h/IGG
Molec. gen. Genet. 169, 337-343 (1979)
© by Springer-Verlag 1979
Genetic Organization of the E. coli Chromosome Around the Structural Gene for Initiation Factor IF3 (infC) M. Springer, M. Graffe, and M. Grunberg-Manago Institut de Biologic Physico-Chimique, 13, rue Pierre et Marie Curie, F-75005 Paris, France
Summary. A set of 2 transducing phages carrying varying lengths of the E. coli chromosome around the structural gene for initiation factor IF3 (infC) was derived from 2p2 which is known to carry, besides infC, the structural genes for the c~ subunit of phenylalanyl-tRNA synthetase (pheS), the fl subunit of phenylalanyl-tRNA synthetase (pheT) and the structural gene for threonyl-tRNA synthetase (thrS). The E. coli coding content of these derived phages was analysed by genetic complementation of a set of mutants and by SDS-polyacrylamide gel analysis of the proteins synthesized in UV irradiated cells infected with these phages. The segregation pattern of the different genes among these derived phages indicates that the order of the genes is p h e T - p h e S - "'P12"' - (infC, thrS) where infC is probably between " P 1 2 " and thrS. "'P12" is the structural gene of a 12,000 molecular weight unidentified protein.
Introduction Many of the genes coding for proteins involved in translation are clustered on the E. coli chromosome (Nomura et al., 1977). Some of these clusters are organized in transcription units carrying the structural genes for proteins playing very different roles in the translation machinery;e.g, the ribosomal proteins S 7 and S 12 are expressed from genes located on the same transcription unit as the structural genes for elongation factors Tu and G (Jaskunas et al., 1975). For offprints contact: M. Springer Abbreviations: phenylalanyl-tRNA synthetase: PRS (EC 6.1,l.20), threonyl-tRNA synthetase: TRS (EC 6.1.1.3), Initiation factor IF3: IF3, SDS: Sodium dodecyl sulfate, ppR: pyrophosphate resistant, ppS: pyrophosphate sensitive
A 2 transducing phage (2p2) has been isolated which carries the structural genes for initiation factor IF3 (Springer et al., 1977b), for the ~ and/3 subunits of phenylalanyl-tRNA synthetase (PRS) (Hennecke etal., 1977a) and for threonyl-tRNA synthetase (TRS) (Hennecke et al., 1977b). This phage thus carries the structural genes for four proteins belonging to the translational apparatus of the cell. This clustering of translational genes raises the question whether these genes are expressed as a single or separate transcription units. The aim of the present work was to isolate and characterize a set of transducing phages to provide a way to map the structural genes which are on that 2 transducing phage and to determine whether they belong to separate transcription units or not. To do so, we derived from the original transducing phage a set of phages containing deletions of varying lengths in their E. coli D N A component. The original 2p2 transducing phage was ideal for this purpose because almost all the non essential parts of 2 are absent or have been replaced by E. coli DNA (Springer et al., 1977b). Thus selecting for deletions which did not affect the phage's viability gave phages where E. coli D N A is deleted. This set of phages was studied genetically by complementation of pheS, pheT, and thrS mutants and biochemically by SDS polyacrylamide gel analysis of the labelled proteins synthesized in infected UV irradiated cells.
Materials and Methods The E. coli strains used throughout the present work are listed in Table 1. Besides 2p2 which has been described (Springer et al,, 1977b), all the 2 phages used were derived from 2p2 as described below. Transductions using P l v i r w e r e performed as described by Miller (1972).
0026-8925/79/0169/0337/$01.40
M. Springer et al. : Genetic Organization of the E. coli Chromosome
338 Table 1. List of E. coli strains used Strain
Genotype
Origin or Reference
AB1365
F , th~l, argE3, his-4, proA2, lacY1, galK2, mt~l, xyl-5, tsx-29, supE44, 2pheS5, 2 + lysogen; all other markers
Springer et al. (1977a)
AB1601 (2 +) AB1701 (2 +) AB4610 (2 +) 2K401 159 (2ind-) AB1360 NP37 JPII16 GT342 AB1380 (2 +)
like AB 1365 pheT354, 2 + lysogen; all other markers like AB 1365 thrS1029, aroD-6 +; all other markers like AB 1380 (2 +)
thi-1, rha-4, lacZ82, ga133, kdpABC5, trkD1, trkA401, 2rpsL, su-, gal-, UVs, 2ind lysogen aroD-6; all other markers like AB1365 HfrC, pheS5, rel-1, tonA22, 7"2R HfrH, pheT354, thi, galE-PL5, rel-1, 2thrS1029, trp, his, proC, rpsL, ara, lae, gal, real, 2 r aroD-6, rpoB, 2+ lysogen; all other markers like AB1365
aroD-6 + pheS5 transductant of AB1360. Plvir grown on NP37
aroD-6 + pheT354 transductant of AB 1360. Plvir grown on JP 1116
aroD-6 + thrS transductant of AB1380 (2+). Plvir grown on GT342 Epstein and Kim (1971) Springer et al. (1977b) Taylor and Thoman (1964) B6ck and Neidhardt (1967) Russel and Pittard (i971) Derived from GT302 Johnson et al. (1977) RifR )~+lysogen derivative of AB1360
Isolation of the Pyrophosphate Resistant (ppR) Derivatives of 21)2 Three lysates of 2p2 made from different clones were treated with pyrophosphate as follows: 10 gl of lysate (at about 10l° p.f.u. ml-1) was mixed with 1 ml of 10 mM sodium pyrophosphate and 15 mM Tris-HC1 pH 7.5 (the final pH of the mixture is 8.5). After 30 min at 45 ° C, 0.1 ml of 1 M MgSO 4 was added and the mixture preadsorbed to 0.2 ml of a stock of AB1365 prepared as described by Shrenk and Weisberg (1975) for 15 min at 30°. The mixture was then plated on R plates (Miller, 1972) supplemented with 10.2 MgSO4 and 0.2% maltose (RMM plates), and incubated for 12 hours at 44 ° C. The lysates obtained as described by Miller (1972) have a titer of about 109 to 10 l° p.f.u. ml- 1. The pyrophosphate treatment was repeated two more times. The survival frequency was 10 -4, 10 -3 and 5x 10 1 after the first, second, and third pyrophosphate treatment, respectively.
(Springer et al., 1977b). After isotopic dilution, the cells were centrifuged (20 min at 20,000 x g), washed with an excess of 10 mM Tris-HC1 buffer pH 6.8 and resuspended in the minimal volume of the same buffer, then frozen. The cells were thawed and refrozen three times and then treated with DNAse I (from Worthington) at a final concentration of 20 ~tg ml- ~ for 20 min at 4 ° C before resuspending in the SDS polyacrylamide gel sample buffer (Laemmli, 1970). The samples were kept at - 2 0 ° and an aliquot containing 10,000 cpm was heated 2 min at 100° before electrophoresis, performed as described previously (Springer etal., 1977b). The gel was then treated successively with 7% (v/v) acetic acid for 1 hour; with 10% (v/v) acetic acid, 50% (v/v) methanol and 0.25% (w/v) coomassie brilliant blue for 2 hours; with 10% (v/v) acetic acid and 50% (v/v) methanol for 15 min; with 7% (v/v) acetic acid and 10% (v/v) methanol for 24 hours. The gel was further treated as in Springer et al. (1977b) and the films exposed to the dried gels for 440 hours.
