scholarly journals Restoration of wild-type motility to flagellin-knockout Escherichia coli

2019 ◽  
Author(s):  
Nicholas M. Thomson ◽  
Mark J. Pallen
Keyword(s):  
Escherichia Coli ◽  
Copy Number ◽  
Plasmid Copy Number ◽  
Major Constituent ◽  
Strongly Correlated ◽  
Wild Type ◽  
Plasmid Copy ◽  
Inducible Promoters ◽  
E Coli ◽  
Flagellar Filament

AbstractFlagellin is the major constituent of the flagellar filament and faithful restoration of wild-type motility to flagellin mutants may be beneficial for studies of flagellar biology and biotechnological exploitation of the flagellar system. Therefore, we explored the restoration of motility by flagellin expressed from a variety of combinations of promoter, plasmid copy number and induction strength. Motility was only partially restored using the tightly regulated rhamnose promoter, but wild-type motility was achieved with the T5 promoter, which, although leaky, allowed titration of induction strength. Motility was little affected by plasmid copy number when dependent on inducible promoters. However, plasmid copy number was important when expression was controlled by the native E. coli flagellin promoter. Motility was poorly correlated with flagellin transcription levels, but strongly correlated with the amount of flagellin associated with the flagellar filament, suggesting that excess monomers are either not exported or not assembled into filaments. This study provides a useful reference for further studies of flagellar function and a simple blueprint for similar studies with other proteins.

scholarly journals Escherichia coli O157:H7 Strains Isolated from High-Event Period Beef Contamination Have Strong Biofilm-Forming Ability and Low Sanitizer Susceptibility, Which Are Associated with High pO157 Plasmid Copy Number

2016 ◽  
Vol 79 (11) ◽  
pp. 1875-1883 ◽  
Author(s):  
RONG WANG ◽  
BRANDON E. LUEDTKE ◽  
JOSEPH M. BOSILEVAC ◽  
JOHN W. SCHMIDT ◽  
NORASAK KALCHAYANAND ◽  
...  
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Biofilm Formation ◽  
Copy Number ◽  
Plasmid Copy Number ◽  
Bacterial Biofilm ◽  
Escherichia Coli O157 ◽  
Plasmid Copy ◽  
E Coli ◽  
High Event

ABSTRACT In the meat industry, a high-event period (HEP) is defined as a time period when beef processing establishments experience an increased occurrence of product contamination by Escherichia coli O157:H7. Our previous studies suggested that bacterial biofilm formation and sanitizer resistance might contribute to HEPs. We conducted the present study to further characterize E. coli O157:H7 strains isolated during HEPs for their potential to cause contamination and to investigate the genetic basis for their strong biofilm-forming ability and high sanitizer resistance. Our results show that, compared with the E. coli O157:H7 diversity control panel strains, the HEP strains had a significantly higher biofilm-forming ability on contact surfaces and a lower susceptibility to common sanitizers. No difference in the presence of disinfectant-resistant genes or the prevalence of antibiotic resistance was observed between the HEP and control strains. However, the HEP strains retained significantly higher copy numbers of the pO157 plasmid. A positive correlation was observed among a strain's high plasmid copy number, strong biofilm-forming ability, low sanitizer susceptibility, and high survival and recovery capability after sanitization, suggesting that these specific phenotypes could be either directly correlated to gene expression on the pO157 plasmid or indirectly regulated via chromosomal gene expression influenced by the presence of the plasmid. Our data highlight the potential risk of biofilm formation and sanitizer resistance in HEP contamination by E. coli O157:H7, and our results call for increased attention to proper and effective sanitization practices in meat processing facilities.

scholarly journals Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli

2006 ◽  
Vol 123 (3) ◽  
pp. 273-280 ◽  
Author(s):  
Changsoo Lee ◽  
Jaai Kim ◽  
Seung Gu Shin ◽  
Seokhwan Hwang
Keyword(s):  
Escherichia Coli ◽  
Copy Number ◽  
Plasmid Copy Number ◽  
Plasmid Copy ◽  
Qpcr Quantification

scholarly journals RepB C-terminus mutation of an ori pRi vector affects plasmid copy number inAgrobacteriumand transgene copy number in plants

