Abstract
The molecular mechanisms controlling expression of the long polar fimbriae 2 (Lpf2) of enterohemorrhagic Escherichia coli (EHEC) O157:H7 were evaluated. Primer extension was used to locate the lpfA2 transcriptional start site in EHEC strain EDL933 at 171 bp upstream of the lpfA2 start codon. Semi-quantitative RT-PCR demonstrated that the highest lpfA2 expression occurs between an OD<inf>600</inf> of 1.0 and 1.2 in DMEM at pH 6.5 and 37 °C. The level of lpfA2 transcription at OD<inf>600</inf> 1.2 and pH 6.5 was four times greater than that at pH 7.2. Although lpfA2 expression was decreased under iron-depleted conditions, its expression was increased in a ferric-uptake-regulator (Fur) mutant strain. The lpfA2 transcript was 0.7 and 2 times more abundant in wt EHEC grown in DMEM pH 6.5 plus iron and MacConkey broth at 25 °C, respectively, than in DMEM at pH 6.5. The lpf2 expression in DMEM pH 6.5 plus iron and bile salts was 2.7 times more abundant than baseline conditions. Further, transcription in the EDL933{increment}fur was 0.6 and 0.8 times higher as compared with the wt strain grown in DMEM pH 6.5 plus iron and MacConkey broth, respectively. Electrophoretic mobility shift assays showed that purified Fur interacts with the lpf2 regulatory region, indicating that Fur repression is exerted by direct binding to the promoter region. In summary, we demonstrated that the EHEC lpf2 operon is regulated in response to temperature, pH, bile salts and iron, during the exponential phase of growth, and is controlled by Fur.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 105-114 |
| Number of pages | 10 |
| Journal | FEMS Microbiology Letters |
| Volume | 357 |
| Issue number | 2 |
| DOIs | |
| State | Published - 2014 |
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Keywords
- Ferric-uptake-regulator
- Long polar 2 fimbriae
- lpf2 operon
ASJC Scopus subject areas
- Microbiology
- Genetics
- Molecular Biology
- Medicine(all)
Cite this
Environmental regulation of the long polar fimbriae 2 of enterohemorrhagic Escherichia coli O157 : H7. / Arenas-Hernández, Margarita M.; Rojas-López, Maricarmen; Medrano-López, Abraham; Nuñez-Reza, Karen J.; Puente, José Luis; Martínez-Laguna, Ygnacio; Torres, Alfredo.
In: FEMS Microbiology Letters, Vol. 357, No. 2, 2014, p. 105-114.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Environmental regulation of the long polar fimbriae 2 of enterohemorrhagic Escherichia coli O157
T2 - H7
AU - Arenas-Hernández, Margarita M.
AU - Rojas-López, Maricarmen
AU - Medrano-López, Abraham
AU - Nuñez-Reza, Karen J.
AU - Puente, José Luis
AU - Martínez-Laguna, Ygnacio
AU - Torres, Alfredo
PY - 2014
Y1 - 2014
N2 - The molecular mechanisms controlling expression of the long polar fimbriae 2 (Lpf2) of enterohemorrhagic Escherichia coli (EHEC) O157:H7 were evaluated. Primer extension was used to locate the lpfA2 transcriptional start site in EHEC strain EDL933 at 171 bp upstream of the lpfA2 start codon. Semi-quantitative RT-PCR demonstrated that the highest lpfA2 expression occurs between an OD600 of 1.0 and 1.2 in DMEM at pH 6.5 and 37 °C. The level of lpfA2 transcription at OD600 1.2 and pH 6.5 was four times greater than that at pH 7.2. Although lpfA2 expression was decreased under iron-depleted conditions, its expression was increased in a ferric-uptake-regulator (Fur) mutant strain. The lpfA2 transcript was 0.7 and 2 times more abundant in wt EHEC grown in DMEM pH 6.5 plus iron and MacConkey broth at 25 °C, respectively, than in DMEM at pH 6.5. The lpf2 expression in DMEM pH 6.5 plus iron and bile salts was 2.7 times more abundant than baseline conditions. Further, transcription in the EDL933{increment}fur was 0.6 and 0.8 times higher as compared with the wt strain grown in DMEM pH 6.5 plus iron and MacConkey broth, respectively. Electrophoretic mobility shift assays showed that purified Fur interacts with the lpf2 regulatory region, indicating that Fur repression is exerted by direct binding to the promoter region. In summary, we demonstrated that the EHEC lpf2 operon is regulated in response to temperature, pH, bile salts and iron, during the exponential phase of growth, and is controlled by Fur.
AB - The molecular mechanisms controlling expression of the long polar fimbriae 2 (Lpf2) of enterohemorrhagic Escherichia coli (EHEC) O157:H7 were evaluated. Primer extension was used to locate the lpfA2 transcriptional start site in EHEC strain EDL933 at 171 bp upstream of the lpfA2 start codon. Semi-quantitative RT-PCR demonstrated that the highest lpfA2 expression occurs between an OD600 of 1.0 and 1.2 in DMEM at pH 6.5 and 37 °C. The level of lpfA2 transcription at OD600 1.2 and pH 6.5 was four times greater than that at pH 7.2. Although lpfA2 expression was decreased under iron-depleted conditions, its expression was increased in a ferric-uptake-regulator (Fur) mutant strain. The lpfA2 transcript was 0.7 and 2 times more abundant in wt EHEC grown in DMEM pH 6.5 plus iron and MacConkey broth at 25 °C, respectively, than in DMEM at pH 6.5. The lpf2 expression in DMEM pH 6.5 plus iron and bile salts was 2.7 times more abundant than baseline conditions. Further, transcription in the EDL933{increment}fur was 0.6 and 0.8 times higher as compared with the wt strain grown in DMEM pH 6.5 plus iron and MacConkey broth, respectively. Electrophoretic mobility shift assays showed that purified Fur interacts with the lpf2 regulatory region, indicating that Fur repression is exerted by direct binding to the promoter region. In summary, we demonstrated that the EHEC lpf2 operon is regulated in response to temperature, pH, bile salts and iron, during the exponential phase of growth, and is controlled by Fur.
KW - Ferric-uptake-regulator
KW - Long polar 2 fimbriae
KW - lpf2 operon
UR - http://www.scopus.com/inward/record.url?scp=84906078922&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84906078922&partnerID=8YFLogxK
U2 - 10.1111/1574-6968.12513
DO - 10.1111/1574-6968.12513
M3 - Article
C2 - 24966050
AN - SCOPUS:84906078922
VL - 357
SP - 105
EP - 114
JO - FEMS Microbiology Letters
JF - FEMS Microbiology Letters
SN - 0378-1097
IS - 2
ER -