Preliminary Characterization of the ppR Derivatives of 21)2 The ppR lysates were plated with 2K401 on RMM plates supplemented with 10 1 M KC1 (RMMK plates) at dilutions to obtain isolated plaques. The plaques were diluted into a drop of 10 -2 MgSO4 with a toothpick. A platinum wire was then used to transfer phages to RMM plates covered with a lawn of the Ts- mutants. Transduction was made at 44 ° C. 2K401 was used as indicator strain because it grows on RMMK plates but not on RMM plates so that the bacteria taken from the RMMK plate together with the phage will not grow on the RMM transduction plates. The only bacterial mutants used at this preliminary stage were pheS (AB1601 (2)) and pheT (AB1701(2)) thermosensitive mutants. Phages with a particular transduction pattern were purified three times, and lysates were made as described by Miller (1972).
Results Genetic Characterization o f the P h a g e s D e r i v e d f r o m 21o2 R e s i s t a n c e o f a 2 s t r a i n t o c h e l a t i n g a g e n t s s u c h as p y r o p h o s p h a t e d e p e n d s u p o n t h e size o f t h e D N A p a c k e d in t h e h e a d o f t h e b a c t e r i o p h a g e . U n d e r s p e cific c o n d i t i o n s , p h a g e s w i t h s h o r t e r s i z e d D N A a r e p y r o p h o s p h a t e r e s i s t a n t (ppR) w h e r e a s p h a g e s w i t h
UV h'radiated Cells System and Gel Electrophoresis
longer D N A are sensitive to t h a t c h e l a t i n g a g e n t (Park i n s o n a n d H u s k e y , 1971). T h u s , p r e e x i s t i n g d e l e t e d p h a g e s in t h r e e i n d e p e n d e n t s t o c k s o f t h e p y r o p h o s p h a t e s e n s i t i v e (ppS) s t r a i n 2p2 c o u l d b e i s o l a t e d as
The lysates were extensively dialyzed against TM buffer (Tris-HCl pH 7.5, 10 mM; MgSO4, 10 mM). The growth of 159(2), infection and radioactive labelling were carried out as described previously
described under Material and Methods. After a prel i m i n a r y c h a r a c t e r i z a t i o n , a s p e c i f i c set o f p h a g e s from each stock was further studied by complementa-
M. S p r i n g e r et al. : G e n e t i c O r g a n i z a t i o n o f the
E. coli C h r o m o s o m e
339
T a b l e 2. C o d i n g c o n t e n t o f the p y r o p h o s p h a t e resistant p h a g e s d e r i v e d f r o m 2p2 Phage
A:Genetic complementation
B : G e n e s expressed in the U V i r r a d i a t e d cells
pheT
~
~
P12
IF3
TRS
+
+
+
--
--
+
+
pheS
thrS
2p2
+
+
+
+
+
2ppl~
+
--
--
+
--
2ppl-9 2 p p l 16 2ppl~6 2 p p l 103 2 p p l 104 2pp1-125 2 p p l 141
-. + +
-. + + + -
+ . + + +
--
2pp2-3 2pp2-34
+ -
2pp3 1 2pp3-3 2pp3 6
. + -
.
.
.
.
.
.
+
.
.
.
+ +
+ + + -
+ + + +
+ + + + +
+ + + -
+ -
+ -
+
+
+
+
+
.
-+ .
--
+
. +
. + -
. . -
.
.
.
.
.
A : Genetic complementation : m a r k e r : 5 gl of the ppR Iysates (at a b o u t 101° p.f.u, m 1 - 1 ) were s p o t t e d with 1 d r o p of AB1601 (2) stock diluted 102 times with 10 . 2 M M g S O 4 on R M M plates i n c u b a t e d at 44 ° o v e r n i g h t . pheT m a r k e r : s a m e m e t h o d as for pheS except t h a t AB1701 (2) was used diluted 104 times. thrS m a r k e r : s a m e m e t h o d as for pheS except t h a t A B 4 6 1 0 (2) was used diluted 10 times on m i n i m a l A glucose plates (Miller, 1972) s u p p l e m e n t e d with arginine, histidine a n d proline: i n c u b a t i o n was at 37 ° for 24 hours. + m e a n s c o n t i n u o u s g r o w t h on the spot
phe S
B: + -
means Genes means means
discontinuous growth (varying expressed in the U V i r r a d i a t e d t h a t the c o r r e s p o n d i n g b a n d is t h a t the c o r r e s p o n d i n g b a n d is
b e t w e e n < 10 to > 100 colonies). cells: p r e s e n t on the gel a b s e n t f r o m the gel
tion of pheS, pheT and thrS mutants. The complementation properties of the three independent sets of ppR phages given in Table 2 can already be interpreted to order these three genes. Assuming that each ppR resistant phage carries a single deletion (the validity of this assumption is considered in the Discussion) the existence of a specific phage with two genes missing and one (or more) present means that the expressed gene(s) cannot be located in the interval between the two missing ones. If the expressed gene is within that interval then two deletions on either side of the expressed gene are necessary. So the existence of 2ppl-4 shows that pheT is outside the pheS thrS interval and the existence of 2ppl-9 shows that thrS is outside the pheT - pheS interval, thus the order of these genes is pheT pheS thrS or thrS - pheS - pheT.
