Genetic restriction in Escherichia Coli strain W. : a thesis ... presented for the degree of Master of Science in Microbiology

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GENETIC RESTRICTION IN ESCHERICHIA COLI STRAIN W

A thesis

presented in partial fulfilment of the requirements for the degree of

Master of Science in Microbiology

at

Massey University

A.F. JAMIESON

.1fil

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/1.BSTRACT

The ability of various pha~os to propagate on Escherichia coli strain

l ,r

^w^us invostig;:i.ted, Phagos P1 , T2, ¹

r3 ,

^T4^, ^T5^, ^{T6 and}^Ty

coulcl not be shown to form plnqv.cs on this strain. Phagcs T2, T3, T5 and T6 prevented tho development of a bactcri,'.ll la~,m when ;:i.dded to a plate at an input ratio of about throe phage per bactcriu~; it appears t]103e phagos exerted a killing effect on strain W. Phage P1 and phage T4 did not exhibit this killin,1 effect . Plugo T1 formed

- 4

atypicnl plnques on strain U with an efficiency of plnting of 10 ; it appearc these plaques are due to mutants occurin1 in the T1 population nble to propagate on strain ·r.

ill of tho :.i.bovo phagos adsorbed efficiently to strain W with the exception of T4, explaining its inability to either

propagatG on or kill strQ.in ;'J. 'l'he infection of sti·ain U by P1 was similar in most respects to that of °/\ but in ordGr to 0stablish the occurence of conventional restriction, D~'...'1. degradation would need to be demonstrated.

Phages were isolated which propngatc on strain W; they are similar in morphology to phagcs T5 ;md ~ ,'.l.nd do not readily adsorb to ]. coli strains

n ,

^{C or}^K.

The supernatant from broth cul tu.res of strain W ,ms shown to contain two closely related phages, one plating on 111.£2.ll. C, and the other on .m, .£Qll K, Ea.ch possesses a characteristic pattern of plating efficiencies on strains C and IC when propagated alternately in these two hosts but the two phages were shown to be co-immune and identical with respect to heat sensitivity, morphology and serolOffY•

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Both tended to lose tho .::i.bility to exclude phage P1 on lysogeniDing strain C once havinc mutated to plate on strain K. Thin mn.y be due to the inte:;ration of tho mutated phage at altern.:i.tc ¹¹non-restricting"

sites on the~ coli C chromosome.

A serie:., or conjuGal crosses was cr..iployed to doter:nine tho sites of integration of the pha;;os on t:ho chromosomes of]. coli

strains U ,:md C. 'i'ho phage present in tho :,; supernatant ·which plated on K uas found to ii"tegrate close to the proline loci on the

chromosome of

_g .£ill

^W^but^thephage plating on C -1:ppearod to have more tban one locus, one of eThich !:lay !llap close to tho 85 minute m.J.rk on the linkago oap of Q coli ¹!!

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J?i0m·e 22). ifo inforrnntion has so far been obt.:1i;1ed concornine ti1e 8i'Los of i:nte:;re.tion of -t:he w ph.:.ges in restrictive and non-restrictive l:rsoge.:ns of .m_ ccli C. Tho failure to ob t.ain 2. 'cared' strc.:.in of _i1 coli H by oli1:1inntion or the

proph;:,ge inh'cratod nt the two in::-!.1m.::d situs leaves opon the possibility of tl:o existence of noro than one integration si to of phage u. C on the

J.

colt 1:l chromosome.

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A OOT OWL.sDG EHEf

'££

Tho author wishes to express his sincere thanks to Professor D. F. Bacon for his invaluablG c-uidanco thro1.¹.ghout the course of this ,-rork and also for tho supply of many of the bacterial and b~ctoriophage strains.

Thanks are also due to the otaff of tho Pal1:1erston Horth D.S. I .R. electron wicroscope unit for their assistance in the preparation of tho electron o.icrographs, to r-Iiss J. Quigan for her expert typine; nnd to all staff in tho fiicrobiolo:;y ::md Genetics DG})D.rtinent ,;-rho have helped in work loading to tho presonte.tion of this thesj_s.

