferi sensu lato in Dutch Ixodes ricinus ticks by characterization of the amplified intergenic spacer region between 5S and 23S rRNA genes.

Journal of Clinical Microbiology ◽

10.1128/jcm.33.12.3091-3095.1995 ◽

1995 ◽

Vol 33 (12) ◽

pp. 3091-3095 ◽

Cited By ~ 173

Author(s):

S G Rijpkema ◽

M J Molkenboer ◽

L M Schouls ◽

F Jongejan ◽

J F Schellekens

Keyword(s):

Borrelia Burgdorferi ◽

Simultaneous Detection ◽

Intergenic Spacer ◽

Rrna Genes ◽

23S Rrna ◽

Borrelia Burgdorferi Sensu Lato ◽

Intergenic Spacer Region ◽

Ixodes Ricinus Ticks ◽

23S Rrna Genes

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Molecular characterization of three mutations in katG affecting the activity of hydroperoxidase I of Escherichia coli

Biochemistry and Cell Biology ◽

10.1139/o90-153 ◽

1990 ◽

Vol 68 (7-8) ◽

pp. 1037-1044 ◽

Cited By ~ 14

Author(s):

Peter C. Loewen ◽

Jacek Switala ◽

Mark Smolenski ◽

Barbara L. Triggs-Raine

Keyword(s):

Escherichia Coli ◽

Specific Activity ◽

Polymerase Chain Reaction Amplification ◽

Protein A ◽

Wild Type ◽

Wild Type Enzyme ◽

Gene Encoding ◽

Reaction Amplification ◽

Mutant Genes

Hydroperoxidase I (HPI) of Escherichia coli is a bifunctional enzyme exhibiting both catalase and peroxidase activities. Mutants lacking appreciable HPI have been generated using nitrosoguanidine and the gene encoding HPI, katG, has been cloned from three of these mutants using either classical probing methods or polymerase chain reaction amplification. The mutant genes were sequenced and the changes from wild-type sequence identified. Two mutants contained G to A changes in the coding strand, resulting in glycine to aspartate changes at residues 119 (katG15) and 314 (katG16) in the deduced amino acid sequence of the protein. A third mutant contained a C to T change resulting in a leucine to phenylalanine change at residue 139 (katG14). The Phe139-, Asp119-, and Asp314-containing mutants exhibited 13, < 1, and 18%, respectively, of the wild-type catalase specific activity and 43, 4, and 45% of the wild-type peroxidase specific activity. All mutant enzymes bound less protoheme IX than the wild-type enzyme. The sensitivities of the mutant enzymes to the inhibitors hydroxylamine, azide, and cyanide and the activators imidazole and Tris were similar to those of the wild-type enzyme. The mutant enzymes were more sensitive to high temperature and to β-mercaptoethanol than the wild-type enzyme. The pH profiles of the mutant catalases were unchanged from the wild-type enzyme.Key words: catalase, hydroperoxidase I, mutants, sequence analysis.

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Demonstration of the absence of intervening sequences within 23S rRNA genes fromCampylobacter lari

Journal of Basic Microbiology ◽

10.1002/jobm.200800232 ◽

2009 ◽

Vol 49 (4) ◽

pp. 386-394 ◽

Cited By ~ 1

Author(s):

Akihiro Tazumi ◽

Yuki Kakinuma ◽

John E. Moore ◽

Cherie B. Millar ◽

Ikue Taneike ◽

...

Keyword(s):

Rrna Genes ◽

23S Rrna ◽

Intervening Sequences ◽

23S Rrna Genes

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EDL933 Strains of Escherichia coli O157 can Demonstrate Genetic Diversity and Differential Adherence to Bovine Recto-Anal Junction Squamous Epithelial Cells

The Open Microbiology Journal ◽

10.2174/1874285802115010129 ◽

2021 ◽

Vol 15 (1) ◽

pp. 129-138

Author(s):

Raegan S. Hoefler ◽

Indira T. Kudva

Keyword(s):

Escherichia Coli ◽

Wild Type ◽

Cell Adherence ◽

O157 Strain ◽

Shiga Toxins ◽

The Us ◽

Strain Type ◽

Enterocyte Effacement ◽

Different Sources

Background: Differences between Escherichia coli O157 (O157) strains are well-established with some of these strains being associated with major outbreaks in the US. EDL933 is one such O157 strain that caused a multistate outbreak in 1982 and has since been used as a prototype in various O157-related experiments. Objective: As O157 can readily acquire genetic mutations, we sought to determine if the genetic and phenotypic profiles of EDL933 strains from different sources would be consistent. Methods: We evaluated wild-type O157 strains stocked as EDL933 from three different laboratories, in the strain typing Polymorphic Amplified Typing Sequence (PATS) and the bovine rectal-anal junction squamous epithelial (RSE) cell- and HEp-2 cell- adherence assays. In addition, we also verified if Shiga toxins (Stx), the Locus of Enterocyte Effacement (LEE) or curli fimbriae contributed to the adherence phenotypes observed using mutant and wild-type EDL933 isolates. Results: Our results showed differences in PATS profiles and RSE cell-adherence phenotype, with no influence from the Stx or LEE genes, between EDL933 from different sources. Interestingly, the EDL933 strain that demonstrated the most contrasting diffuse adherence phenotype on RSE cells, EDL933-T, had decreased curli production that may have contributed to this phenotype. Conclusion: Our observations suggest that a comprehensive characterization of bacterial isolates, even if assigned to the same strain type prior to use in experiments, is warranted to ensure consistency and reproducibility of results.

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