Biochemical Characterization of the ppR phages Derived from 2p2
When irradiated under specific conditions with UV light, E. coli strains still incorporate labelled amino acids in response to 2 infection. SDS polyacrylamide
gel analysis of the labelled material shows bands corresponding to the different gene products of 2, but when the UV irradiated cell is a 2 lysogen, the 2 repressor, still active under the conditions employed (Springer et al., 1977b), inactivates the expression of the genes transcribed from the two main early promoters of the infecting 2:p~ and Pr- However, if the infecting 2 is a transducing phage the main genes expressed are transcribed from E. coli promoters which are not repressed by the 2 repressor. Thus proteins corresponding to the E. coli content of the infecting phage can be seen by SDS polyacrylamide gel analysis of the labelled material from a UV irradiated lysogen infected with a 2 transducing phage. After a preliminary genetic screening (see Material and Methods), several pyrophosphate resistant phages derived from 2p2 were studied using this UV irradiated cells system. 2p2 infection of UV irradiated 159 (2 ind-) strain stimulates the incorporation of (lgc) leucine into five proteins (Fig. 1 sample 2), these proteins are, in order of decreasing molecular weights: ~ subunit of phenylalanyl-tRNA synthetase (PRS) (Hennecke et al., 1977a), threonyl-tRNA synthetase (TRS) (Hennecke et al., 1977b), ~ subunit of PRS (Hennecke et al., 1977a), initiation factor IF3
340
M. Springer et al. : Genetic Organization of the E. coli Chromosome
., ,,
f3 PRS TRS
--
~ PRS
-
IF3
P12
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Fig. 1. SDS polyacrylamide gel fluorogram from extracts of UV irradiated cells infected with ppR phages derived from )@2. The samples were treated as indicated under Material and Methods. Sample 1: no infecting phage. Sample 2: 2p2. Sample 3: 2pplM. Sample 4: 2ppl-9. Sample 5: 2ppl 16. Sample 6: 2pp1-23. Sample 7: 2ppl 25. Sample 8: 2ppl 30. Sample 9: 2ppl~0. Sample 10: 2pp1-46. Sample 11: 2pp3-3. Sample 12:2pp3 6. Sample 13: 2pp348. Sample 14: 2ppl 103. Sample 15: 2ppl 104. Sample 16: 2ppl 139. Sample 17:2pp2 3. Sample 18: 2pp24. The molecular weights are: fiPRS, 94.000; TRS, 76.000; c~PRS, 38.000; IF3; 22.000; P12, 12.000
(Springer et al., 1977 b), and a 12,000 molecular weight unidentified protein which we call P12. As seen in Fig. 1 (samples 3 to 18) each of ppR phage shows a specific pattern of bands which corresponds to the genes expressed from the E. coli promoters present on each of these phages. Many of these phages do not synthesize one or more proteins which are synthesized by 2p2 because the corresponding genes have been deleted. Some of the ppR phages synthesize proteins not made by 2p2. In particular 2ppl-4 and 2ppl-30 (Fig. 1 samples 3 and 8) synthesize a 34,000 molecular weight protein (under the c~PRS position) and 2pp3-6 (Fig. 1 sample 12) synthesizes a 60,000 molecular weight protein (under the TRS position). These new proteins may be fragments caused by deletions starting within the promoter distal part of a gene or fusion products between two genes put together by a deletion. The antigenic sites of these new proteins should be studied before any further conclusions can be drawn. In Fig. 1, IF3 when present, gives a double band; the relative intensity of the upper and lower bands both associated with IF3 is strongly dependent on the cell lysis conditions but both species are generally observed (Springer et al., 1977b). Whether these two species correspond to those observed by Suryana-
rayana and Subramanian (1977) is not known. Slots 3 and 11 show a faint band migrating to the IF3 position; the possibility exists that these proteins correspond to residual expression of the infC gene or to another unidentified product of the same molecular weight. The (14C) leucine incorporation is much stronger for IF3 and P12 (P12 protein is not easily seen in Fig. 1 because of the strong background incorporation into low molecular weight proteins seen in UV irradiated cells) than it is for the other higher molecular weight proteins. It is not known whether this reflects a true physiological phenomenon (e.g. infC or "P12"' have stronger promoters or their products are more stable) or if it is a property of the UV irradiated cells (which can synthesize only low molecular weight proteins at high yield). Among the set of phages which was studied in the UV irradiated cells system (only a part of this set is shown in Fig. 1) we selected a subset of independent phages which are described in Table 2. The phages were selected as independent on the basis of two criteria: either they were derived from independent lysates, or within a single lysate, they show a different gel pattern. Firstly it can be seen that, as expected, for the
M. Springer et al. : Genetic Organization of the E. coli Chromosome and fi subunits of PRS and for TRS, the biochemical and genetical data coincide: when a phage complements a given mutant it also expresses the corresponding gene product in the UV irradiated cells system. However it is only with the biochemical method that the phages carrying the structural genes for initiation factor IF3 and the P12 protein are identified. Further information about the gene order in this region can be obtained from the biochemical data using the same rationale as for pheT, pheS and thrS. Indeed 2ppl-4 shows that pheT is at one end of the genes on 2p2 and 2ppl-9 shows that "P12", infC and thrS do not lie between pheT and pheS. Thus the order is pheT p h e S - ("P12", infC, thrS) or ("P12", infC, thrS) pheS pheT, where genes in parenthesis are still not ordered. 2pp1-125 shows that infC and thrS cannot be between pheT, pheS and "P12" so that the order is p h e T - pheS "P12""- (infC, thrS) or (thrS, infC) - " P 1 2 " pheS-pheT. Unfortunately no phage was isolated carrying only infC or thrS to order infC and thrS. The above order is not contradicted by any characterized phage except 2ppl 141. If the order of the genes on 2p2 is to be consistent with 2pp 1-141, thrS can be located in three alternative positions (1) outside the pheT - pheS interval on the side of pheT which would be contradicted by 2ppl-4, 2pp2-3 and 2pp3 3, (2) between pheT and pheS which would be contradicted by 2ppl-9, 2pp1-125, 2pp2 34 and 2pp3-6 or (3) between pheS and "P12" which would be contradicted by 2pp1-125 and 2pp3-6. Thus any order compatible with 2pp1-141 is contradicted by at least two other independent phages. This probably means that 2pp1-141 contains two deletions as will be discussed later.
Discussion
Mapping of the Genes Around infC Order of the Genes Both genetic and biochemical data fit reasonably well with the following order of the genes on 2p2:pheT pheS "P12" (infC, thrS) or (infC, thrS) - "P12" - p h e S - p h e T . The deletions in the E. coli component of the ppR phages usually cover several genes (Table 2): in that respect 2ppl 46 and 2ppl 103 are exceptional, as only one gene seems to be deleted. 2 p p l ~ 6 is deleted for the terminal pheT gene and it is possible that this deletion extends into remaining non-essential regions of 2 D N A or into non-characterized regions of E. coli D N A (between pheT and 2DNA). Thus in spite of the fact that 2 p p l ~ 6 is deleted only for
341
pheT, it could be that deletions extend over a distance equivalent to several genes. In the case of 2ppl-103 where only thrS is deleted this might imply that the deletion covers a terminal gene and non-essential regions of 2DNA adjacent to that gene which in turn implies that thrS is also a terminal gene. It thus seems plausible that the order is pheT - pheS - "P12" infC thrS. However the possibility remains that thrS is between "P12" and infC and in that case 2ppl 103 carries a deletion covering only the thrS gene and not the surrounding genes. This type of deletion mapping cannot order the genes on the phage respective to other E. coli genes which are not on the phage. However thrS (Hennecke et al., 1977b) and pheS pheT (Comer and B6ck, 1976) have been mapped respective to nearby genes and the order is: aroD -pheT pheS - thrS. Thus the order of the genes at 38 rain on the E. coli chromosome is aroD - pps - pheT - pheS "P12'" (infC, thrS) where infC is probably between "P12" and thrS. Except for P12 all the main E. coli gene products synthesized by 2p2 infection of UV irradiated cells are characterized. This P12 protein is very probably a product of a "P12" gene and not a degradation product of a protein synthesized by 2p2 because the "P12" gene segregates among the different ppR phages as an independent gene, and is clearly visible on SDS gels in amounts too great to be consistent with its being a breakdown product.
Validity of the Main Mapping Hypothesis The mapping rationale is based on the hypothesis that the deletions on the ppR phages are single. Here we show that this assumption is justified although a few doubly deleted phages may appear by crossingover of two singly deleted ones. The number of spontaneously deleted phages in a lysate (N) is between 10 .6 and l0 7 (Davis and Parkinson, 1971). Under the condition used, the deleted phages are a l m o s t insensitive to the pyrophosphate treatment and the survival frequency of 2p2 is 10 -4. As the derived ppR phages grow at about the same rate as 2p2 under lyric conditions, we assume that each amplification step leaves the ratio of deleted to non deleted phages constant. Starting with l0 s 2p2, if N - - 1 0 -6, the first pyrophosphate treatment gives 10 4 2p2 and 102 deleted phages (called 2p2~). The first amplification maintains constant the ratio of 2p2 to 2p2~ populations but new deletions appear both in 2p2 (called 2p2a) and in 2p2~ (called 2p2Aa). 2p2~ is a population of doubly deleted phages and 2p2da/2p2~ =N. The second pyrophosphate treatment preserves the subpopulations of deleted phages (2p2d 2p2a and 2p2aa)
342 but the number of 2p2 is now very low because the third treatment has almost no killing effect, i.e. after the second pyrophosphate treatment the whole population is essentially ppR. Subsequent amplifications do not modify the relative sizes of the existing subpopulations, however new deletions will appear in all subpopulations but always so that the ratio of doubly deleted to singly deleted phages is N and for triply deleted to singly deleted it is N 2. Thus, although several cycles have been used to enrich sufficiently the lysates in ppR phages, the yield of spontaneous doubly deleted phages is negligible (of the order of N). However doubly deleted phages may appear by crossing over as is discussed now.