2018 ◽  
Author(s):  
Zarir Vaghchhipawala ◽  
Sharon Radke ◽  
Ervin Nagy ◽  
Mary L. Russell ◽  
Susan Johnson ◽  
...  
Keyword(s):  
Transgenic Plants ◽  
Copy Number ◽  
Binary Vector ◽  
Single Copy ◽  
Plasmid Copy Number ◽  
Plant Production ◽  
Silent Mutation ◽  
Wild Type ◽  
Plasmid Copy ◽  
C Terminus

AbstractA nativerepABCreplication origin, ori pRi, was previously reported as a single copy plasmid inAgrobacterium tumefaciensand can improve the production of transgenic plants with a single copy insertion of transgenes when it is used in binary vectors forAgrobacterium-mediatedtransformation. A high copy ori pRi variant plasmid, pTF::Ri, which does not improve the frequency of single copy transgenic plants, has been reported in the literature. Sequencing the high copy pTF::RirepABCoperon revealed the presence of two mutations: one silent mutation and one missense mutation that changes a tyrosine to a histidine (Y299H) in a highly conserved area of the C-terminus of the RepB protein (RepBY299H). Reproducing these mutations in the wild-type oriRi binary vector showed thatAgrobacteriumcells with the RepBY299Hmutation grow faster on both solidified and in liquid medium, and have higher plasmid copy number as determined by ddPCR. In order to investigate the impact of the RepBY299Hmutation on transformation and quality plant production, the RepBY299Hmutated ori pRi binary vector was compared with the original wild-type ori pRi binary vector and a multi-copy oriV binary vector in canola transformation. Molecular analyses of the canola transgenic plants demonstrated that the multi-copy ori pRi with the RepBY299Hmutation inAgrobacteriumcells lost the advantage of generating high frequency single copy, backbone-free transgenic plants compared to using the single copy wild-type ori pRi binary vector.

scholarly journals Pyruvate Kinase-Deficient Escherichia coli Exhibits Increased Plasmid Copy Number and Cyclic AMP Levels

2009 ◽  
Vol 191 (9) ◽  
pp. 3041-3049 ◽  
Author(s):  
Drew S. Cunningham ◽  
Zhu Liu ◽  
Nathan Domagalski ◽  
Richard R. Koepsel ◽  
Mohammad M. Ataai ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Pyruvate Kinase ◽  
Copy Number ◽  
Plasmid Copy Number ◽  
Phosphotransferase System ◽  
Plasmid Copy ◽  
Copy Numbers ◽  
High Copy Number Plasmid ◽  
Pathway Flux

ABSTRACT Previously established consequences of abolishing pyruvate kinase (Pyk) activity in Escherichia coli during aerobic growth on glucose include reduced acetate production, elevated hexose monophosphate (HMP) pathway flux, elevated phosphoenolpyruvate carboxylase (Ppc) flux, and an increased ratio of phosphoenolpyruvate (PEP) to pyruvate. These traits inspired two hypotheses. First, the mutant (PB25) may maintain more plasmid than the wild type (JM101) by combining traits reported to facilitate plasmid DNA synthesis (i.e., decreased Pyk flux and increased HMP pathway and Ppc fluxes). Second, PB25 likely possesses a higher level of cyclic AMP (cAMP) than JM101. This is based on reports that connect elevated PEP/pyruvate ratios to phosphotransferase system signaling and adenylate cyclase activation. To test the first hypothesis, the strains were transformed with a pUC-based, high-copy-number plasmid (pGFPuv), and copy numbers were measured. PB25 exhibited a fourfold-higher copy number than JM101 when grown at 37°C. At 42°C, its plasmid content was ninefold higher than JM101 at 37°C. To test the second hypothesis, cAMP was measured, and the results confirmed it to be higher in PB25 than JM101. This elevation was not enough to elicit a strong regulatory effect, however, as indicated by the comparative expression of the pGFPuv-based reporter gene, gfp uv , under the control of the cAMP-responsive lac promoter. The elevated cAMP in PB25 suggests that Pyk may participate in glucose catabolite repression by serving among all of the factors that tighten gene expression.

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