e coli o157 h7 strain edl933 atcc 700927  (ATCC)

Journal: BMC Microbiology

Article Title: SspA up-regulates gene expression of the LEE pathogenicity island by decreasing H-NS levels in enterohemorrhagic Escherichia coli

doi: 10.1186/1471-2180-12-231

Figure Lengend Snippet: SspA positively affects LEE expression in stationary phase cells. Primer extension analyses on total RNA extracted from wild type EHEC EDL933 (lane 1), the sspA mutant (lane 2) and the sspA mutant complemented with wild type sspA (lane 3) or mutant sspA84-86 (lane 4) as indicated, grown in LB at 37°C to stationary phase (OD 600 ~ 3.0). The Labeled DNA oligos specific to the transcripts of LEE1/ler ( A and I ) , LEE2/espZ ( B ) , LEE3/mpc ( C ) , LEE4/sepL ( D ), LEE5/tir ( E ), map ( F ), grlRA ( G ) and stcE ( H ) were used. The ompA transcripts, detected with a labeled ompA -specific DNA oligo, served as internal control for the primer extension reaction. Wild type and mutant SspA were expressed from pQE sspA and pQE sspA84-86 respectively in the absence of induction at similar levels. The transcripts LEE1-5, map, grlRA, stcE and the control transcript ompA are indicated. The relative transcript levels of target genes normalized to that of ompA are indicated by the numbers in parenthesis.

Article Snippet: To evaluate the effect of sspA on virulence gene expression in EHEC during the stationary phase we constructed an in-frame deletion of sspA in the E. coli O157:H7 strain EDL933 ATCC 700927 [ ] and measured transcription of LEE- ( LEE1-5 , grlRA and map ) and non-LEE-encoded ( stcE encoded by pO157) genes (Figure ).

Techniques: Expressing, Mutagenesis, Labeling

Journal: BMC Microbiology

Article Title: SspA up-regulates gene expression of the LEE pathogenicity island by decreasing H-NS levels in enterohemorrhagic Escherichia coli

doi: 10.1186/1471-2180-12-231

Figure Lengend Snippet: Increased ler expression overcomes repression of LEE in an sspA mutant. The expressions of virulence genes in wild type EHEC EDL933 (lane 1), the sspA mutant harboring the empty vector pACYC184 (pVector) (lane 2) and pACYC ler (p ler ) expressing ler from its native promoters (lane 3) were determined by primer extension analyses using labeled DNA oligos specific to LEE1/ler ( A ) , LEE2/espZ ( B ), LEE4/sepL ( C ), grlRA ( D ) and stcE ( E ) along with the ompA- specific oligo as a control. Samples were prepared and analyzed as described in the legend of Figure . The relative transcript levels of target genes normalized to that of ompA are indicated by the numbers in parenthesis.

Article Snippet: To evaluate the effect of sspA on virulence gene expression in EHEC during the stationary phase we constructed an in-frame deletion of sspA in the E. coli O157:H7 strain EDL933 ATCC 700927 [ ] and measured transcription of LEE- ( LEE1-5 , grlRA and map ) and non-LEE-encoded ( stcE encoded by pO157) genes (Figure ).

Techniques: Expressing, Mutagenesis, Plasmid Preparation, Labeling

Journal: BMC Microbiology

Article Title: SspA up-regulates gene expression of the LEE pathogenicity island by decreasing H-NS levels in enterohemorrhagic Escherichia coli

doi: 10.1186/1471-2180-12-231

Figure Lengend Snippet: SspA negatively affects H-NS levels in EHEC. The levels of H-NS were determined in wild type (lane 1) and sspA mutant (lane 2) derivatives of EHEC EDL933 grown to stationary phase cells by western blot. Equal amounts of total protein were resolved on a 10% Bis-Tris SDS-PAGE gel and transferred to a nitrocellulose membrane. Levels of H-NS and Fis were detected using polyclonal antibodies against the respective proteins. Fis served as an internal control for total protein levels. The relative amounts of H-NS normalized to that of Fis are indicated by the numbers in parenthesis.

Article Snippet: To evaluate the effect of sspA on virulence gene expression in EHEC during the stationary phase we constructed an in-frame deletion of sspA in the E. coli O157:H7 strain EDL933 ATCC 700927 [ ] and measured transcription of LEE- ( LEE1-5 , grlRA and map ) and non-LEE-encoded ( stcE encoded by pO157) genes (Figure ).

Techniques: Mutagenesis, Western Blot, SDS Page

Journal: BMC Microbiology

Article Title: SspA up-regulates gene expression of the LEE pathogenicity island by decreasing H-NS levels in enterohemorrhagic Escherichia coli

doi: 10.1186/1471-2180-12-231

Figure Lengend Snippet: SspA is upstream of H-NS in the regulatory network of virulence gene expression in EHEC. The expression of virulence genes in wild type EHEC EDL933 (lane 1), sspA (lane 2) , hns (lane 3) and hns sspA (lane 4) mutant derivatives was determined by primer extension analyses using labeled DNA oligos specific to the transcripts of LEE1/ler ( A ) , LEE2/espZ ( B ) , LEE3/mpc ( C ) , LEE4/sepL ( D ), LEE5/tir ( E ), map ( F ), grlRA ( G ) and stcE ( H ). In each reaction, the ompA transcript served as an internal control. Samples were prepared and analyzed as described in the legend of Figure . The relative transcript levels of target genes normalized to that of ompA are indicated by the numbers in parenthesis.

Article Snippet: To evaluate the effect of sspA on virulence gene expression in EHEC during the stationary phase we constructed an in-frame deletion of sspA in the E. coli O157:H7 strain EDL933 ATCC 700927 [ ] and measured transcription of LEE- ( LEE1-5 , grlRA and map ) and non-LEE-encoded ( stcE encoded by pO157) genes (Figure ).

Techniques: Expressing, Mutagenesis, Labeling

Journal: BMC Microbiology

Article Title: SspA up-regulates gene expression of the LEE pathogenicity island by decreasing H-NS levels in enterohemorrhagic Escherichia coli

doi: 10.1186/1471-2180-12-231

Figure Lengend Snippet: SspA is required for cell adherence and A/E lesion formation. HEp-2 cells were infected by wild type EHEC EDL933 ( A ) and its mutant derivatives of sspA ( B ), sspA pQE sspA ( C ), sspA pQE sspA84-86 ( D ), hns ( E ) and hns sspA ( F ). Bacterial adherence was examined by phase-contrast images (left panels) and the actin cytoskeleton of infected HEp-2 cells by fluorescent microscopic images (right panels). Representative images are shown. Black and white arrowheads indicate bacteria and A/E lesions, respectively.

Article Snippet: To evaluate the effect of sspA on virulence gene expression in EHEC during the stationary phase we constructed an in-frame deletion of sspA in the E. coli O157:H7 strain EDL933 ATCC 700927 [ ] and measured transcription of LEE- ( LEE1-5 , grlRA and map ) and non-LEE-encoded ( stcE encoded by pO157) genes (Figure ).

Techniques: Infection, Mutagenesis