Before the first amplification the ratio of 2p2~ to 2p2 is 10 -2 and the absolute number of 2p2A is lOW SO that crossing over will arise mainly within the 2p2 population or between 2p2 and 2p2~ populations which does not give doubly deleted phages. However after the second pyrophosphate treatment all the phages are deleted and crossing overs occur between singly deleted phages to yield doubly deleted phages. As the population at this stage is already ppR the doubly deleted phages are not selectively enriched for during the following cycle. Thus the frequency of doubly deleted phages should not exceed the crossing over frequency of two close genes of 4, that is a few percent. As any gene order compatible with 2pp1-141 is contradicted by at least two other independent phages (as described in Results), it is possible that this phage is doubly deleted. If 2ppl 141 arose through crossing over between two singly deleted phages, these parental phages should be found in the same lysate. Indeed 2ppl-141 could arise from a cross between 2 p p l - 9 and 2ppl 103 which were isolated from the same lysate. A total of 28 phages were studied and of these 2pp1-141 is the only one showing the apparent double deletion phenomenon. The selected subset of phages described in Table 2 are all classed as independent because they belong to different original lysates or because (if they are derived from the same lysate), their deletion pattern is different as judged by the SDS polyacrylamide gel analysis of the proteins synthesized in UV irradiated infected cells. However this biochemical method only differentiates deletions extending over a varying number of whole genes but not deletions extending differently within the same gene. Thus the subset of twelve phages in Table 2 (2ppl-141 excluded) is the minimum number of independent phages, so the frequency of doubly deleted phages is 1/12 or lower which is not in contradiction with the few percent expected.
M. Springer et aI. : Genetic Organization of the E. coli Chromosome Conclusions
The deletion mapping used in this work indicates that the order of the genes on 2p2 is p h e T - p h e S " P 1 2 " - (infC, thrS) where infC is probably between " P 1 2 " and thrS. A m o n g these genes oniy pheT, p h e S and thrS are genetically well characterized and previous mapping indicated for these three genes the same order as found here (Hennecke et al., 1977b). The fact that the deletion method and classical genetic mapping agree, confirms the validity of the former method, which also permitted the localisation of the structural gene for the initiation factor IF3 (infC) and of a still unidentified gene "P12". The present data are insufficient to decide whether some of these genes are transcribed together or each one independently. The precise identification of the transcription units around infC might be possible with the set of phages described here if the size and the exact position of their deletions were known. Therefore analysis of the structure of the D N A of these phages is under way using restriction endonucleases and heteroduplex mapping. Acknowledgements. We wish to thank Dr. J. Plumbridge and Dr. A. Danchin for many helpful suggestions and for careful reading of the manuscript. We are grateful to Dr. I. Saint-Girons for providing us with the thrS strain. This work was supported by the followinggrants : Centre National de la Recherche Scientifique (Groupe de Recherche n° 18); Delegation Generale ~tla Recherche Scientifique et Technique (74.7.0356; 76.7.1178), C.E.A. and Ligue Nationale Frangaise contre le Cancer (Comit6 de la Seine).
References B6ck, A., Neidhardt, F.C.: Genetic mapping of phenylalanylsRNA synthetase in E. coli. Science 157, 78-79 (1967) Comer, M.M., B6ck, A.: Genes for the ct and /3 subunits of the phenylalanyl transfer ribonucleic Acid Synthetase of Escherichia Coli. J. Bacteriol. 127, 923-933 (1976) Davis, R.W., Parkinson, J.S.: Deletion mutants of bacteriophage 4. III Physical structure of att 4~. J. Mol. Biol. 56, 403423 (1971) Epstein,' W., Kim, B.S. : Potassium transport loci in E. coli K12. J. Bacteriol. 108, 639 644 (i971) Hennecke, H., B6ck, A., Thomale, J., Nass, G. : Threonyl-transfer ribonucleic acid synthetase from E. coli: subunit structure and genetic analysis of the structural gene by means of a mutated enzyme and of a specialized transducing )~ bacteriophage. J. Bacteriol. 131, 943-950 (1977b) Hennecke, H., Springer, M., B6ck, A.: A specialized transducing phage carrying the E. coli genes for phenylalanyl-tRNA synthetase. Mol. Gen. Genet. 152, 205-210 (1977a) Jaskunas, S.R., Lindahl, L., Nomura, M., Burgess, R.R.: Identification of two copies of the gene for the elongation factor EF-Tu in E. coli. Nature 257, 458 462 (1975) Johnson, E.J., Cohen, G., Saint-Girons, I. : Threonyl-transferribonucleic acid synthetase and the regulation of the threonine operon in E. coll. J. Bacteriol. 129, 66 70 (1977)
M. Springer et al. : Genetic Organization of the E. coli Chromosome Laemmli, U.K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680 685 (1970) Miller, J.H.: Experiments in molecular genetics. Cold spring Harbor, N.Y. Cold spring Harbor laboratory 1972 Nomura, M., Morgan, E.A., Jaskunas, S.R. : Genetics of bacterial ribosomes. In: Ann. Rev. Genet. Vol. i1, pp. 297 347. Palo Alto: Annual reviews inc. 1977 Parkinson, J.S., Huskey, R.J. : Deletion mutants of bacteriophage 2. I. Isolation and initial characterization. J. Mol. Biol. 56, 369-384 (1971) Russel, R.R.B., Pittard, A.J. : Mutants of E. coli unable to make protein at 42 ° C. J. Bacteriol. 108, 790-798 (1971) Shrenk, W.J., Weisberg, R.A. : A simple method for making new transducing lines of coliphage lambda. Mol. Gen. Genet. 137, 10i-I07 (1975) Springer, M., Graffe, M., Grunberg-Manago, M. : Characterization
343 of an E. coli mutant with a thermolabile initiation factor IF3 activity. Mol. Gen. Genet. 151, 17-26 (1977a) Springer, M., Graffe, M., Hennecke, H.: Specialized transducing phage for the initiation factor IF3 gene in E. coli. Proc. Natl. Acad. Sci. USA 74, 3970-3974 (1977b) Suryanarayana, T., Subramanian, A.R. : Separation of two forms of IF3 in Escherichia Coli by two-dimensional gel electrophoresis. FEBS Letters 79, 264-268 (1977) Taylor, A.L., Thoman, M.S.: The genetic map of E. coli KI2 Genetics 50, 659-677 (i964)
Communicated
by H.G.
