coli 0157:H7 (ATCC 35150) contains both slt-l and slt-ll coding sequences (section

3.5.2). Studies have shown that the sit-1 and s/MI operons are both transcribed from polycistronic mRNA from a promoter upstream of the sit A gene (section 1.4.2). The size of the polycistronic mRNA species produced from each operon was estimated from published sit-1 and s/f-II sequence data (Calderwood et al., 1987; Jackson et al., 1987b respectively) to be approximately 1.6 Kb (for slt-l) and 1.7 Kb (for slt-U).

Figure 4.2 (tracks B & C) shows electrophoresed total RNA prepared from a stationary

Figure 4.2 Northern blot analysis o f E. c o li 0 1 5 7 :H 7 (ATCC 35150)

Total cellular RNA was denatured in formaldehyde loading buffer and electrophoresed on a 1.5% (w/v) agarose, 2.2 M formaldehyde gel in 1 XLMOPSJbuffer at 80V. The RNA was transferred to nitrocellulose and probed with ^PdCTP labelled VT1 and VT2 D N A fragments as described in materials and methods.

Key to tracks

A: RNA markers (BRL). Fragment sizes were 9.49, 7.46, 4.4, 2.37, 1.4 & 0.24 Kb. B: Total RNA isolated from E. coli 0157:H7 (ATCC 35150) - concentration unknown. C: Total RNA isolated from E. coli 0157:H7 (ATCC 35150) - concentration unknown.

Tracks D & E correspond to tracks A and B respectively probed with a ^PdCTP labelled 0.75 Kb H indi fragment from plasmid NTP705 which contant VT1 B subunit coding sequences. Track F corresponds to track C probed with a ^PdCTP labelled 0.85 Kb Smal-Pstl fragment from plasmid NTP707 which contains VT2 A subunit coding sequences. Filters were washed in 1 x SSC at 65°C for 1 hour and exposed to autoradiographic film for 21 days at -70°C.

phase culture of E. coli 0157:H7 (ATCC 35150) and the subsequent blots obtained after probing Northern filters with the ^P-dCl'P labelled VT1 and VT2 probes (tracks E & F respectively). RNA was prepared as described in section 3.30 except that in this case attempts were made to extract the RNA from a 50 ml cell pellet. The cell concentration was, however, too high and a small aliquot of this was removed for RNA preparation. It can be seen that upon electrophoresis of 5 /A of the 0157:H7 (ATCC 35150) cellular RNA, the 23S, 16S and 5S ribosomal RNA (rRNA) bands are clearly visible, indicating that there has been no gross degradation of the RNA. The corresponding Northern blots, after washing with 2 x SSC, 0.1 % (w /v) SDS and then 1 x SSC, 0.1% (w/v) SDS at 65°C for 1 hour, showed evidence of hybridisation to the labelled DNA probes (tracks E & F). Non-specific hybridisation with material within the RNA marker lane was also observed (track D). There was no apparent difference in the pattern of the hybridised material in tracks E & F. The arrowed fragments, estimated to be approximately 1.5 Kb, were thought to correspond to the polycistronic mRNA produced by transcription of the sit operons in this strain. In an attempt to wash away the non-specifically bound material, filters were washed more stringently with 0.1 x SSC, 0.1% (w/v) SDS at 65°C for 1 hour. After 9 days exposure to autoradiographic film at -70°C, however, the filters showed no evidence of hybridisation. This could have been a consequence of

(a) The filters were over 4 weeks old and it is possible that any ^P-labelled material had decayed to such an extent as to be undetectable, or, (b) the labelled probes had been removed by the stringent washing conditions.