A. I'. J.ru~IESON

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_TABLE OF COETEliTTS

HITRODUCTION

I Superinfection immunity

II Superinfection exclusion

.. . .

III Inhibition by ^F+

.. . .

IV Restriction

.. . .

V Abortive infection by T5 and the T-even phQgcs

AHIS OF TIIE INVESTI';ATIOE

BACTERE

BJ.CTERIOPHAGES ••

HEDii,.

METHODS

RESULTS AHD DISCUSSIOl~

I Investigation of the abortive infection of

. .

9 9 9

10 11 26

31

32 36

37

40

47

~scllerichia coli W by various pho.gcs 47 II Isolation of phage able to p:ropa:;ate on

Escherichia coli W 63

III Plating efficiencies of phage won

.fil

coli strains

C and IC • • 69

IV Comparison of the properties of phages w.KC and u. CKC 75 V Mapping of the sites of integration of thew prophages

in the chronosomos of E, coli strains C and i;T 87

CONCLUSIONS

. . .. • • 100

BIBLIOGRAPHY 106

"~*********

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Table 1 •

2.

3.

4.

5.

6.

7.

8.

9.

1

o.

11.

12.

1 3.

1 4.

1 5.

1 6.

17. 18.

LIST OF TABLES

Efficiency of plating of phngo won Dsch0richin coli

strains K and C ••

Efficiency of plating of phage u on Escherichi.:t g_oli

strains Kand C •• ••

Bacterial strains Bactoriophne;os

Plating effects at different input levels of phage w on

!

coli strain 1J • • • • Dotormina tion of the frcq_1.wncv of producti vo infection of

!

coli ¹'I cells by phage T7. B

i'::fficioncios of platin.~ of phagos on Eschorichin coli strains TT, Band C

Adsorption coefficionts of the 'J" pha::;es o.nd P1 on adsorption to ~ coli strains 1·J, B and K12

Scroonine of ~ .coli isolutcsand sewage samples for tho presence of phai~C .'3.blc to propagntc on strain W

Dimensions of the V-pho.ces ••

The r0lative efficiencies of i)latin:~ of phage from. an ] co]j._ W supernatant on

!

coli str.:~ins C and K ••

Adsorption coefficients of w phage to_£ coli strains

C .'.lnd K ••

Hc.'.lt sensitivities of ph.agos 11.CKC and w.KC in O. HI NaCl + O. 5E Cc1..Cl

2

Heat sensitivities of phages w.CKC and w.KC iE

0.1M NaCl ••

Adsorption of pho.ges w e.nd P1 to l~rsogens of

!

coli C Ability of

.fil.

coli K and C lysogcnisod. with pho.ge w to support the propagation of various phages ••

Aborti vc infection of phage w lysogcms of C by phage P1 Presence of phage able to plate on strains Kand C in the supornatants of broth cultures of substrains of

lJ.

coli i'l ••

22

22 32 36

48

51

55 57

63 66

71

72

75

75 81

82 83

88

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Table

19. li'rcquG11cy of seloct0d a~d unselected m.::i.rkers in the

cross }lfr H X D2-18-1-0

..

⁹⁰

20. ?rcquoncy of scloc·i;od ::ind unselected narkers in tho

cross Hfr H x D2-8

..

⁹¹

21 . Frequency of soloctcd and unsoloctcd mark0rs in the

cross Hfr F2-3-27 x C1-a/50

93

22. Frequency of selected and unselected narkors in crosses of Efr H with lysogens of E, ~oli

strainn C and K • • 96

23. }roquency of.' rvcovery of s0lcctei and+ unsc!octod markers in the cross Efr 808 x D2-18-1-0 (pro 1·r .K ) . • 98

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Figure 1 •

2.

3.

4.

5.

6.

7.

8.

9.

1

o.

11.