Wittmann
Received August4 / October 20, 1978
Molec. gen. Genet. 169, 337-343 (1979)
© by Springer-Verlag 1979
Genetic Organization of the E. coli Chromosome Around the Structural Gene for Initiation Factor IF3 (infC) M. Springer, M. Graffe, and M. Grunberg-Manago Institut de Biologic Physico-Chimique, 13, rue Pierre et Marie Curie, F-75005 Paris, France
Summary. A set of 2 transducing phages carrying varying lengths of the E. coli chromosome around the structural gene for initiation factor IF3 (infC) was derived from 2p2 which is known to carry, besides infC, the structural genes for the c~ subunit of phenylalanyl-tRNA synthetase (pheS), the fl subunit of phenylalanyl-tRNA synthetase (pheT) and the structural gene for threonyl-tRNA synthetase (thrS). The E. coli coding content of these derived phages was analysed by genetic complementation of a set of mutants and by SDS-polyacrylamide gel analysis of the proteins synthesized in UV irradiated cells infected with these phages. The segregation pattern of the different genes among these derived phages indicates that the order of the genes is p h e T - p h e S - "'P12"' - (infC, thrS) where infC is probably between " P 1 2 " and thrS. "'P12" is the structural gene of a 12,000 molecular weight unidentified protein.
Introduction Many of the genes coding for proteins involved in translation are clustered on the E. coli chromosome (Nomura et al., 1977). Some of these clusters are organized in transcription units carrying the structural genes for proteins playing very different roles in the translation machinery;e.g, the ribosomal proteins S 7 and S 12 are expressed from genes located on the same transcription unit as the structural genes for elongation factors Tu and G (Jaskunas et al., 1975). For offprints contact: M. Springer Abbreviations: phenylalanyl-tRNA synthetase: PRS (EC 6.1,l.20), threonyl-tRNA synthetase: TRS (EC 6.1.1.3), Initiation factor IF3: IF3, SDS: Sodium dodecyl sulfate, ppR: pyrophosphate resistant, ppS: pyrophosphate sensitive
A 2 transducing phage (2p2) has been isolated which carries the structural genes for initiation factor IF3 (Springer et al., 1977b), for the ~ and/3 subunits of phenylalanyl-tRNA synthetase (PRS) (Hennecke etal., 1977a) and for threonyl-tRNA synthetase (TRS) (Hennecke et al., 1977b). This phage thus carries the structural genes for four proteins belonging to the translational apparatus of the cell. This clustering of translational genes raises the question whether these genes are expressed as a single or separate transcription units. The aim of the present work was to isolate and characterize a set of transducing phages to provide a way to map the structural genes which are on that 2 transducing phage and to determine whether they belong to separate transcription units or not. To do so, we derived from the original transducing phage a set of phages containing deletions of varying lengths in their E. coli D N A component. The original 2p2 transducing phage was ideal for this purpose because almost all the non essential parts of 2 are absent or have been replaced by E. coli DNA (Springer et al., 1977b). Thus selecting for deletions which did not affect the phage's viability gave phages where E. coli D N A is deleted. This set of phages was studied genetically by complementation of pheS, pheT, and thrS mutants and biochemically by SDS polyacrylamide gel analysis of the labelled proteins synthesized in infected UV irradiated cells.
Materials and Methods The E. coli strains used throughout the present work are listed in Table 1. Besides 2p2 which has been described (Springer et al,, 1977b), all the 2 phages used were derived from 2p2 as described below. Transductions using P l v i r w e r e performed as described by Miller (1972).
0026-8925/79/0169/0337/$01.40
M. Springer et al. : Genetic Organization of the E. coli Chromosome
338 Table 1. List of E. coli strains used Strain
Genotype
Origin or Reference
AB1365
F , th~l, argE3, his-4, proA2, lacY1, galK2, mt~l, xyl-5, tsx-29, supE44, 2pheS5, 2 + lysogen; all other markers
Springer et al. (1977a)
AB1601 (2 +) AB1701 (2 +) AB4610 (2 +) 2K401 159 (2ind-) AB1360 NP37 JPII16 GT342 AB1380 (2 +)
like AB 1365 pheT354, 2 + lysogen; all other markers like AB 1365 thrS1029, aroD-6 +; all other markers like AB 1380 (2 +)
thi-1, rha-4, lacZ82, ga133, kdpABC5, trkD1, trkA401, 2rpsL, su-, gal-, UVs, 2ind lysogen aroD-6; all other markers like AB1365 HfrC, pheS5, rel-1, tonA22, 7"2R HfrH, pheT354, thi, galE-PL5, rel-1, 2thrS1029, trp, his, proC, rpsL, ara, lae, gal, real, 2 r aroD-6, rpoB, 2+ lysogen; all other markers like AB1365
aroD-6 + pheS5 transductant of AB1360. Plvir grown on NP37
aroD-6 + pheT354 transductant of AB 1360. Plvir grown on JP 1116
aroD-6 + thrS transductant of AB1380 (2+). Plvir grown on GT342 Epstein and Kim (1971) Springer et al. (1977b) Taylor and Thoman (1964) B6ck and Neidhardt (1967) Russel and Pittard (i971) Derived from GT302 Johnson et al. (1977) RifR )~+lysogen derivative of AB1360
Isolation of the Pyrophosphate Resistant (ppR) Derivatives of 21)2 Three lysates of 2p2 made from different clones were treated with pyrophosphate as follows: 10 gl of lysate (at about 10l° p.f.u. ml-1) was mixed with 1 ml of 10 mM sodium pyrophosphate and 15 mM Tris-HC1 pH 7.5 (the final pH of the mixture is 8.5). After 30 min at 45 ° C, 0.1 ml of 1 M MgSO 4 was added and the mixture preadsorbed to 0.2 ml of a stock of AB1365 prepared as described by Shrenk and Weisberg (1975) for 15 min at 30°. The mixture was then plated on R plates (Miller, 1972) supplemented with 10.2 MgSO4 and 0.2% maltose (RMM plates), and incubated for 12 hours at 44 ° C. The lysates obtained as described by Miller (1972) have a titer of about 109 to 10 l° p.f.u. ml- 1. The pyrophosphate treatment was repeated two more times. The survival frequency was 10 -4, 10 -3 and 5x 10 1 after the first, second, and third pyrophosphate treatment, respectively.
(Springer et al., 1977b). After isotopic dilution, the cells were centrifuged (20 min at 20,000 x g), washed with an excess of 10 mM Tris-HC1 buffer pH 6.8 and resuspended in the minimal volume of the same buffer, then frozen. The cells were thawed and refrozen three times and then treated with DNAse I (from Worthington) at a final concentration of 20 ~tg ml- ~ for 20 min at 4 ° C before resuspending in the SDS polyacrylamide gel sample buffer (Laemmli, 1970). The samples were kept at - 2 0 ° and an aliquot containing 10,000 cpm was heated 2 min at 100° before electrophoresis, performed as described previously (Springer etal., 1977b). The gel was then treated successively with 7% (v/v) acetic acid for 1 hour; with 10% (v/v) acetic acid, 50% (v/v) methanol and 0.25% (w/v) coomassie brilliant blue for 2 hours; with 10% (v/v) acetic acid and 50% (v/v) methanol for 15 min; with 7% (v/v) acetic acid and 10% (v/v) methanol for 24 hours. The gel was further treated as in Springer et al. (1977b) and the films exposed to the dried gels for 440 hours.
