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Thèse de doctorat/ PhD Thesis Citation APA:
Lathe, R. (1976, September). The RNA polymerase of E. coli and the four minute region of the E. coli chromosome (Unpublished doctoral dissertation).
Université libre de Bruxelles, Faculté des sciences, Bruxelles.
Disponible à / Available at permalink : https://dipot.ulb.ac.be/dspace/bitstream/2013/214430/3/103f8a6b-caa0-426d-8401-af9c1a0d654b.txt
(English version below)
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UNIVERSITÉ LIBRE DE BRUXELLES
' FACULTÉ DES SCIENCES
DÉPARTEMENT DE BIOLOGIE MOLÉCULAIRE
•LABORATOIRE DE GÉNÉTIQUE
The RNA polymerase of E. coli and the four minute région
of the E. coli chromosome
THÈSE PRÉSENTÉE POUR L’OBTENTION DU GRADE DE DOCTEUR ÈS SCIENCES
SECTION : BIOLOGIE MOLÉCULAIRE RICHARD LATHE
SEPTEMBRE 1976
FACULTÉ DES SCIENCES
DÉPARTEMENT DE BIOLOGIE MOLÉCULAIRE LABORATOIRE DE GÉNÉTIQUE
The RNA polymerase of E. coli and the four minute région
of the E. coli chromosome
THÈSE PRÉSENTÉE POUR L’OBTENTION DU GRADE DE DOCTEUR ÈS SCIENCES
SECTION : BIOLOGIE MOLÉCULAIRE RICHARD LATHE
SEPTEMBRE 1976
my greetings to ail of you, Yeasts, Bacteria, Viruses,
Aérobics and Anaérobies:
A Very Happy New Year to ail for whom my ectoderm
is as niddle Earth to me.
From 'A New Year Greeting W.H. Auden
I would liKe to express my thanKs to everyone who has contributed directly or indirectly to this thesis. In particular, I must thanK R. Thomas, in whose laboratory thls worK was performed, for advice and many interesting discussions.
My thanks are due to Alex, Robert, Theresa, Arianne, Annette, Jean DL, Jean P and Michel for invaluable advice on the many problems encountered in this work and more often than not for stimulating discussions.
I am also grateful to Lucie, Anne-Marie, Joseph, Marie-Jeanne and Yolande without whom the lab would hâve ground to a hait long ago, to those of the Service Technique for converting my esoteric designs into apparatus, to Jacques and Yvette for teaching me fluorography and to Claude for teaching me the préparation of antiserum.
Richard H and John S deserve a spécial mention for introducing me to RNA polymerase, Noreen M, Bill B and John D for introducing me to lambda. Without the aid of Noreen and Bill the latter part of this work would not hâve been possible.
My thanks are also due to ail those at Berkeley, Madison, Heidelberg, Munich and Edinburgh who hâve corne forward with their ideas and unpublished data.
I must gratefully acknowledge the contribution of the following people : Annette for electronmicroscopy, W. Zillig and H. Weissbach for antisera, R. Calendar and others for bacterial and phage strains, Alex and Robert for running some of the gels, Huevon for continued good humour and impeccable colony-picking, Daniel and those at the Atelier de Photographie who produced the photographe for this thesis, Percy for some of the figures and Mme Valentin for typing part of the manuscript.
I need not mention Chris whose interesting discussions at the frontiers of science and beyond hindered me considerably.
Finally I must sincerely thank Jean-Pierre and Christine for their encouragement during this work and for many long discussions.
Their contribution to this work needs no qualification.
I am grateful to the Ciba-Geigy Fellowship Trust for providing financial assistance (1973-1976] to enable me to carry out this work, and to E.M.B.Ü. for a short term fellowship in July 1975.
When this worK was begun in 1973, the genetlcs of the RNA
polymerase of E. coli were stlll at an elementary level, and only the gene coding for the g subunit of the RNA polymerase had been identified.
Several thermosensitive mutations affecting transcription existed at that time, of which ts2ÜO was judged to be the most interesting in view of the ’fir-effect’ and the clear thermosensitivity of RNA synthesis observed in crude extracts. My subséquent research into the in vivo and in vitro characteristics of strains carrying the ts2D0 mutation is presehted in this thesis. I hâve since shown that the ts200 allele does not affect either the g’, a or a subunits of the RNA polymerase, and this is confirmed by reports that the structural genes for these subunits are located elsewhere on the bacterial chromosome Cg’ in June 1974, a in Dctober 1975, a as yet unpublished1.
The worR described in this thesis is presented largely in temporal order. Whilst I hâve tried to describe my research in depth where
necessary, I hâve at the same time done my best to be succinct. Above ail I hâve endeavoured neither to molly-coddle nor to bore the reader, and this should be borne in mind when reading this thesis.
Introduction 1
Materials and Nethods 17
A : Genetic Manipulation of Bactériophage 17
B : " ” " Bacteria 21
C : Large Scale Préparation of Bacteria S Bactériophage 25
□ : In-vivo Labelling Experiments 27
E : Immunological Techniques 29
F : Extraction of Cell Proteins 31
G : Gel Electrophoresis & Related Techniques 33 H : Restriction Enzymes " " ’’ 40 I : Préparation of Columns for Chromatography & Gel Filtration 41
J : RNA Polymerase 42
K : DNA Polymerase 47
L : Media 49
M : Reagents (and abbreviations] 50
N : Bacterial Strains 52
0 : Phage Strains 56
P : Notes 57
Appendix 60
Results and Discussion 63
A: An Introduction to ts200 63
B: PI Transduction Mapping of the ts200 Marker 67 C : Dominance/Recessivity of the ts200 Marker 79 D : Effects of Genetic Background upon the ’fir effect' 81 E : Purification and Properties of the RNA Polymerase 83
from ts200 Strains
F : Purification and Properties of DNA Polymerase III 96 from ts200 Strains
G : Spécifie Activity of Endogenous RNA Polymerase 10.1 H : Stringent Control in ts200 Strains 106 1 : Transduction of the firA Gene by Abnormal Excision 109
of Second Site Lysogens
J : Transduction of the firA Gene by Phage Constructed 122 in vitro
K : The Sigma Mobility Différence and 21fir 135
N : An Application of UV-infection to the Analysis 165 of Lambda Proteins
SUMMARY 171
REFERENCES 173
THE SYNTHESIS DF RNA IN E. COLI
The structure of the enzyme
Only a single major DNA-dependent RNA polymerising activlty is found in E. coli cells. The minimal or ’core’ enzyme consista of three subunits
(TB) and has the ability to synthesise RNA upon nicKed double stranded templates, though for full activity on native DNA a further subunit, the sigma factor, is necessary (1B, 118). The core enzyme complexed with sigma (now referred to as holoenzyme) has the structure (119, 120) the molecular weights of the subunits being about 165,000, 155,000, 40,000 and 90,000 daltons respectively (119, 120). The holoenzyme thus has a total
0
molecular weight of about 4 x 10 daltons (121, 122), though at low ionic strength it exista as a dimer (121, 18). In vivo, the quantity of sigma is generally suboptimal, being présent at about 0.3 to 0.4 copies per molécule of the core enzyme (318, 123).
The pattern of two large subunits (88’) and one small (a) making up the core enzyme has also been found to be the case in other procaryotes such as B. cereua (117), L. curvatus (41) and (T. tuberculosis (124); in most cases additional subunits similar to sigma are also présent.
The reaction catalysed
The élongation reaction catalysed by the core enzyme can be
represented as below : ,
pppNX + nNTP --- ^ pppX(pN)^ + nPP^
where pppX and NTP are ribonucleoside triphosphates complementary to
the DNA template. The release of pyrophosphate (and its subséquent hydrolysis) leads to an irréversible reaction.
