Phage morphology was determined by transmission electron microscopy, allowing differentiation between phages, and classification into Brad ley/Acker mann morphological groups (see Section 1.4.3.1). Phages were prepared on form var coated grids and stained with 2% (w/v) phosphotungstic acid. Three phage groups were observed which were classified according to the Bradley classification system (Fig. A9).
The first group consisted o f eight Ecu phages, possessed isometric heads and contractile tails, and fell into group A (Myoviridae) (Fig. C5a). All the phages in group A were o f similar size (Table C20). Dimensions were obtained by comparison to Lambda on grids containing a m ixture o f phages. This gro u p of phages produced turbid plaques and were the only group of Eca phages to do so (Fig. C l) . Plaques obtained from the group A phages were indistinguishable.
1 4 9
Figure C5. Electron micrographs of Eca and Ecc phages, a) represents group A phages, b) represents group C phages, c) represents M2 which possesses a base plate, and d) represents group B phages, al, b, c, and di are magnified 50,000x, and all and dll are magnified 100,000x.
(Podoviridae). Phages in this group were o f similar size (Fig. C20) and produced clear plaques, with varying diameters (Fig. C l) . The group C phages had isometric heads but possessed only short tails (Fig C5b). In the case o f phage M2 a base plate was clearly visible (Fig. C5c).
The remaining phages, all o f which were Ecc, fell into Bradley group B (Sryloviridae). The group B phages had isometric heads, long non-contractile tails and were o f varying dimensions (Fig. C5d). This group produced turbid plaques which appeared similar in three of the four members (Fig. C2).
On the basis o f plaque morphology and structural morphology the phages were loosely categorised into three groups (Fig. C16). To compare the phages more closely their nucleic acid was submitted to restriction endonuclease (RE) digestion. Up to six enzymes were used including, EcoRI, BglII, BamHl, Clal, Hindlll and HaeIII. The nucleic acid o f all phages was digested by one or more of the enzymes, indicating that each phage contained double stranded DNA. The migration of restriction bands through agarose gels was compared to a Lambda standard restricted with f/i/id lll. A plot of the logarithm (log 10) o f molecular weight (y-axis) against the relative motility (x-axis) was made and band sizes obtained from this. In general it was not possible to obtain values between 1 kb units, due to the accuracy o f the technique, so bands which were obviously intermediate could only be estimated.
The DN As of the group A phages were restricted by EcoRI, BgHl, BamHl and Clal. T he phages within this group, with the exception o f S4S (data not shown),
gave a number o f common restriction bands (Table C3, Fig. C6). On digestion with EcoRI 7-8 bands were seen in each case. A2 and A3 appeared identical (set 1), as did A4S, A6 and S21 (set 2), with only a single band difference between the two sets. S34 and S41, although different, possessed bands common to both sets. Digestion with BgM, producing 17-19 bands, also showed similarities within sets 1 and 2 (Table C4, Fig. C7). S41 differed from set 2 by a single band, with S34 showing several differences. The banding pattern of S41 appeared the same as those in set 2 on digestion with BamHl and Clal (5-6 bands and 6-7 bands respectively) (Tables C5, C6, Fig. C8, C9). S34 showed some differences from both sets but common bands were present. The estimated genome sizes o f S34 and phages in set 1 were 80-90 kb, compared to 75-90 kb of phages in set 2 (including S41). The group A phage S45 had unrelated banding patterns to the rest of this group when digested with the above enzymes (data not shown). The genome size o f S45 was significantly smaller at only 30 kb.
The group C phages were divided into four subgroups on the basis o f their restriction patterns (Fig. C l6).
Subgroup I contained phages M2, M3, M4, S72 and S42. The phages in subgroup I were digested with EcoRI, flg/II and BamHl, producing 3-4 bands, 12-14 bands and 8-15 bands respectively (Tables C7, C8, Figs. CIO, C l l) . The banding patterns o f M2 and M4, after digestion with all these enzymes, appeared identical. On digestion with Bgtll and BamHl, S72 differed from M2 and M4 by 2-3 bands only. All four bands, produced on digestion of S72 with EcoRI, differed from M2 and M4. M3 however, although different from M2, M4 and S72, shared many common bands. The banding patterns o f S42 showed some relation to the other phages in this subgroup. Data on the digestion o f M3 and S42 by EcoRI is not given. The estimated genome sizes o f M2, M4 and M3 were 65-75 kb. S72 appeared slightly smaller at 65-70 kb and S42 contained only 60-65 kb.
Band sizes (kb) o f DNA from group A phages after digestion with EcoRl (E). Row 2 indicates the num ber o f bands produced after digestion with EcoRl.
Figure C6
Electrophoresis in a 0.89c agarose gel o f DNA from group A phages after digestion with EcoRI . (a) Direct comparison, (b) and (c) are S21 DNA and S41 DNA respectively showing more clearly the banding patterns produced.
Band sizes (kb) o f DNA from group A phages after digestion with BglU (B). Row indicates the number o f bands produced after digestion with Bglll.