Preliminary Characterization of the ppR Derivatives of 21)2 The ppR lysates were plated with 2K401 on RMM plates supplemented with 10 1 M KC1 (RMMK plates) at dilutions to obtain isolated plaques. The plaques were diluted into a drop of 10 -2 MgSO4 with a toothpick. A platinum wire was then used to transfer phages to RMM plates covered with a lawn of the Ts- mutants. Transduction was made at 44 ° C. 2K401 was used as indicator strain because it grows on RMMK plates but not on RMM plates so that the bacteria taken from the RMMK plate together with the phage will not grow on the RMM transduction plates. The only bacterial mutants used at this preliminary stage were pheS (AB1601 (2)) and pheT (AB1701(2)) thermosensitive mutants. Phages with a particular transduction pattern were purified three times, and lysates were made as described by Miller (1972).
Results Genetic Characterization o f the P h a g e s D e r i v e d f r o m 21o2 R e s i s t a n c e o f a 2 s t r a i n t o c h e l a t i n g a g e n t s s u c h as p y r o p h o s p h a t e d e p e n d s u p o n t h e size o f t h e D N A p a c k e d in t h e h e a d o f t h e b a c t e r i o p h a g e . U n d e r s p e cific c o n d i t i o n s , p h a g e s w i t h s h o r t e r s i z e d D N A a r e p y r o p h o s p h a t e r e s i s t a n t (ppR) w h e r e a s p h a g e s w i t h
UV h'radiated Cells System and Gel Electrophoresis
longer D N A are sensitive to t h a t c h e l a t i n g a g e n t (Park i n s o n a n d H u s k e y , 1971). T h u s , p r e e x i s t i n g d e l e t e d p h a g e s in t h r e e i n d e p e n d e n t s t o c k s o f t h e p y r o p h o s p h a t e s e n s i t i v e (ppS) s t r a i n 2p2 c o u l d b e i s o l a t e d as
The lysates were extensively dialyzed against TM buffer (Tris-HCl pH 7.5, 10 mM; MgSO4, 10 mM). The growth of 159(2), infection and radioactive labelling were carried out as described previously
described under Material and Methods. After a prel i m i n a r y c h a r a c t e r i z a t i o n , a s p e c i f i c set o f p h a g e s from each stock was further studied by complementa-
M. S p r i n g e r et al. : G e n e t i c O r g a n i z a t i o n o f the
E. coli C h r o m o s o m e
339
T a b l e 2. C o d i n g c o n t e n t o f the p y r o p h o s p h a t e resistant p h a g e s d e r i v e d f r o m 2p2 Phage
A:Genetic complementation
B : G e n e s expressed in the U V i r r a d i a t e d cells
pheT
~
~
P12
IF3
TRS
+
+
+
--
--
+
+
pheS
thrS
2p2
+
+
+
+
+
2ppl~
+
--
--
+
--
2ppl-9 2 p p l 16 2ppl~6 2 p p l 103 2 p p l 104 2pp1-125 2 p p l 141
-. + +
-. + + + -
+ . + + +
--
2pp2-3 2pp2-34
+ -
2pp3 1 2pp3-3 2pp3 6
. + -
.
.
.
.
.
.
+
.
.
.
+ +
+ + + -
+ + + +
+ + + + +
+ + + -
+ -
+ -
+
+
+
+
+
.
-+ .
--
+
. +
. + -
. . -
.
.
.
.
.
A : Genetic complementation : m a r k e r : 5 gl of the ppR Iysates (at a b o u t 101° p.f.u, m 1 - 1 ) were s p o t t e d with 1 d r o p of AB1601 (2) stock diluted 102 times with 10 . 2 M M g S O 4 on R M M plates i n c u b a t e d at 44 ° o v e r n i g h t . pheT m a r k e r : s a m e m e t h o d as for pheS except t h a t AB1701 (2) was used diluted 104 times. thrS m a r k e r : s a m e m e t h o d as for pheS except t h a t A B 4 6 1 0 (2) was used diluted 10 times on m i n i m a l A glucose plates (Miller, 1972) s u p p l e m e n t e d with arginine, histidine a n d proline: i n c u b a t i o n was at 37 ° for 24 hours. + m e a n s c o n t i n u o u s g r o w t h on the spot
phe S
B: + -
means Genes means means
discontinuous growth (varying expressed in the U V i r r a d i a t e d t h a t the c o r r e s p o n d i n g b a n d is t h a t the c o r r e s p o n d i n g b a n d is
b e t w e e n < 10 to > 100 colonies). cells: p r e s e n t on the gel a b s e n t f r o m the gel
tion of pheS, pheT and thrS mutants. The complementation properties of the three independent sets of ppR phages given in Table 2 can already be interpreted to order these three genes. Assuming that each ppR resistant phage carries a single deletion (the validity of this assumption is considered in the Discussion) the existence of a specific phage with two genes missing and one (or more) present means that the expressed gene(s) cannot be located in the interval between the two missing ones. If the expressed gene is within that interval then two deletions on either side of the expressed gene are necessary. So the existence of 2ppl-4 shows that pheT is outside the pheS thrS interval and the existence of 2ppl-9 shows that thrS is outside the pheT - pheS interval, thus the order of these genes is pheT pheS thrS or thrS - pheS - pheT.