On single stranded DNA templates, the RNA polymerase synthesises a complementary antiparallel RNA chain, extending the growing RNA Chain in the 5' ---^ 3' direction (134, 135, 136). On a double stranded naturel DNA template generally only one strand is copied into RNA, though there are cases where the same région may be transcribed in both directions in vivo (see ref. 137).
Although the core enzyme is able to carry out the élongation reaction unaided, sigma factor is necessary for the correct initiation of RNA
synthesis. Not only does sigma stimulate the initiation of transcription at spécifie sites (promoters) but it also reduces the level of non- specific initiation by the core enzyme (10Ü, 101, 125). Dnce the
élongation reaction is underway the sigma factor is released and thus the function of sigma is catalytic rather than stochiometric (126).
The holoenzyme has the ability to form stable complexes at promoter sites in the absence of one or more nucleoside triphosphates, and under these oonditions a localised opening of the DNA-helix has been suggested (127, 128, 129], though at most only a few nucléotides can be exposed (130, 131].
The energy for opening the DNA-helix during translocation is probably provided by cleavage of the a - ^phosphate bond of the nucleoside triph
osphates.
Termination of the élongation reaotion may occur at speoifio sites in the absence of additional protein factors (132, 133], though a protein
’rho’ has been found in extraots of E. ooli (168] which catalyses the termination of transcription at additional speoific sites on naturel DNA templates.
The problems of initiation and termination are discussed in more detail in later sections.
The fonctions of the individuel subunits
The largest subunit 3’ has been implicated in the primary binding of the enzyme to the DNA template (138] partïcularly in view of its basic nature and strong binding to heparin, a compétitive inhibitor for DNA binding (139]. The mixture of 3 and 3' obtained by sucrose gradient centrifugation in the presence of 3.5 M LiCl retains the ability to bind DNA, whllst the a fraction is inaotive (139].
The sigma subunit, although implicated in the primary récognition of promoter DNA sequences, cannot bind to DNA, although both the isolated 3 and 3’ subunits hâve been reported to interact with sigma vitro
(149, 140, 141]. The 3 subunit itself interacts strongly with the antibiotic rifampicin (142], which inhibits transcription by binding to the enzyme.
Although no clear functional rôle can be allocated to the a subunlt, reconstitution of enzymio activity from the isolated subunits has an
absolute requirement for 3. 3' and a(143]; and an a„3 complex has been , .
2 and
reported to be a precursor in the assembly of the enzyme in vitro ^ g ) Furthermore, even though purine nucleoside triphosphates bind effi-
ciently to the holoenzyme, neither the core enzyme nor the isolated subunits show strong substrate .binding (139], suggesting that ail subunits are
necessary for the full activity of the RNA polymerase holoenzyme.
Initiation of transcription
As mentioned earlier, transcription of natural templates initiâtes at certain spécifie sites (promoters] on the DNA. Under certain conditions the DNA fragment protected by the RNA polymerase against nuclease digestion can be isolated and the sequence determined (145, 146, 147, 148]. Although
there is no striKing similarity between the sequences, careful analysis shows that there are structural similaritles (149, 253} which may be sufficient to explain the specificlty of primary promoter récognition.
Recent worK has shown that the 5’ terminal sequences of RNA synthesised in vivo are largely (but not entirely} heterogeneous (163). The formation of a tight complex with a promoter sequence requires sigma factor,
however, it is unclear whether the sigma factor itself carries the specificity déterminants for this reaction. As described earlier, the
tight complex probably involves opening the DNA hélix, but primary sequence récognition probably occurs without unwinding of the hélix, and thus two forms of polymerase-promoter comple x can be envisaged - either "open"
or "closed" with a dynamic equilibrium existing between the two
forms (127). It has recently been demonstrated that at the three different early promoters of phage T7, Chain initiation is promoted with equal
efficiency once stable preinitiation (open?) complexes hâve been formed (150), even though the three sites differ marKedly in their efficiency as promoters. Tnis suggests that transition from the closed to the open form occurs during site sélection and is largely irréversible. (For a
detailed discussion of promoter site sélection see ref. 151). Surprisingly, some evidence has emerged to suggest that the site of tight binding is not identical with the site of primary récognition; it is possible that the polymerase may firstly recognise a nucléotide sequence a short distance away, undergo a conformational change and subsequently drift to the tight binding site where it is then able to initiate RNA synthesis. (See references 253, 149, 321, 151).
nany protein factors hâve been identified ^ vitro which stimulate the initiation of transcription from natural promoters (152, 153, 154 etc.) but their rôle in vivo is unclear. One exceptionis the CAP factor (155) which together with cyclic ANP binds to a site adjacent to the lac promoter, and strongly stimulâtes the initiation of transcription from this site
(156, 157). It seems likely that the CAP-cANP complex acts to specifically déstabilisé the DNA-helix at the lac promoter and to facilitate the transition from the 'closed' to the 'open' form of the polymerase-promoter complex
(see ref. 158).
The rate of initiation by the RNA polymerase may also be regulated by métabolites of low molecular weight. When E. coli cells are starved
for an amino acid, the presence of an uncharged tRNA species in the ribosomal donor site leads to the rapid enzymic production of ppGpp and a concomitant dlminuation in total RNA synthesis (67, 68). It has recently been reported that the synthesis of rilu)oom.)l RNA Jus is stronj'ly inhihited by ppGpp (159) and a direct; intei’acLLon betwutin this mnlucule and the RNA [Mil /merasu Is lieiiee liKely.
Finally, operon-specific repressors seem to function by binding at or adjacent ta the promoter site In such a way as to physically exclude the polymerase from the promoter [160, 1611, even though the binding of the lac repressor to lac DNA is not reduced by prier binding of RNA polymerase
[162].
Terminatlon and Atténuation
Transcription of natural templates vivo gives rise to largely distinct RNA species, with spécifie terminal 3’ sequences [164, 1653;
and the template sequence following the termination site has also been shown to be spécifie in each case examined [166, 1673. There is, however, little apparent sequence homology between the sites. In vitro termination of transcription is generally less efficient and much longer messenger species are synthesised [168, 1693, but these again tend to be of unique length [160, 1703. Thus there are at least two distinct mechanisms for the termination of transcription i3 direct termination-sequence récognition by the RNA polymerase in the absence of additional factors ü3 termination of transcription at additional sites through the intervention of factors normally présent in vivo. Extracts of E. coli contain at least two factors with the ability to induce spécifie termination by the purified RNA
polymerase in vitro : rho [1683 and kappa [1133. Little is known of the rôle of kappa, but rho factor [50,000 daltons] has been shown to cause termination in vitro on X DNA at the same sites as those used vivo
[1713. Rho factor is also implicated in mutational polarity, whereby a
new codon for the termination of translation not only éliminâtes the function of the mutant gene but also reduces expression of distal genes in the same operon. Certain mutations of the rho factor resuit vivo in the reappearance of distal fonctions [172, 173, 174, 1753. According to the model of Adhya et al [1763, rho factor interacts with unprotected [after ribosome release]
nascent RNA behind the polymerase and subsequently causes the RNA polymerase to terminate transcription at the next rho-sensitlve termination site.