Figure C7
Electrophoresis in a 0.8% agarose gel o f DNA from the group A phages S21, S41 and S34 after digestion with B g l l l .
Band sizes o f DNA from group A phages after digestion with BamHl (M). Row 2 indicates the number o f bands produced after digestion with BamHl.
Figure C8
Electrophoresis in a 0.8% agarose gel o f DNA from the group A phages after digestion with B a m H l .
1 5 4 A2 A3 A4s A6 S21 S34 S41 M 5 M 5 M 5 M 5 M 5 M 6 M 5 50-55 50-55 50-55 50-55 50-55 50-55 50-55 (X2) (X2) (X2) (X2) (X2) <X2) <X2) 14.0 14.0 — — 7PART 14.0 — — 9.1 9.1 9.1 — 9.1 — — 8.9 8.9 8.9 — 8.9 — — — 6 .6 — 3.5 3.5 3.5 3.5 3.5 3.5 3.5 2.1 2.1 - -- 2 .3 - A2 A 3 A 4 S A 6 S21 S34 S41
Band sizes o f D N A from group A phages after digestion with Clal (C). Row 2 indicates the n u m b er o f bands produced after digestion with Clal.
Figure C9
Electrophoresis in a 0.8% agarose gel o f DNA from the group A phages after digestion with C l a l .
Band sizes o f DNA from group C phages (subgroup 1) after digestion with BglII (B). Row 2 indicates the number of bands produced after digestion with BgM.
Figure CIO
Electrophoresis in a 0.8% agarose gel of DNA from the subgroup C 1 phages M2, M4 and S72 after digestion with B g / l l and £coRI.
Band sizes o f DNA from group C phages (subgroup 1) after digestion with BamHl (M). Row 2 indicates the number o f bands produced after digestion with BamHl.
Figure C l 1
Electrophoresis in a 0.8% agarose gel of DNA from the subgroup C I p h ag es M2, M4 and M3 after digestion with B a m H l .
Subgroup II contained phages S31, S32, S33 and S71. On digestion with Clal, which produced only 2 bands, the restriction banding patterns appeared the same in each case. S32, S33 and S71 produced a restriction pattern on £coRI digestion (5-6 bands), which differed to that of S31 by 3 bands (T able C9, Fig. C12). The estimated genome sizes o f S32, S33 and S71 were approxim ately 36 kb, while that o f S31 was larger at 42kb.
Subgroup III contained phages A4L, A5, <£M1 and S62. A4L and A5 gave one banding pattern on digestion with EcoRI and another using Clal, producing 9 and 3 bands respectively (Table CIO, Fig. C l 3). On digestion with EcoRl </>Ml shared 7 bands in comm on with the above, while S62 showed on ly 4. <£M1 and S62 appeared similar after digestion with Clal, with 2 bands com igrating with those o f S41 and A5. W hile the estimated genome sizes o f A4L, A5 and M l were approximately 45 kb, that o f S62 was smaller at 39 kb.
O f the rem aining group C phages only subgroup IV phages A1 and S22 were subjected to RE analysis. The phages differed in bo th plaque morphology and restriction pattern but were both group C phages (Table C l l , Fig. C14).
The Eca phages M5 and S61 were placed in a m iscellaneous group since no restriction data was obtained for them.
The group B phages contained phages D2, S65, S75 and 0K P , all o f which were isolated on Ecc SCRI193. Restriction patterns were obtained for only two o f the phages, S65 and 4>KP, which did not appear to be related (Table C12, Fig. C15). The genome size o f S65 was approximately 100 kb com pared to <i>KP at 46 kb.
Table C9
Band sizes o f DNA from group C phages (subgroup II) after digestion with EcoRI (E) and Clal (C). Row 2 in each case indicates the number o f bands produced after digestion with EcoRI and Clal.
Figure C12
Electrophoresis in a 0.8% agarose gel of DNA from the subgroup C II phages S31, S32, S33 and S71 after digestion with (a) EcoRI and (b) C l a l .
Table CIO
Band sizes o f D N A from group C phages (subgroup III) after digestion with £coRI (E) and Clal (C ). Row 2 in each case indicates the number o f bands produced after digestion with £coRI and Clal.
Figure C13
Electrophoresis in a 0.8% agarose gel o f DNA from the subgroup C III phages A4L, A5 and M l after digestion with (a) EcoRI and (b) C l a l .
Table C l l
Band sizes o f DNA from group C phages (subgroup IV) after digestion with EcoRI (E), BglII (B) and Clal (C). Row 2 in each case indicates the number o f bands produced after digestion with EcoRI, Bglll and Clal.
Figure C14
Electrophoresis in a 0.8% agarose gel o f DNA from subgroup IV phages A1 and S22 after digestion with EcoRI and BglII.
A l S22 E 6 B 2 C 2 E 6 B 3 8.1 > 2 5 > 2 5 13.6 19.0 7.8 4.0 2.0 8.3 14.1 7.2 6.0 11.0 4.8 3.0 2.9 2.6 2.2 2.2 E B 5 2 2 S22