Biochemical Characterization of the ppR phages Derived from 2p2
When irradiated under specific conditions with UV light, E. coli strains still incorporate labelled amino acids in response to 2 infection. SDS polyacrylamide
gel analysis of the labelled material shows bands corresponding to the different gene products of 2, but when the UV irradiated cell is a 2 lysogen, the 2 repressor, still active under the conditions employed (Springer et al., 1977b), inactivates the expression of the genes transcribed from the two main early promoters of the infecting 2:p~ and Pr- However, if the infecting 2 is a transducing phage the main genes expressed are transcribed from E. coli promoters which are not repressed by the 2 repressor. Thus proteins corresponding to the E. coli content of the infecting phage can be seen by SDS polyacrylamide gel analysis of the labelled material from a UV irradiated lysogen infected with a 2 transducing phage. After a preliminary genetic screening (see Material and Methods), several pyrophosphate resistant phages derived from 2p2 were studied using this UV irradiated cells system. 2p2 infection of UV irradiated 159 (2 ind-) strain stimulates the incorporation of (lgc) leucine into five proteins (Fig. 1 sample 2), these proteins are, in order of decreasing molecular weights: ~ subunit of phenylalanyl-tRNA synthetase (PRS) (Hennecke et al., 1977a), threonyl-tRNA synthetase (TRS) (Hennecke et al., 1977b), ~ subunit of PRS (Hennecke et al., 1977a), initiation factor IF3
340
M. Springer et al. : Genetic Organization of the E. coli Chromosome
., ,,
f3 PRS TRS
--
~ PRS
-
IF3
P12
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Fig. 1. SDS polyacrylamide gel fluorogram from extracts of UV irradiated cells infected with ppR phages derived from )@2. The samples were treated as indicated under Material and Methods. Sample 1: no infecting phage. Sample 2: 2p2. Sample 3: 2pplM. Sample 4: 2ppl-9. Sample 5: 2ppl 16. Sample 6: 2pp1-23. Sample 7: 2ppl 25. Sample 8: 2ppl 30. Sample 9: 2ppl~0. Sample 10: 2pp1-46. Sample 11: 2pp3-3. Sample 12:2pp3 6. Sample 13: 2pp348. Sample 14: 2ppl 103. Sample 15: 2ppl 104. Sample 16: 2ppl 139. Sample 17:2pp2 3. Sample 18: 2pp24. The molecular weights are: fiPRS, 94.000; TRS, 76.000; c~PRS, 38.000; IF3; 22.000; P12, 12.000
(Springer et al., 1977 b), and a 12,000 molecular weight unidentified protein which we call P12. As seen in Fig. 1 (samples 3 to 18) each of ppR phage shows a specific pattern of bands which corresponds to the genes expressed from the E. coli promoters present on each of these phages. Many of these phages do not synthesize one or more proteins which are synthesized by 2p2 because the corresponding genes have been deleted. Some of the ppR phages synthesize proteins not made by 2p2. In particular 2ppl-4 and 2ppl-30 (Fig. 1 samples 3 and 8) synthesize a 34,000 molecular weight protein (under the c~PRS position) and 2pp3-6 (Fig. 1 sample 12) synthesizes a 60,000 molecular weight protein (under the TRS position). These new proteins may be fragments caused by deletions starting within the promoter distal part of a gene or fusion products between two genes put together by a deletion. The antigenic sites of these new proteins should be studied before any further conclusions can be drawn. In Fig. 1, IF3 when present, gives a double band; the relative intensity of the upper and lower bands both associated with IF3 is strongly dependent on the cell lysis conditions but both species are generally observed (Springer et al., 1977b). Whether these two species correspond to those observed by Suryana-
rayana and Subramanian (1977) is not known. Slots 3 and 11 show a faint band migrating to the IF3 position; the possibility exists that these proteins correspond to residual expression of the infC gene or to another unidentified product of the same molecular weight. The (14C) leucine incorporation is much stronger for IF3 and P12 (P12 protein is not easily seen in Fig. 1 because of the strong background incorporation into low molecular weight proteins seen in UV irradiated cells) than it is for the other higher molecular weight proteins. It is not known whether this reflects a true physiological phenomenon (e.g. infC or "P12"' have stronger promoters or their products are more stable) or if it is a property of the UV irradiated cells (which can synthesize only low molecular weight proteins at high yield). Among the set of phages which was studied in the UV irradiated cells system (only a part of this set is shown in Fig. 1) we selected a subset of independent phages which are described in Table 2. The phages were selected as independent on the basis of two criteria: either they were derived from independent lysates, or within a single lysate, they show a different gel pattern. Firstly it can be seen that, as expected, for the
M. Springer et al. : Genetic Organization of the E. coli Chromosome and fi subunits of PRS and for TRS, the biochemical and genetical data coincide: when a phage complements a given mutant it also expresses the corresponding gene product in the UV irradiated cells system. However it is only with the biochemical method that the phages carrying the structural genes for initiation factor IF3 and the P12 protein are identified. Further information about the gene order in this region can be obtained from the biochemical data using the same rationale as for pheT, pheS and thrS. Indeed 2ppl-4 shows that pheT is at one end of the genes on 2p2 and 2ppl-9 shows that "P12", infC and thrS do not lie between pheT and pheS. Thus the order is pheT p h e S - ("P12", infC, thrS) or ("P12", infC, thrS) pheS pheT, where genes in parenthesis are still not ordered. 2pp1-125 shows that infC and thrS cannot be between pheT, pheS and "P12" so that the order is p h e T - pheS "P12""- (infC, thrS) or (thrS, infC) - " P 1 2 " pheS-pheT. Unfortunately no phage was isolated carrying only infC or thrS to order infC and thrS. The above order is not contradicted by any characterized phage except 2ppl 141. If the order of the genes on 2p2 is to be consistent with 2pp 1-141, thrS can be located in three alternative positions (1) outside the pheT - pheS interval on the side of pheT which would be contradicted by 2ppl-4, 2pp2-3 and 2pp3 3, (2) between pheT and pheS which would be contradicted by 2ppl-9, 2pp1-125, 2pp2 34 and 2pp3-6 or (3) between pheS and "P12" which would be contradicted by 2pp1-125 and 2pp3-6. Thus any order compatible with 2pp1-141 is contradicted by at least two other independent phages. This probably means that 2pp1-141 contains two deletions as will be discussed later.
Discussion
Mapping of the Genes Around infC Order of the Genes Both genetic and biochemical data fit reasonably well with the following order of the genes on 2p2:pheT pheS "P12" (infC, thrS) or (infC, thrS) - "P12" - p h e S - p h e T . The deletions in the E. coli component of the ppR phages usually cover several genes (Table 2): in that respect 2ppl 46 and 2ppl 103 are exceptional, as only one gene seems to be deleted. 2 p p l ~ 6 is deleted for the terminal pheT gene and it is possible that this deletion extends into remaining non-essential regions of 2 D N A or into non-characterized regions of E. coli D N A (between pheT and 2DNA). Thus in spite of the fact that 2 p p l ~ 6 is deleted only for
341
pheT, it could be that deletions extend over a distance equivalent to several genes. In the case of 2ppl-103 where only thrS is deleted this might imply that the deletion covers a terminal gene and non-essential regions of 2DNA adjacent to that gene which in turn implies that thrS is also a terminal gene. It thus seems plausible that the order is pheT - pheS - "P12" infC thrS. However the possibility remains that thrS is between "P12" and infC and in that case 2ppl 103 carries a deletion covering only the thrS gene and not the surrounding genes. This type of deletion mapping cannot order the genes on the phage respective to other E. coli genes which are not on the phage. However thrS (Hennecke et al., 1977b) and pheS pheT (Comer and B6ck, 1976) have been mapped respective to nearby genes and the order is: aroD -pheT pheS - thrS. Thus the order of the genes at 38 rain on the E. coli chromosome is aroD - pps - pheT - pheS "P12'" (infC, thrS) where infC is probably between "P12" and thrS. Except for P12 all the main E. coli gene products synthesized by 2p2 infection of UV irradiated cells are characterized. This P12 protein is very probably a product of a "P12" gene and not a degradation product of a protein synthesized by 2p2 because the "P12" gene segregates among the different ppR phages as an independent gene, and is clearly visible on SDS gels in amounts too great to be consistent with its being a breakdown product.
Validity of the Main Mapping Hypothesis The mapping rationale is based on the hypothesis that the deletions on the ppR phages are single. Here we show that this assumption is justified although a few doubly deleted phages may appear by crossingover of two singly deleted ones. The number of spontaneously deleted phages in a lysate (N) is between 10 .6 and l0 7 (Davis and Parkinson, 1971). Under the condition used, the deleted phages are a l m o s t insensitive to the pyrophosphate treatment and the survival frequency of 2p2 is 10 -4. As the derived ppR phages grow at about the same rate as 2p2 under lyric conditions, we assume that each amplification step leaves the ratio of deleted to non deleted phages constant. Starting with l0 s 2p2, if N - - 1 0 -6, the first pyrophosphate treatment gives 10 4 2p2 and 102 deleted phages (called 2p2~). The first amplification maintains constant the ratio of 2p2 to 2p2~ populations but new deletions appear both in 2p2 (called 2p2a) and in 2p2~ (called 2p2Aa). 2p2~ is a population of doubly deleted phages and 2p2da/2p2~ =N. The second pyrophosphate treatment preserves the subpopulations of deleted phages (2p2d 2p2a and 2p2aa)
342 but the number of 2p2 is now very low because the third treatment has almost no killing effect, i.e. after the second pyrophosphate treatment the whole population is essentially ppR. Subsequent amplifications do not modify the relative sizes of the existing subpopulations, however new deletions will appear in all subpopulations but always so that the ratio of doubly deleted to singly deleted phages is N and for triply deleted to singly deleted it is N 2. Thus, although several cycles have been used to enrich sufficiently the lysates in ppR phages, the yield of spontaneous doubly deleted phages is negligible (of the order of N). However doubly deleted phages may appear by crossing over as is discussed now.