Atténuation of transcription is a closely related phenomenon, whereby in vivo only a fraction of transcribing RNA polymerases terminate at the attenuator site. In the case of the tryptophan operon, only about 10% of transcription initiations continue beyond a spécifie attenuator site into the structural genes for the tryptophan synthesising enzymes, and this
fraction can be varied in response to the cellular requirement for tryptophan [1773. [This subject is discussed in more detail in a later section]. The involvement of the N gene product of phage X in inhibiting the action of
rho factor [160, 170, 1793 can also be considered to be an aspect of atténuation, and the flnriing that mnny hactnrial opérons contain sites sensitive to
various levais of rho factor C180) suggests that the level of transcription of an operon may commonly be regulated not only at the level of initiation but also by atténuation once initiation bas taKen place (258). The exact molecular mechanism of termination, however, remains unclearj for although rho factor is able to arrest transcription in vitro,no enzyme release occurs under these conditions and the intervention of additional factors is thus implicated (165).
Antibiotics specifically inhibiting the RNA polymerase
1) Rifamycins : the antibiotic rifamycin was first identified in the 1950's, and its Chemical structure was determined by Oppolzer et al (181) in 1964. The semi-synthetic dérivative rifampicin is. however, the most
effective of the rifamycins in terms of antibacterial activity (see ref. 182) and this form of the antibiotic has been most commonly used for bio-
chemical studies. In vitro the antibiotic strongly inhibits DNA dépendent RNA synthesis, whereas DNA dépendent DNA synthesis is unimpaired (183, 184).
The antibiotic at low concentrations specifically inhibits the initiation of transcription (185), and exerts its effect by binding to the g subunit of the RNA polymerase (142). The complex structure of rifampicin suggests that it is not a simple substrate or template analogue, and the observation that it is the formation of the second phosphodlester bond, rather than the first, which is blocKed by rifampicin (186) shows that the mechanism of action of rifampicin is also complex. The vast majority of procaryotlc species are sensitive to low concentrations of this drug, and thus the
binding site for rifampicin has been strongly conserved throughout évolution.
Although for the most part euKaryotes are insensitive to rifampicin, dérivatives exist which hâve the ability to inhibit certain eukaryotic RNA polymerases, and thus the rifampicin binding site must exist in a modified form (see ref. 182).
2) Streptolydigin : RNA synthesis is far less sensitive to
streptolydlgin than to rifampicin (190) and at the concentrations used élongation of the RNA chain is blocKed (190, 191). As do rifamycin and streptovaricin, the drug blnds to the g subunit of the RNA polymerase (192).
3) Streptovaricin : this antibiotic is chemically very similar to the rifamycin group of antibiotics. (187), it Inhibits RNA synthesis by binding to the g subunit of the RNA polymerase, and blocKs initiation rather than élongation (188, 189).
4) Thlolutin : this small molécule (194) inhibits RNA synthesis both in vivo and lun vitro (195) and a recent report has suggested that the antibiotic inhibits the Initiation of RNA synthesis rather than élongation
(196). The subunit(s) to which thi3 drug binris is unKnown.
Coupling between transcription and translation
It Is well established in bacteria that nascent RNA is found associated wlth ribosomes [197, 198, 199, 2001 and the observation that the speed
of translation matches very closely the speed of transcription (201, 202, 2031 strongly suggests that vivo, translation of the messenger RNA occurs as it is being synthesised. As described earlier, arrest of translation at oM.ouubM- codon results in an arrest of transcription, unless translation is able to recommence at an adjacent initiator codon
(204, 2051. However, transcription of natural templates in vitro occurs without concomitant translation of the messenger RNA so produced; and ribosomal and transfer RNA's are synthesised in vivo without concomitant translation. Thus translation is not necessarily a prerequisite for continued transcription.
On the other hand, studies with thermosensitive mutants of translation hâve shown that whilst the transcription of ribosomal and transfer (bulKI RNA continues at the non-permissive température, the synthesis of messenger RNA is strongly reduced (206, 2071j^coupling is a property of spécifie genes, thus and implies that ’coupling-recognition' sequences are associated with these genes.
Since transcription of the trp operon from the trp promoter is sensitive to translation inhibition whilst transcription of the same operon from the P(_ promoter of phage lambda (in the presence of the N gene of phage lambdal is insensitive (208, 2091 it appears liKely that the 'coupling-recognition' sequences are rho-dependent termination sites;
and that the intervention of rho factor enforces the coupling between
transcription and translation (see earlier sections and ref. 1761. Presumably ribosomal and transfer RNA genes do not contain rho-dependent termination sites.
The involvement of RNA synthesis in the réplication of DNA
Nuch evidence has accumulated to demonstrate that DNA synthesis at the réplication fork is primed by short fragments of newly synthesised RNA
(less than 100 nucléotides longl which is subsequently removed by a 5' ---^ 3' exonucleolytic activity (210 - 2161. Nevertheless, the enzyme(s1 responsible for this synthesis hâve not been clearly identified.
Although continued DNA réplication in bacteria is largely unaffected by the presence of rifampicin in the growth medium (217, 2181, the réplication of DNA phage 0X174 is blocked even though conversion of the single stranded form to the double stranded réplicative form occurs under these conditions
(219, 2201. Thus an involvement of the RNA polymerase (or at least the 8 subuniti in DNA réplication is implied. The conversion of the single
stranded tü the réplicative form has been suggested to taKe place through the intervention of a new rifampicin-insensitive RNA polymerase, the product of the dnaG gene (221K Nevertheless, it has been shown that the conversion occurs either in the presence of rifampicin, or in a dnaC^_ host at the
non-permissive température, but not when both conditions are met simultaneously (222); the dnaC gene product and the RNA polymerase would thus be expected to hâve overlapping rôles. Furthermore, a protein factor has been identified which not only inhibits transcription by the normal RNA polymerase but
allows the polymerase to discriminate between N13 and 0X174 phage DNA s in the priming of DNA synthesis (223).
In vivo it is probable that the synthesis of RNA at the réplication fork is not carried out by the RNA polymerase holoenzyme; rather the enzyme must be modified to eliminate its rifampicin sensitivity or
alternatively a new enzyme, such as the product of the dnaC or dna G genes, must be seriously considered. Not enough data is available at présent to discriminate between these possibilities.
The RNA polymerase and the RNA phage réplication
Many authors hâve reported that the antibiotic rifampicin interfères with the growth of RNA phages, and that the effect disappears when
the host RNA polymerase has mutated to rifampicin résistance (224, 225, 226, 227). Thus the host RNA polymerase is involved in the life-cycle of RNA phage, however, from this evidence alone it cannot be concluded
that the intracellular réplication of RNA phage requires the host polymerase.
In the case of phage 06 it has been shown that the involvement is indirect, and that phage RNA synthesis continues normally in the presence of the
drug (226). This is due to the appearance of a new RNA synthesising activity in 06 infected cells which contains no subunit of the host RNA polymerase (228, 229, 230).
On the other hand, the réplication of RNA phages MS2, f2 and R17 is blocked by rifampicin; however, the involvement of the host RNA polymerase is again likely to be indirect,for RNA réplication proceeds in a normal manner for longer than 20 minutes after addition of the
antibiotic to the oulture medium (227, 231), and the block is not complété (232). It has been suggested thet RNA phage growth may be dépendent upon the concomitant synthesis of a labile bacterial protein, or that the host RNA polymerase in the presence of rifampicin remalns bound to the DNA and is unable to release certain accessory factors essentlal for RNA phage
réplication (232).
The tryptophan operon of E. coli
The tryptophan operon of E. coli consists of 5 genes, A to E,
coding for enzymes synthesising tryptophan in several steps from chorismic acid (2333. The genes are transcribed as an operon in the direction of E to A and expression from the trp promoter is repressed by the product of the unlinKed trpR gene in the presence of tryptophan (234, 235). A minor promoter, insensitive to repression, is located within the trpD gene and leads to low level constitutive transcription of the C, B and A genes (236). After derepression of the trp operon the A, B, C, □, and E gene products are produced in approximately equimolar amounts, (237, 230), although under certain conditions an excess of the E gene product has been reported (239) and is presumably due to the prématuré termination of
transcription within the trp operon referred to as ’natural polarity'.