LIST OF FIGURES

Restriction und modification of phage

'.A

in Escherichia coli strains Kand C ••

The specificity site nucleotide ::;equence of Haemophilus influenzac

Derivation of substrains of Escherichia coli strain W (D.F. i3acon)

Linkage map of Escherichia coli K12

..

. .

Adsorption kinetics of phage T1 to Escherichia .2.Q.ll.

strains B and i'l

Ldsorption kine tics of phage T2 to Eschericlg_?._ coli strains B and W

Adsorption kinetics of phage T3 to JschericJ.iia coli strains Band W

Adsorption kinetics of phage

T4

to ]Escherichia coli strains B .:md W • • • • • •

Adsorption kinetics o"! phae;o T4 to _;:.:sc}lcrichia coli st rains B and. C

Adsorption kinetics of phage T5 to Escherichia coli strains B and '.J

Adsorption kinetics of phat;o T6 to ~sche:richi.'.l coli r:Jtra.ins :S and ;1

12. Adsorption kinetics of phage T7 to pscherichia coli strains B and W

1 3.

14.

1 5.

16.

17 .

Adsorption kinetics of phage P1 to J.!scherichia .901i strains IC and W • • • • • •

Plating efficiencies of phage from the supernatant of ]1 coli W on strains K and C

Adsorption kinetics of phage w.K to

.fil

coli strains C and K

Adsorption kinetics of phage w.KC to

.fil

coli strains C and K •• ••

Adsorption kinetics of phage w.C to

.fil £ill

^W

strains C and K •• ••

. .

12

16

33 34

58

59

60

61

62

70

73

74

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Figure Page 18. Kinetics of Heat Inactivation of phage w. CKC at

70°c in 0.1i'I NaCl +

o.

5li CaC12 76 1 9. Kinetics of Heat Inactivation of phage w.KC at

70°C in 0.1 M NaCl + 0.5M CaC12

..

⁷⁶

20. Inactivation of phage w. CKC nnd phage 1-1.KC by w. CKC

antiserum

..

⁷⁹

21. Inactivation of phage w.CKC and phage w .I~C by w.KC

antiserum

. . ..

⁸⁰

22. The tentative locations on the chromosome of ]l coli W of: i) tho W genome hs site; ii) two possible

integration sites of phage w 95

';~*·*-*******

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Plate 1 •

2.

3.

4.

5.

6.

1. 8.

9.

1

o .

11 •

1 2.

1 3.

1 4,

1 J. 16.

17 .

18.

19.

LIST OF PLATES

Plaques formed by phage •r1 . B on .:~. coli R (x3 Plaques formed by ~ uha""e 0 T1. B on

!

.coli W (x3 Plaques formed by phage T2,B on

f

coli 13 (x3 Plaques formed by phar,..e

- u T2.B on ^.)l;_ coli_ C (x3

'l'he scrGening method tcsed to detrJct ·:;hagc and active against

1

^coli

W

Plaques formed by phage Phage V7 (x116,000) Phage V1 0 (x116, 000)

V1 i

..

on

.fil

coli W (x3 )

) )

..

)

..

)

colicins

Phage V11 ai:tnchcd to bacterial de:)ris (x11 G, 000) Pha50 V11 attacl~ed to cell debris (x116, 000) Pha,r,e V1 I adsorbed to _m, coli -·!; ap1'"Jro:drnntc phage input ratio= 50:1 (x85,000) .• ..

Phage V1 I and E coli I:; Uimroxim:1t0 ^pli-:1ze input ratio= 50:1 (x43,000) ^Co^{• •}

A rose Ho of phage w (::116, GOO) Pl1age w. KC (x11 !; , 000)

Pha1;c w. C::.ZC (x11 6,000)

Plaques of pha,gG w.CKC on _~9oli C (x3)

Screening of lysogens for ability to exclude ptnge P1 A lysogen of

g

coli K K(w.CK) spot ted on a plate previously spread with phage P1 pc1rticles

The method employed to screen for phage w lysogens of

£J, coli strains K and C • • • •

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53 53 56 56

64

67 67 67 67

68

68 78 78 78

85 05

86