Before the first amplification the ratio of 2p2~ to 2p2 is 10 -2 and the absolute number of 2p2A is lOW SO that crossing over will arise mainly within the 2p2 population or between 2p2 and 2p2~ populations which does not give doubly deleted phages. However after the second pyrophosphate treatment all the phages are deleted and crossing overs occur between singly deleted phages to yield doubly deleted phages. As the population at this stage is already ppR the doubly deleted phages are not selectively enriched for during the following cycle. Thus the frequency of doubly deleted phages should not exceed the crossing over frequency of two close genes of 4, that is a few percent. As any gene order compatible with 2pp1-141 is contradicted by at least two other independent phages (as described in Results), it is possible that this phage is doubly deleted. If 2ppl 141 arose through crossing over between two singly deleted phages, these parental phages should be found in the same lysate. Indeed 2ppl-141 could arise from a cross between 2 p p l - 9 and 2ppl 103 which were isolated from the same lysate. A total of 28 phages were studied and of these 2pp1-141 is the only one showing the apparent double deletion phenomenon. The selected subset of phages described in Table 2 are all classed as independent because they belong to different original lysates or because (if they are derived from the same lysate), their deletion pattern is different as judged by the SDS polyacrylamide gel analysis of the proteins synthesized in UV irradiated infected cells. However this biochemical method only differentiates deletions extending over a varying number of whole genes but not deletions extending differently within the same gene. Thus the subset of twelve phages in Table 2 (2ppl-141 excluded) is the minimum number of independent phages, so the frequency of doubly deleted phages is 1/12 or lower which is not in contradiction with the few percent expected.
M. Springer et aI. : Genetic Organization of the E. coli Chromosome Conclusions
The deletion mapping used in this work indicates that the order of the genes on 2p2 is p h e T - p h e S " P 1 2 " - (infC, thrS) where infC is probably between " P 1 2 " and thrS. A m o n g these genes oniy pheT, p h e S and thrS are genetically well characterized and previous mapping indicated for these three genes the same order as found here (Hennecke et al., 1977b). The fact that the deletion method and classical genetic mapping agree, confirms the validity of the former method, which also permitted the localisation of the structural gene for the initiation factor IF3 (infC) and of a still unidentified gene "P12". The present data are insufficient to decide whether some of these genes are transcribed together or each one independently. The precise identification of the transcription units around infC might be possible with the set of phages described here if the size and the exact position of their deletions were known. Therefore analysis of the structure of the D N A of these phages is under way using restriction endonucleases and heteroduplex mapping. Acknowledgements. We wish to thank Dr. J. Plumbridge and Dr. A. Danchin for many helpful suggestions and for careful reading of the manuscript. We are grateful to Dr. I. Saint-Girons for providing us with the thrS strain. This work was supported by the followinggrants : Centre National de la Recherche Scientifique (Groupe de Recherche n° 18); Delegation Generale ~tla Recherche Scientifique et Technique (74.7.0356; 76.7.1178), C.E.A. and Ligue Nationale Frangaise contre le Cancer (Comit6 de la Seine).
References B6ck, A., Neidhardt, F.C.: Genetic mapping of phenylalanylsRNA synthetase in E. coli. Science 157, 78-79 (1967) Comer, M.M., B6ck, A.: Genes for the ct and /3 subunits of the phenylalanyl transfer ribonucleic Acid Synthetase of Escherichia Coli. J. Bacteriol. 127, 923-933 (1976) Davis, R.W., Parkinson, J.S.: Deletion mutants of bacteriophage 4. III Physical structure of att 4~. J. Mol. Biol. 56, 403423 (1971) Epstein,' W., Kim, B.S. : Potassium transport loci in E. coli K12. J. Bacteriol. 108, 639 644 (i971) Hennecke, H., B6ck, A., Thomale, J., Nass, G. : Threonyl-transfer ribonucleic acid synthetase from E. coli: subunit structure and genetic analysis of the structural gene by means of a mutated enzyme and of a specialized transducing )~ bacteriophage. J. Bacteriol. 131, 943-950 (1977b) Hennecke, H., Springer, M., B6ck, A.: A specialized transducing phage carrying the E. coli genes for phenylalanyl-tRNA synthetase. Mol. Gen. Genet. 152, 205-210 (1977a) Jaskunas, S.R., Lindahl, L., Nomura, M., Burgess, R.R.: Identification of two copies of the gene for the elongation factor EF-Tu in E. coli. Nature 257, 458 462 (1975) Johnson, E.J., Cohen, G., Saint-Girons, I. : Threonyl-transferribonucleic acid synthetase and the regulation of the threonine operon in E. coll. J. Bacteriol. 129, 66 70 (1977)
M. Springer et al. : Genetic Organization of the E. coli Chromosome Laemmli, U.K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680 685 (1970) Miller, J.H.: Experiments in molecular genetics. Cold spring Harbor, N.Y. Cold spring Harbor laboratory 1972 Nomura, M., Morgan, E.A., Jaskunas, S.R. : Genetics of bacterial ribosomes. In: Ann. Rev. Genet. Vol. i1, pp. 297 347. Palo Alto: Annual reviews inc. 1977 Parkinson, J.S., Huskey, R.J. : Deletion mutants of bacteriophage 2. I. Isolation and initial characterization. J. Mol. Biol. 56, 369-384 (1971) Russel, R.R.B., Pittard, A.J. : Mutants of E. coli unable to make protein at 42 ° C. J. Bacteriol. 108, 790-798 (1971) Shrenk, W.J., Weisberg, R.A. : A simple method for making new transducing lines of coliphage lambda. Mol. Gen. Genet. 137, 10i-I07 (1975) Springer, M., Graffe, M., Grunberg-Manago, M. : Characterization
343 of an E. coli mutant with a thermolabile initiation factor IF3 activity. Mol. Gen. Genet. 151, 17-26 (1977a) Springer, M., Graffe, M., Hennecke, H.: Specialized transducing phage for the initiation factor IF3 gene in E. coli. Proc. Natl. Acad. Sci. USA 74, 3970-3974 (1977b) Suryanarayana, T., Subramanian, A.R. : Separation of two forms of IF3 in Escherichia Coli by two-dimensional gel electrophoresis. FEBS Letters 79, 264-268 (1977) Taylor, A.L., Thoman, M.S.: The genetic map of E. coli KI2 Genetics 50, 659-677 (i964)
Communicated
by H.G.
Wittmann
Received August4 / October 20, 1978