Surprisingly, certain délétions within the trp operon lead to an increased transcription of the trp genes under derepressed conditions
(240) and it was proposed that such délétions remove a site whose normal fonction is to reduce expression of the trp genes (240). Further analysis of this phenomenon has shown that there exists an ’attenuator' région between the promoter and the first structural gene, and that the RNA transcript of this région may exist in a 10-fold molar excess over that of the structural genes (241). The excess 'leader' RNA transcript has been sequenced and shown to possess the same 5' terminus as the transcript of the whole operon (242, 177). Transcription is initiated about 165
nucléotides before the beginning of the first structural gene, and for the most part terminâtes about 20 nucléotides prier to the first gene
(243, 241, 244). The proportion of transcripts which arc elongated past the attenuator site has been shown to be a fonction of the cellular requirement for tryptophan,(241, 245), and is probably directly related to the levels of uncharged amino—acyl tRNA^^^ (245). It has been reported that, ^ vitro, the purified RNA polymerase will efficiently terminate
transcription within the leader région and thus an antitermination mechanism must exist in vivo (244). The nature of this antitermination is not yet clear, but as the leader RNA sequence contains two stretches of two-fold rotational symmetry (242) these may represent récognition sites for antitermination factors (244) . In seeming contradiction to this resuit, it has been reported that mutations of termination factor rho reduce atténuation within the leader région vivo, strongly suggesting that the observed termination is factor dépendent (247). A further observation is that the leader sequence contains two efficient ribosome binding sites, ono corrQS[Jonding to Uio AUG coilon at the bog.inning, of tho E gene, the other corresponding to an AUG codon Just under 30 nucléotides from the 5'
terminus (248]. From the Known sequence, a short peptide ('v 30 amino acids] cüuld be synthesised from the leader région, terminating at a pair of stop codons immediately before the attenuator région (242, 248].
This peptide has not been detected vivo, however, it has been reported that the ribosome binding site within the leader sequence is occupied in vivo (249]. The rôle of this additional binding site is as yet unknown.
The structure of the promoter-attenuator région is represented below.
AAGUU
IAUG
J
promoter
tt uuuuuu
t = translation stop codon a,b: ribosome binding sites /,//= zones of symmetry
The leftwards operon of phage lambda
The leftwards lambda operon comprises at least 8 genes involved with various aspects of recombination and lysogeny. The first gene transcribed, the N gene, codes for a protein whose presence is not only necessary
for normal growth of the phage but is also necessary for the transcription of those non-essential genes in the leftwards operon downstream from the N gene (see ref. 137]. The level of transcription initiation at is determined by the binding of the lambda repressor to this site, and various studies hâve shown that the promoter is located to the left of a sériés of repressor binding sites, the affinity of each for the
repressor diminishing rightwards away from the N gene (250, 251, 252, 254].
Each binding site contains an axis of 2-fold rotational symmetry, presumably reflecting the dimer form of the active repressor (255, 256]. The existence of additional inter-digitating rotational symmetries within this zone
(overlapping the first and second repressor binding sites] suggests that these may represent récognition sequences for other proteins, such as the product of the cro gene (257].
In the absence of the N gene product, transcription originating at continues to a site (t^^) immediately after the N gene, where factor rho induces termination of the growing RNA chain [168). In the presence of the N protein, transcription continues past this site and it has been postulated that the N gene product counteracts termination by interacting with the RNA polymerase [168). Nuch evidence has now accumulated to suggest that the interaction is direct rather than indirect [259, 260, 261),
howBver, the observation that the N proteins of the various lambdoid phages are not necessarily interchangeable shows that the fonction of the N gene product has an additional specificity [262) which must most probably be associated with the promoter région [263). Current models propose that only those polymerase molécules complexed with the N gene product can comtinuetranscription beyond t , and that the DNA sequence in the promoter région defines in the same way whether the polymerase is able to form a complex with N proteins of a particular specificity [264, 265). On the other hand, it has been reported that N protein can actually stimulate transcription from the leftward promoter [266); if the action of
termination factor rho is stochiometric then models can be formulated whereby the proportion of transcrlpts terminated at t^^ varies inversely with^the rate of initiation from P^. In other words, if the frequency of initiation is sufficiently high the termination mechanism may become saturated.
Most models for the action of the N gene product nevertheless require a récognition event between the N protein and the polymerase-promoter complex, and one possibility is that this involves the additional zones of symmetry mentioned earlier.
The recent observation that inversion of a distal segment of the leftwards operon [att - Ea22) does not affect the expression of the last gene [Int) in the operon [267) demonstrates that the major expression of this gene is under the control of a secondary promoter within the inverted région [267). It has been proposed that initiation at the promoter
[268) requires the presence of the CII and CIII gene products [269), and hence remalns under the control of the N gene product, which is required for the expression of these two proteins [see ref. 137).
The approximate structure of the leftwards operon is represented on the next page.
A _^int rho
--- J--- i
3’ FL \nl
--- — 1--- _______ !____ !
îatt int xis Ea22 lÊâ/Olfil N 1 I II in \
p::polymerase binding site s=zone of multiple symme tries IJIJII - repressor binding sites
Genes coding for RNA polymerase subunlts and the control of their expression Mutations conferrlng résistance to the antibiotics rifampicin and streptolydigin are located in a very small zone of the chromosome [referred to as the rif région] and probably represent alterations of the 3 subunit of the RNA polymerase (46. 142, 143, 108]. Gther authors hâve shown that the gene for the other large subunit of the RNA polymerase, 3’. maps adjacent to and is transcribed together with the gene coding for the 3 subunit (270, 271, 272, 273]. The direction of transcription is 3--- ^6’
(270, 272] and the synthèses of these two proteins are under coordinate control (274, 275]. There is also some evidence to suggest that the 33’
operon may include other cistrons, possibly coding for ribosomal genes (104, 286] although equimolar synthesis of the 3 and 3’ proteins with adjacent ribosomal proteins has not been observed (287].
The gene coding for the a subunit of the RNA polymerase is located in a different région of the chromosome (106, 280, 209], and is transcribed along with several ribosomal proteins (290] thus explaining the observed oversynthesis of this subunit (2-3 fold excess] to that required for core enzyme assembly (291].
At présent, the location of the gene coding for the sigma subunit has not been identified, and the available evidence suggests that it is not located close to either the 33’ operon or the gene coding for the a subunit (104, 105, 288. 291].
A tentative localisation has, however, been proposée! (S. Pedersen;
Personal communication3. Other components of the transcription machinery may be identified by the various existing thermosensitive mutations (see next section}.
It was at first thought that the expression of the 33' operon might be subject to simple feedback control by the 3 and/or 3’ subunitCsI, since partial inhibition of the RNA polymerase by rifampicin results in increased expression of this operon (2933. However, streptolydigin inhibition does not hâve a similar effect (2943 and an artificially increased level of active 3 and 3’ does not reduce the expression of this operon (2953.
Nonetheless it is clear that a réduction in the level of active polymerase can resuit in an increased expression of this operon (293, 296, 2973.
A positive control mechanism has been implicated in the control of 33’ expression (298, 2973, in particular by the existance of an amber mutant (am1003 which in the absence of suppression leads to a 4-fold réduction in the rate of synthesis of 3 and 3’ (2993. The synthesis of 0 is unaffected and that of a is only slightly reduced under such
conditions (2993. Mutants hâve also been isolated which primarily modify the expression of the sigma subunit (3G03. In particular, the rate of synthesis of the sigma subunit may be increased 10-fold and that of the a subunit 5-fold (3003. Thus négative control is likely to be dominant in the expression of the a and a genes, rather than positive control as in the case of the 3 and 3’ operon.