Figure 4.3 shows electrophoresed total RNA prepared from 1.5 ml overnight cultures of VT producing strains E3787, E32511, 0157:H7 (ATCC 35150) and 0157:H7 (Cl), (section 2.30). Gel B contains double the amount of RNA per track (i.e. 10 /A per track) as gel A. Northern filters were washed in either 2 x SSC, 0.1% (w/v) SDS, at 65°C for 45 min (gel A) or 2 x SSC, 0.1% (w/v) SDS, at room temperature for 45 min (gel B). Other than the D NA fragment controls, autoradiographs showed no evidence

Figure 4 3 Northern blot analysis o f VT-producing E . c o li

Duplicate samples of total cellular RNA from the strains indicated were denatured in formaldehyde loading buffer and electrophoresed on a 1.5% (w/v) agarose, 2.2 M formaldehyde gel in 1 X MOPS buffer at 80V (gel A) and 30V (gelB). Gel B contained double the amount of RNA as gel A (concentration unknown). RNA was transferred to nitrocellulose and probed with ^PdCTP labelled VT1 and VT2 DNA fragments as described in materials and methods. Only one half of each gel is illustrated. Key to tracks (Gels A & B)

1 & 6 : RNA markers (BRL). Fragment sizes were 9.49, 7.46, 4.4, 2.37, 1.4 & 0.24 Kb. 2 & 7 : Total RNA isolated from E. coli E3787.

3 & 8 : Total RNA isolated from E. coli E32511.

4 & 9 : Total RNA isolated from E. coli 0157:H7 (ATCC 35150). 5 & 10 : Total RNA isolated from£. coli 0157:H7 (Cl).

8 fil o f VT1 H indi DNA probe fragment and VT2 Smal-Pstl DNA probe fragment were included on each gel as controls.

Gel A Gel B

1 2 3 4 5

6 7 8 9

10

of hybridisation with either probe after exposure to the Northern filters for 4 days and 27 days at -70°C (autoradiographs not shown). DNA control probe fragments hybridised specifically with the homologous probes as a dark smear from the well of the track due to the denaturing conditions employed.

In another experiment RNA was prepared from E. coli strains 60R363 and 60R746 from which the VT1 and VT2 probes had been prepared (figure 4.4). Upon probing of Northern filters with the labelled VT1 and VT2 probes and washes in 2 x SSC, 0.1% (w/v) SDS and 1 x SSC, 0.1% (w/v) SDS at room temperature for 1 hour each, the control probe fragments were seen to hybridise with the homologous probes as expected. In addition, bands in the wells of tracks 2 & 6 hybridised with the VT1 probe and the VT2 probe respectively. This was thought to be due to plasmid DNA contamination and to confirm this, RNA samples were treated with deoxyribonuclease (DNase) as described in section 2.34. 20 n\ of the DNase treated RNA was denatured and electrophoresed as previously described. Subsequent autoradiographs only showed hybridisation to the control DNA probe fragments, confirming that contamination with plasmid DNA had occurred in figure 4.4 (autoradiographs not shown).

4.2.3 Probing or RNA from different points in the growth cycle o f VT-producing strains

It was not known whether VT mRNA synthesis occurred throughout growth of the organisms or only at specific points in the growth cycle. In an attempt to detect VT mRNA transcripts, R N A was prepared from E. coli strains E3787, E32511, 60R746 and 60R363 at 2 (exponential), 4 (early stationary) and 6 (stationary) hours culture as described in section 2.36. Samples, adjusted to contain 15 /¿g o f cellular RNA were denatured, electrophoresed on formaldehyde gels and Northern blotted as described above (figure 4.5). Filters were washed in 2 x SSC, 0.1% (w/v) SDS and 1 x SSC, 0.1% (w/v) SDS at room temperature for 1 hour each. After three weeks exposure at -70°C,

Figure 4.4 Northern blot analysis o f E. c o li strains 60R746 & 60R363

Total cellular RNA was denatured in formaldehyde loading buffer and electrophoresed on a 1.5% (w/v) agarose, 2.2 M formaldehyde gel in 1 XlMOPSbuffer at 60V. The RNA was transferred to nitrocellulose and probed with •}ZPdCTP labelled VT1 and VT2 DNA fragments as described in materials and methods.

Key to tracks

1 RNA markers (BRL). Fragment sizes were 9.49, 7.46, 4.4, 2.37,1.4 & 0.24 Kb. 2 & 5 : 5 n\ of total RNA isolated from E. coli 60R746.

3 & 6 : 5 /il total RNA isolated from E. coli 60R363. 4 : 8 /d 0.75 kb H indi VT1 DNA probe fragment. 7 : 8 fi\ 0.85 kb Smal-Pstl VT2 D NA probe fragment.

Tracks 8-11 and 12-14 correspond to tracks 1-4 and 5-7 after probing with a ^PdCTP