PMC

Bacteria were incubated in LB with different pH values as indicated at 37°C and 23°C with agitation (+) and under static growth conditions (-). Proteins were immunoblotted and EibG signals were measured using chemiluminescence. The influence of temperature (A), pH values (B) and both in combination (C) are shown for wild-type strain 2875/96. Marker sizes (M) are provided.

Strain 2875/96 was inoculated for 20h at 37°C with and without shaking. Bacteria grown under agitated conditions grew homogenously and were turbid without biofilm formation (A). However, statically grown bacteria aggregated and deposited a biofilm (A). Aggregates and deposits were stained with crystal violet (B). Strain 659/97 was used as EibG negative control. Microscopically, shaken bacteria demonstrated single and non-aggregated cells whereas static grown bacteria formed coherent chains (C). The figures exemplify microscopic images of strain 2875/96 and 0520/99, magnified as indicated.

Strains 2875/96 and 0520/99 were inoculated into agitated (+) or static conditions (-) in jars under aerobic, microaerophilic and anaerobic conditions at 37°C for 18h. After cell harvesting, proteins were loaded and immunoblotted. (A) EibG proteins were visualized by chemiluminescence and (B) EibG signals were densitometrically quantified and are reported as computer internal units. Values are typical of those from repeated growth conditions and independent gel runs. (C) Cells were harvested from LB plates. Proteins of 5 and 15 μg per lane were loaded for 2875/96 and 0520/99, respectively, and EibG proteins were immunologically detected.

STEC strain 2875/96 was grown in LB medium at varying revolutions per minute (rpm), as indicated. After overnight inoculation at 37°C, bacteria were harvested by centrifugation. For comparison, identical protein quantities, ranging from 3.0 to 0.1 μg per lane were separated by SDS-PAGE and immunoblotted. EibG signals were visualized using mab human Fc fragment and chemiluminescence (A). Marker sizes are indicated (M). EibG signals were quantified densitometrically and intensities are provided (B; n.d. = not determined). To analyse differences in signal intensities among protein amounts loaded on the gel and among gel runs, EibG signals of three independent immunoblots were quantified (C). The highest signal intensity was defined as 1.0 and the ratios of lower signals were estimated and reported as means (± standard deviations). The ratios of EibG signals are provided for 3 and 1 μg protein (black bars and grey bars, respectively).

Cells of several STEC strains carrying the eibG gene were inoculated in LB medium with (+) and without (-) shaking at 37°C for 16h. Proteins were separated by SDS-PAGE and immunoblotted. (A, B) To compare expression levels, identical protein quantities of 7.5 μg were loaded in each lane. EibG was detected with human IgG Fc conjugated with HRP on immunoblots and visualized by chemiluminescence. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as marker protein and as control for loading. Marker sizes (M) and EibG proteins are indicated. (C) Proteins of strain 0520/99 were diluted serially as indicated and immunoblotted to demonstrate specifity and sensitivity to the detection platform. To standardize between immunoblots, the highest intensities were defined as 1.0 and the ratios of the diluted signals of three independent gel runs were calculated as means (± standard deviations of the means). Intensities of static grown bacteria and agitated cultures are shown by black and grey bars, respectively.

Single colonies of strain 2875/96 were grown as starting culture (SC) in LB for 18 h with (+) or without (-) shaking. Cells were inoculated at a 1:100 dilution indicated as main culture (MC) to fresh LB medium followed by incubation with or without shaking, respectively. Proteins of grown cells (1 μg per lane) were separated by SDS-PAGE and immunoblotted. (A) EibG signals were visualized using human IgG Fc fragment conjugated to HRP. (B) EibG signals were quantified densitometrically and are reported as computer internal units. Values presented are representative of those from repeated growth conditions and independent gel runs. (C) After inoculation RNA was isolated for RT-PCR. The bars represent the relative levels of EibG mRNA normalized to GapA expression. Values for bacteria grown under agitated conditions (+) were defined as 1.0 and the increase of EibG mRNA was calculated after static growth (-). Samples were analysed in triplicate and results represent the means and standard deviations of the mean.