The genetic locations of these genes are presented in the next section.
The fir effect and other mutations affecting transcription
As described in previous sections, the antibiotic rifampicin binds to the 3 subunit of the RIMA polymerase and blocKs the initiation of transcription. Whilst no mutations conferring rifampicin résistance hâve been reported to occur except within the 3 subunit of the RNA polymerase, mutations at several loci hâve been reported to reduce the level of
résistance to rifampicin of an already rifampicin-resistant strain. This phenomenon, known as the ’fir-effect’ (fir is rif in reverse3 has been reported to occur primarily in diploids for the rif région, whereby the rifampicin résistance of a strain carrying the rif allele is eliminatedr by the introduction into the cell of the wild type rif gene (3013. The3
g
mechanism of the dominance of the rif allele is not understood, however, mutations of the rif région exist, which, although they do not alter the
g
rif phenotype of the haploid strain, are no longer dominant in their sensitivity (3013. Furthermore, dominant rif alleles do exist (6, 3023P though they appear only rarely (3U23. One possibility is that the sensitive
enzyme complexée! with rifampicin binds Irreversibly to promoter sites and blocKs further transcription. This is supported by the observation that inactivation of the rif allele by mutation leads to a return ofS rifampicin—résistance in the rif /rif merodiploid (320). The récessive S r
S r
rif alleles and dominant rif alleles are presumably altered in their relative affinities for the promoter site. Note that the dimer form of the RNA polymerase is probably the active form in vivo (121, 18), and the behaviour of mixed dimers (rif /rif ) may be substantially different r s from the behaviour.of either of the parental forms.
Mutations at other sites on the bacterial chromosome may also give rise to the fir-effect. These are i] recA ii) ts200 (firA) iii) ts29D (firB) iv) the pseudo-rif'" allele of strain RCB25C (fIrC) (51, 44, 303, 43).
The fIr phenotype of the recA allele only appears in low sait media;
when NaCl is added to a rifampicin-inhibited culture growth recommences immediately (51), however, a permeability effect can be eliminated. It has also been reported that the recA allele strongly affects the recovery of RNA polymerase from the E. coli nucleoid (0. Klessig; personal
communication).
The ts^QQ mutation (firA200) originally described by Babinet (43) renders RNA synthesis thermosensitive both ^ vivo and vitro and moreover, éliminâtes the rifampicin résistance associâtes with the rifg r allele (43, 44, and this thesis). When the wlld-type fIrA gene is
introduced into the double mutant, the abnormal sensitivity disappears (this thesis), suggesting that the wild-type firA protein oan protect the RNA polymprase in some way.
There is some evidence to suggest that the protein affected by the
—200 Kappa factor of Scnâfer & Zillig (this thesis, 113);
nevertheless the mechanism of the fir-effect with ts^^g is not yet understood.
The ts290 (firB290) mutation of Patterson et al (303) behaves in a very similar manner to the ts^^^ (firA200) mutation, both with respect
to the thermosensitivity of RNA synthesis and its interaction with rifampicin résistance. In virtue of its genetic location it is distinct from the other fir genes and genes for the RNA polymerase subunits (303, 115, 43 and
this thesis). The fonction affected is unknown.
The pseudo-rif marKer (firC) of strain RCB25C (43) is less dramatic P in its fir phenotype and does not render the strain thermosensitive for growth (43, 115). Again it is distinct from other fir and polymerase genes
(J-P Lecocq; personal communication).
Finally the ts3 and ts25 mutations, which are not associated with
the fir phenotype, render RNA synthesis thermosensitive both ^ vivo and in vitro and must represent additional transcriptional fonctions (115]. These mutations do not affect previously described genes (115, J-P Lecocq; personal communication).
It is not Known whether the mutations described above affect previously described components of the transcription machinery such as sigma (118), M factor (304), H^, (305) and others.
The many genes coding fo'r RNA polymerase subunits, additional components of the transcription machinery and the various mutations affecting transcription are summarised below.
Gene map mutation location *
(mins)
remarks references
firA ts200 ts RNA synthesis, fir 43,44 this
phenotype (Kappa termination thesis factor?)
16 ts25 ts RNA synthesis 43
firC (55) see ref 43 fir phenotype 43
recA 58 various general recombination, fir phenotype
306,307,51
relA 59 various stringent control of RNA synthesis
317,69
ait 'V67 altl CAP-independent expression of catabolite-sensitive genes
315
rpoA»^ 71 gro109 a subunit of the RNA polymerase (growth of phage P2)
106,288,289
erp 72 various CAP protein; positive control (with cANP) for expression of catabolite-sensitive genes
155,314
72 ^3 ts RNA synthesis 115
rho 83 suA(various) P termination factor (release 168,308,309 hiitA of polarity, N indépendant 244,174,322 sun growth of phage lambda]
- mes ami 00 positive control of 299
86’ expression
- mflB sig3 ts growth; overproduction 300
'V87 sig2 of a and a subunits 'V89 sigi of RNA polymerase
- 'v89 ts underproduction of 86' 298
at high température
rpo^ 89 rif'"(various] 8 subunit of the RNA 142,188,259 stl polymerase (rifampicin 312,260,261
ron résistance, interaction 272
groN etc. with N protein of phage X etc.]
rpocf^ 89 tsX 8' subunit of RNA 310,272,273
polymerase [ts RNA synthesis]
firB 90 ts290 ts RNA synthesis, fir 303,115?
phenotype
* Conforming to Bachman et al (316]
** Nomenclature of Hayward S Scaife (313).
Redundant remarks
As we hâve seen above, transcription is controlled largely at the level of initiation (and termination) rather than at the level of élongation. The précisé biochemical nature of these Controls is not, however, understood; for instance, at the level of initiation there is evidence both for and against a compétition between the repressor and the polymerase for promoter site binding. A good compromise is the
suggestion that the site at which the polymerase undergoes a preliminary récognition event may be quite distinct from the tight binding site.
however, this raises numerous fundamental questions about the nature of nucleic acid sequence récognition and also about drift phenomena in general. This is a field yet to be properly explored.
Other questions which still await an answer concern the nature of the interaction which allows certain proteins (e.g. CAP] to activate spécifie promoters and also the mechanism of transcription termination.
In the latter case, functional termination requires arrest of élongation of the RNA transcript followed by release of both the polymerase and the messenger. As yet this has not been observed ijn vitro.
There are without doubt other important aspects of the transcription process which remain concealed, and the existence of mutations, which obviously directly affect the transcription process iun vivo (but without any clear in vitro biochemical defect], and of a multitude of transcription- associated protein factors vitro (with no defined vivo rôle] suggests that the overall picture of gene expression and its control may be many- fold more complex than envisaged at présent.
The élucidation of the three-dimensional structure of the RNA polymerase holoenzyme is at the moment well behind timej when this finally arrives
it can only be hoped that a much-needed insight will be provided into the molecular mechanisms of initiation, élongation and termination which make up the transcription process in ail organisms.
A : GENETIC MANIPULATION OF BACTERIOPHAGE
1. Plvir lysatesln liquid media
0
A sensitive E.coli strain is grown te 2x10 cells/ml in 853 containing
—3 10
1°/o glucose and 5x10"" W1 CaCl^. 0,1 mis of a Plvir lysate at 10 pfa/ml is added and the culture aerated vigourously until lysis is completed about 3 1/2 hours later. The lysate is treated with chloroform and centrifuged to remove débris.
2. Sélection of Plcmlclr Lysogens
An overnight cell culture is resuspended in the same volume of 10 M_2
•3 8
MgSO^, 5x10~ M CaCl^ and a 0,1 ml portion treated with 10 Plcmlclr.
After 30' adsorption at room température the mixture is struck out onto TA12 containing 12.5 ng/ml of chloramphenicol and incubated overnight at 32°C.
Survivors of this treatment will be lysogensoF Plcmlclr and should be tested for thermosensitivity (l).
3. Lysâtes by beat treatment of thermoinducible lysogens
Exponentiel cultures of lysogens at 2x10 cells/ml are transferredto 40°C 8 for 20 min. with vigorous aération, and then incubated at 37°C for 60 min. befo- re adding CHCl„ and centrifuging. N.B. : Phage yields are in many cases strongly
«.J
reduced by continuai incubation at températures above 37°C.
4. Phage lysâtes by the plate method
0,1 mis of an overnight cell culture resuspended in 10 M MgSO is pre--2 adsorbed with 10 — 10 phage for 20 min. at room température. 3mls TA3,5 are then added and the mixture poured upon a fresh wet TA12 plate and incubated at 37°C until lysis is complété. N.B. : For PI and f it is préférable to preadsorb in the presence of 10 _2 M CaCl^ and plate upon Liebig plates at 35 — 37°C.
It is essential to use a Hfr [or f''"] host for phage f^. When confluent lysis is achieved, the overlay is removed to a stérile tube, the plate washed with 1 ml TBIMg, and the mixture vortexed and treated with CHCl^. After 30 min. at 4°C the agar and débris is removed by centrifugation.
5. Phage lysâtes by UV-induction of lysogens
0
Lysogens of UV—inducible phages are grown to 2x10 cells/ml in rich medium. Cultures are resuspended in l/5 volume 10 M MgSO^ and 2 ml portions _2 irradiated in standard 10 cm diameter glass pétri dishes for 30 secs, at
10 ergs/mm /sec. The irradiated cell suspension is now diluted 5-fold into fresh rich medium (eg 869) and aerated in the dark until lysis is complété [about 2 1/2 hours). The lysâtes are CHCl^ treated and clarified by centrifugation.
6. Phage lysâtes by mitomycin C induction of lysoqens
Lysogens of UV—inducible phages are grown to 2x10 cells/ml in rich medium, when mitomycin C is added to lüfig/ml. Incubation is continued for 10 min.
and the cultures centrifuged and resuspended in the same volume of fresh rich medium eg 869. After 3 l/2 hours aération, the cultures are treated with chlo- roform and centrifuged to clarify. It is préférable to dialyse such lysâtes against TM buffer to remove residual mitomycin 0 since prolonged exposure to low concen
trations of the drug can also induce lysogens.
7..EolB lysâtes in liquid medium
Sol B lysâtes can be prepared from a Sol b"*” strain by following the protocols for UV induction of mitomycin 0 induction of a phage lysogen. Trace phage in such lysâtes may be inactivated by UV irradiation - 2 ml portions in a standard glass pétri dish are irradiated for 10' at 10 ergs/mm /sec. The 2 activity of the colicin does not appear to be affected by such treatment.
8. General note on the préparation of phage lysâtes in liquid medium
Phage yields are considerably improved if the medium is rich [869 ra- ther than TBIMg) and if strong aération is carried out. In liquid media it is préférable to use higher multiplicities of infection in the case of the "weak"
phage (P1, f^) or to reduce the bacterial concentration at infection so as to allow a two step infection. In ail cases the bacteria should be almost totally lysed by the phage since lysis seems to reduce readsorption of free phage.
In the case of the "strong" phage (T5,T6) these précautions need not be taken, and for the "extra-strong" phage [T?) the highest titre lysâtes are obtained by infection of late log cultures at low multiplicities (eg 0,l).
The presence of MgSO^ at 10 M considerably stabilises many phage,_2 9. Formation and curing of lambdoid lysogens
Lysogens may be prepared by spotting a lawn of the relevant sensitive strain with a lysate at about 10 /ml of the phage in question. After overnight incubation the infected zone is resuspended in a droplet of 10 M MgSO^ and —2 struck out on TA12. A large fraction of such colonies will be immune to the phage in question. Curing is carried out by spotting a lawn of such lysogens with a lysate of a heteroimmune 0+ intégration déficient phage. For immX lysogens phage 21hyb2 is idéal. The infected zone is struck out as before and generally a large fraction of bacterial colonies will hâve lost bcth X immunity and 21—immunity.
10, Phage x Phage cross
0
The host to the cross is grown to 2x10 cells/ml in TBIMg and resus- pended in l/5 volume 10 M MgSO^, 1 oc of the suspension is infected at a m.o.i—2 of 3 into each of the parental phages and adsorption allowed for 20' at room température. The infected cells are now diluted 5 fold with 10 M MgSO^, _2 pelleted and resuspended in the original volume [Smls] of TBIMg. The suspen
sion is now diluted 100-fold into TBIMg and aerated for 90’ before treating with CHCl^. Dilutions are plated upon a sélective lawn.
11, Phage x lysogen cross
0
The lysogen is grown in TBIMg to 2x10 cells/ml and resuspended in l/S
-2 +
volume 10 M MgSO . In the case of C lysogens the prophage is induced by UV irradiation ( 30 secs, at 10 ergs/mm /sec. in a standard glass pétri dish]
before infection with the second phage at am.o.i of 3. Adsorption is allowed for 15' at room température, when the infected culture is diluted 5-fold with
10 M MgSO^, centrifuged, resuspended in the original volume TBIMg, and in- _2 cubated with aération until lysis.
12, UV mutagenesis of phage
ijPreparation of competent cells : A uvr\‘ec'*' strain (such as 594) 0
is grown to 2x10 cells/ml in TBIMg, centrifuged and resuspended in l/5 vo-
"2 2 lume 10 M MgSO^. 2 mis of this suspension are irradiated with 700 ergs/mm of*UV light in a standard glass pétri dish, and diluted 5-fold in fresh TBIMg.
After incubation at 37°C for 25 min. the culture is again centrifuged and resuspended in l/2 volume 10 M MgSO , This suspension is used directly.
^ 8 —2
II] Phage irradiation ; Phage stocks are diluted to 10 /ml in 10 M MgSO^ and 2 ml portions UV irradiated to a total dose of 2000 ergs/mm .2
This reduces viability to 1°/o.
III] Plating : 0,1 ml of dilutions in TBIMg are preadsorbed with 0,1 ml of competent cells for 20' at room température. 0,05 ml of an ove3>-
night culture of sensitive cells resuspended in 10 M MgSO^ is added to —2 each tube and the mixture poured in 2,5 mis TA7 upon TA12. Under these con
ditions clear plaque mutants represent 0,5 - 1% of the survivors. See (2).
14, NG mutagenesis of lambdoid phage
Exponential cultures of lysogens are induced by UV-irradiation [as described before] 25 min. before NG treatment or thermoinduced at 42°C for 15' before NG treatment. After the induction period, the cultures are cen- trifuged and resuspended in l/5 volume 10 M MgSO^, before diluting 5-fold —2 into TMS containing N0 at 300|ig/ml. After 20' without aération at room température (or 42°C for thermoinducible phage] the N0 treated cultures are centrifuged, washed with TBIMg, and resuspended in the original volume of TBIMg, before diluting 20-100 fold into fresh prewarmed TBIMg. After 2 hours
aération the lysâtes are chloroform treated and dilutions plated out on a sensitive host. Under these conditions clear plaque mutants represent 5 — 9% of the survivors.
15, ICR191 mutaqenesis of lambdoid phage
Exponential cultures of lysogens are centrifuged, washed to remove fræ phage, resuspended in l/5 volume 10 Wl MgSO^, and induced by UV irra—2 diation as described before, 1 ml aliquots are diluted 5-fold into TBIMg in foil-covered flasks and ICR191 solution [ made up in the dark) added to give a final concentration of 75iig/ml. After 3 hours aération the mutagenised lysate is CHC1„ treated and centrifuged to clarify, N.B. : Ail steps must
O
be carried out in the dark since ICR191 can probably be photoactivated, leading to lésions similar tothose induced by UV irradiation. In the absence of light frameshift mutations are prédominant (see (4) ), Under these condi
tions clear - plaque mutants represent 1% of survivors. (Contrary to ref.
4, proflavin was not found to be an effective mutagen of lambdoid phage either after infection or after induction).
B ; GENETIC MANIPULATION OF BACTERIA 1. Hfr X F cross
0
Both the donor and récipient are grown to 2x10 cells/ml in 853, 1 ml of the récipient culture is mixed with 0,2 ml of the donor culture in the botbom of a 25 ml conical flask, and incubated without agitation for 60 min. at 32°C or 37°C. 4 mis 10 M WlgSO are added, the mixture centri--2
^ _2
fuged, and after resuspension of the pellet in 1 ml 10 M WIgSO , 0,1 ml
0-4 ^
of dilutions 10 to 10 are plated out onto sélective media.
2. F~*~ X F cross
Mating is performed exactly as for the Hfr x F cross, except that the donor F+ culture is grown in minimal medium to select against sponta- neous loss of the episome,
3. PL transduction
An overnight culture of the strain to be transduced is resuspended
—2 —3
in 10 M MgSO^ 5x10 M CaCl^. 0,1 ml portions are treated with 0.1, 0,05, 0.02, 0,01 ml of a Pl lysate grown on the relevant donor strain, and adsorp—
tion allowed for 20' at room température. After this time 0,2 ml 1 M triso- dium citrate is added to each tube and the mixture plated upon sélective plates, which are subsequently incubated for 2-3 days. N.B. : Wild type pl is not used since lysogens are difficult to cure and moreover lysogens possess a new Pl associated restriction System. This problem is eliminated by the use of Pl mutants (Plvir, PlKc) which cannot lysogenise or by the use of thermoinducible F’icmlclr where cured cells can be isolated by their capacity for growth at 42°C. (see Ref, s).
4. UV mutagenesis of bacteria
Uvr rec strains are grown to 2x10 cells/ml in rich medium and resuspended in l/5 volume 10 M MgSO^. 1 ml aliquots are irradiated in a—2 standard glass pétri dish to total doses of 800, 1400, 2,000, 2,600 ergs/
2
mm /sec, and serial 10-fold dilutions either plated directly upon sélec
tive media or grown for at least 2 hours in rich (unselective) media be—
fore commencing ampicillin enrichment for mutants in cases where a posi
tive sélection is not available.
5. "Second Site" lysogénisation by lambdoid phage
An overnight culture of an att X deleted strain is grown in TBIMg 0
to 2x10 cells/ml, resuspended in l/5 volume MgSO^, and aerated for 15' at 37°C . Phage (XC^ , XCl^^^ etc] is now added to a m,cii of 5 and after 15 min, adsorption at room température the mixture diluted 5-fold into fresh TBIMg and aerated. for 90 min. at 32°C. The infected culture is now centrifuged, washed in 10 M MgSO^ , and resuspended in 2x the original volume of_2
TBIMg. Aération is continuée! at 37°C for a further 3 hours, when the cul- ture is centrifuged and resuspended in l/20th volume of 10 M MgSQ^, Aliquots —2 may now be directly treated with phage to select for immunity or résistance
(see below), alternatively they may be diluted into fresh medium for ampicillin enrichment (see below).
6. Ampicillin enrichment for Auxotrophs
An overnight culture of bacteria in non-selective minimal medium (after mutagenesis or phage treatment etc..) is resuspended in l/l0 volume 10 M MgSO^, and 0,1 ml aliquots diluted into 10 ml portions of starvation _2 medium—lacking the relevant amino acid/vitamin etc. (N.B. : In most cases minimal medium must be used, supplemented with the other requirements of the strain in question. Nevertheless for tryptophan requiring auxotrophs acid- casein hydrolysate may be used). Aération is continued for 60 min. at 37°C, and ampicillin added to a final concentration of 25|ig/ml, Incubation is conti
nued for 2-2 1/2 hours at 37°C and the cultures centrifuged, the pellet washed twice in 10 M WlgSO^ and resuspended in 1 ml of the same. Oilutions may be _2 plated out directly onto minimal agar containing the necessary suppléments plus the amino acid/vitamin in question, and survivors screened for auxotro- phy. The enrichment may be repeated 2-3 times, diluting the final suspen- sion in 10 M MgSO^ 100-fold into non-selective medium and growing overnight _2 before each step, This method can be modified to enrich for antibiotic-sen
sitive or thermosensitive strains.
7. Sélection for phage résistance or immunity
Aliquots of the bacterial culture are spread upon fresh EMBO plates preseeded with 5x10 phage. Where for instance lambda lysogens are to be se- g lected it is préférable to use XCIh" or XCIh^^ ^ simultaneous phage ré
sistance and X immunity the plates are preseeded with boih XCUtT and the phage in question, After overnight incubation at 32°0 or 37°C colonies insensitive to the phage are characterised by their lighter coloration and their smooth morphology. In many cases a number of genetically sensitive colonies will appear in the presence of phage and these can be recognized by their darker coloration and irregular shape.
Such preseeded plates may also be used for "replica-spot" phage résistance testing.
8. Testing for multiple lambda lysogens
Single and multiple X lysogens may be discriminated by their dif- fering sensitivities to phage XCI90C17. Lawns of the test strains are
Q g
with XCI90C17 lysâtes at 10 /ml and 10 /ml and inoubated at 32°C or 37°C,
g 8
Résistance to the lysate at 10 /ml but sensitivity at 10 /ml indicates a
multiple lysogen, wheras sensitivity tolxith concentrations is characteristic of monolysogens (or non-lysogens), [Asecond test involves determining the frequency ofcQϕ^laque mutant phage in the supernatent of an overnight cul
ture of the lysogen in question. Both the phage titre and the frequency of clear plaque mutants are strongly diminished in supernatents of multiple lysogens),
9. Transduction by phage lambda frecovery of the transducing phage]
Non defective phage : Aliquots of the transducing lysate are pre- adsorbed with 0,2 ml of an overnight cell culture resuspended in 10 M MgSO^, -2 and plated upon sélective media. Best results are obtained under conditions where slightgrowth of the background lawn occurs. For instance a few drops of rich
medium may be added to a cell suspension before plating on minimal medium.
Non-defective transducing phage may be recognised by the appearance of haloed centred plaques. Where sélection is carried out for suspension of thermosen- sitivity a cluster of colonies [or "star") is more common than a haloed plaque,
Defective phage : Aliquots of the transducing lysate are mixed with 0,2 ml of an overnight recA culture resuspended in 10 M MgSO^, and homoim- _2 mune non-defective ["helper") phage added to a m.Qi.of2 - 3. After 20' adsorp—
tion at room température the infected cells are plated upon sélective media.
If rec"*” cultures are used the vast majority ofclonæ surviving the sélection will be substitution transductants rather than lysogens for the transducing phage (see Ref. 6).
10. The "Blu" test
Pgl” ( 6 — phospho—glucouslactonase) mutants accumulated a polysac
charide which gives the "starch-iodine" reaction. The strafit to be tested is strtlck out onto minimal maltose agar, and after overnight grown the plate is overlaid with WA7 containing 1 ml 0,1 % I in 1 % Kl. A deep blue coloration develops at the edges of Pgl"” [blu) colonies but not with pgl colonies 11. The "Nue R" test
Inosine and Guanosine do not induce ail the enzymes necessary for their dégradation , and hence strains which are constitutive for the synthesis of these enzymes [nucR~) will grow better under conditions where the sole source of carbon is inosine or guanosine.
Minimal agar plates containing 0,1 % guanosine as the cartxn source are struck out with the strains in question. Good growtiT after 2 days at 32°C indicates a nue r” strain.
12. Nitrate Reductase testing
Chlorate agar : chl'*' strains do not grow on chlorate agar in the absence of oxygen, wheras chl strains grow well, Oxygen is removed by pla—
cing the agar plates in a dessicator, evacuating to 0,1% atmosphère for 5', refilling with N^, repeating twice. The dessicator is incubated overnight 37°C, the plates removed and incubated overnight in air to enlarge the co
lonies (Soth chlA and chlD mutants survive this treatment (l0) ).
Nitrate agar ; single colonies of the strains to be tested are
stabbed into nitrate agar and incubated overnight at 37°C. To each tube is added 0,5 ml 0,0% sulphanilic acid in 5 M acetic acid followed by 0,1 ml 0,5%
a—napthylamine in 5 M acetic acid. The development of a deep red coloration indicates that the strain is nitrate-reductase proficient, ChlD mutations alone do. mot totally abolish this activity
13. Sex testing
Hfr and f"*" strains are sensitive to the male—spécifie phage f .
Lawns of the strains to be tested on Liebig plates are spotted with f^ at 10 pfu/fnl ^ 8 and incubated overnight at 37°C. A turbid zone of lysis indicates that the strain is or Hfr.
14.Isolation of thyA strains
Bacterial strains are grown to saturation in minimal medium + 100jj,g/ml thymine and diluted 50-fold into the same medium. Separate samples are trans-
ferred to stérile flasks and trômetfcpiim added to 10 , 20 , 30[j,g/ml. Incubation is continued for 1-3 days until saturation is reached, and the cultures struck out onto minimal agar + 100pg/ml thymine.
A large proportion of the survivors are thyA (see note later) 15. Sélection for a récessive résistance marker feg UraPl
Mating mixtures or bacteria treated with transducing phage are poured in 2 mis WA7 upon minimal agar plates and incubated overnight at room température or for 6 hoursat 32°C. A futher 2 ml of WA7 containing 600ng/ml 6 - azauracil is poured upon the plate (to give a final concentration of 30|ig/ml after diffu
sion) and the plates incubated for a further 1 - 3 days at 32°C, Surviving colonies cannot take up exogenous uracil or 6-azauracil and are hence résistant.
G ; LARGE SCALE PREPARATION OF BACTERIA AND BACTERIOPHAGE 1, Large Scale Bacterial Growth
A preculture of 50 - 250 mis 069 is inoculated with a single colony of the bacterial strain and grown ouernight at 32°C or 37°C. The precultu
re is then diluted into 2-15 liters Burgess's Growth Medium and grown ei- ther to stationary phase or to late log phase(0D_„ = 1,ll with aération,
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3 ml antifoam A (Sigma] is added to large cultures. Cells are harv/ested by low speed centrifugation, washed in .01 volumes 10 M MgSO^ , repelleted _2 and stored at -20°C.
2, Large Scale Phage Growth
A 100 ml preculture of a lysogen is diluted into 2 litres Burgess's Growth Medium containing 10 M MgSO^. ( BURMg] in a 5 litres flask, and—P incubated at 32° or 37° to 0D„^ = 0,22 - 0,25.
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Thermoinduction ; 450 mis of boiling BURMg medium is added to the 2 litres culture at 32°0, raising the température to 42°C. The flask is then incu
bated in air at 37°C for 2 hours,
UV — induction ; The culture is centrifuged at room température and the pel- lets resuspended in a combined total of 100 mis 10 M MgSO^. The suspension_2 is divided into 4 x 25 mis in 18 cm diameter glass pétri dishes, and each
2 0
UV irradiated to a dose of 3300 ergs/mm”^ (Total dose = 3 x 10 ergs), The irradiated cultures are repooled and diluted into a fresh prewarmed 2 litres portion of BURMg, and incubated at 37°C in the dark for 3 hours,
(Note : the^phage may be prepared by the plate method and the combined su—
pernatents pooled and precipitated with PEG 6000 as described below) 3, Large Scale Phage harvesting
Lysis defective phage (S7) : The culture is subjected to low speed centrifugation at 4°C, and the pellet resuspended in a small volume of TM buffer (6 mis for a 2 liter culture). DNA'se is added to 10 ng/ml and RNA'se to 5 ng/ml follbwed by 1 ml CHCl„ to lyse the cells. The mixture is well
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mixed and held at 4°C for 1 hour before centrifuging to remove débris, Lysis proficient phage ; (Method of Yamamoto et al, ref. 15) 5 mis CHC1„ is added to the 2 litre lysate, followed by DNA'se to 1 ng/ml.
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The mixture is briefly stirred and allowed to stand in the cold room for 1 hour, when NaCl ( solid) is added to 0,625 M, and débris removed by low speed centrifugation. 0,25 vols of 50°/o W/V PEG 6000 are added slowly with stirring ( final concentrations ; 10% PEG 0,5 M NaCl) and the mixture allowed to stand at 4°C for 2 days, The upper 2 litres are siphoned off, the remai-
ning 5G0 mis + sédiment centrifuged, and the pellet resuspended in a small volume of supernatent and recentrifuged at 10,000 g for IG min. The pellet is now resuspended in 4 volumes of TM buffer ( 10 mis for a 2 litre lysate).
4, CsCl Préparative Gradients
CsCl gradients are preformed in 40 ml cellulose nitrate tubes, oare—
fully pouring 5 layers of 3 mis each of CsCl at 1.7, 1.6, 1.5, 1.4 and
1.3 gm/ml in TM buffer. The gradient is overlaid with the concentrated phage sample in TM (see above), followed by 4 mis liquid paraffin to balance
Tubes are centrifuged overnight (20 hr) at 22,5krpm in the SW27 rotor ( 7,5°C) and the phage bands extracted through the side of the tube with a stan
dard 5 ml syringe with needle. Note : One précaution which should be taken is to stick a small piece of adhesive tape ( eg "elastoplast") on the sur- faoe of the tube at the point at which the needle is to be inserted.
5, Unformed CsCl gradient for phage séparation
Phage bands from the préparative gradients which contain two phage of slightly differing densities are diluted with CsCl 1,5 gm/ml in TM to 10 mis, transferred to 12 ml cellulose nitrate tubes and overlaid with liquid paraf
fin to balance. These are then centrifuged at 7,5°C in the SW40 rotor at 25,000 rpm for 60 hours. Phage which differ in DNA content by 5°/o or more may be separately by this method and are extracted by syringe as described above.
6, CsCl solutions
The required amount of CsCl is made up to a volume of 95 mis with ditilled H^O, and 1 ml of each Tris/HCl pH 7,5 and 1 M MgCl^ added. The solutions are then autoclaved and when cool stérile distilled water is added until the correct refractive index is attained.
Density (gm.ml) gms CsCl/100 ml Refractive index
1,3 40 ,24 1,3625
1,4 54,83 1,3718
1,5 67,36 1,3810
1,6 81 ,65 1,3902
1,7 95,14 1,3994
Dialysis of phage bands from CsCl gradients
The phage bands are dialysed overnight versus TM + 0 ,5 M NaCl for 6 hours against TM +0,1 M NaCl, and finally for 6 hours against TM + 0,01 M NaCl before use.
