Gene,86 (1998) 19-25 Elsevier

19

GENE 03359

Mutational analysis of the phage T4 morphogenetic 31 gene, whose product interacts with the Escherichiu cdi GroEL protein (RecombinantDNA; nucleotide sequence; ambermutations; phage head assembly;heat shock proteins; protein-protein interactions)

France KeppeP, Barbara Lipinskab, DebbieAngb and Costa Georgopoulosb QDepartment of Molecular Biology, Sciences II, University of Geneva, 30, Quai Ernest-Ansmnet, Geneva 1211 (Swiftcnland) Tel. 41-22-702-6118: bDepartmentof Cellular, Viraland MolecularBiology,Univers&yof Utah Medical Centet, Salt Lake C&V,UT 84132 (U.S.A.) Received by AJ. Podhajska:20 July 1989 Revised: 23 September1989 Accepted:24 September1989

SUMMARY

The phage T4 morphogenetic gene 32 has been sequenced. Its deduced gene product is a polypeptide of 111aa, with a predicted Mt of 12064 and a p1 of 4.88. The proof that the assigned open reading frame (ORF) encodes Gp31 rests on the sequencing of two known gene 31 amber mutations, amN54 and NG71, demonstrating that these mutations result in translational termination within the assigned ORF. Furthermore, the sequencing of four different T4s mutations, isolated on the basis of allowingthe phage to propagate on EschericcliacollgroEL - hosts, showed that they are either missense mutations or 39bpdeletions in the gene 31 reading frame. The sequencing of neighboring DNA revealed the presence of five other ORFs, one of which overlaps gene 31 substantially, but in the opposite orientation.

INTRODUCTION The morphogenesis of the phage T4 head proceeds in discrete stages under the control of many phage-coded gene products (reviewed in Black and Showe, 1983). The gene 31 product, Gp31, acts at an early stage of prohead Corrqonde?tceto: Dr. C.P.Georgopoulos,Departmentof Cellular,Viral and Molecular Biology, University of Utah MedicalCenter, Salt Lake City, UT 84132 (U.S.A.) Tel. (801)581-3831;Fax (801)581-3607. Abbreviations:aa, amino acid(s); am, amber, i.e., nonsense codon; bp, base pair(s);A,deletion;5,mutationsin T4 which enablephageto bypass the host g&█ cop, efnciency of plating;0~31, gene productof T4 31 gene; kb, kilobase pair(s);LB, Luria-Bertani broth; moi, multiplicity of infection; nt, nucleotide(s); oligo, oliBodeoxyribonucleotide;ORF, open readingframe;PCR, polymerasechain reaction; pl, isoelectric point; RF, replicatingform;Tr +, temperature=resistant; Tr‘(ts), temperature-sensitive; wt, wild type;[ 1,denotesplasmid-carrier state. 0378-l119/90/$03.50 0 1990ElsevierSciencePublishersB.V. (BiomedicalDivision)

assembly, allowing the orderly assembly of the Gp23 capsid protein. Gp3 1 must exert its effect transiently, since it is not part of the final prohead structure. In the absence of functional Gp31, the Gp23 capsid protein aggregates in amorphous ‘lumps’ on the bacterial membrane (Laemmli et al., 1970).The same phenotype of T4 infection has been observed in the absence of a functional GroEL protein (identical to infection of mopA -, tabB’ and hdh’ hosts; Georgopoulos et al., 1972; Takano and Kakefuda, 1972; Coppo et al., 1973; Revel et al., 1980). Phage T4 mutants able to bypass the GroEL-imposed block map in gene 31, suggesting that the GroEL and Gp3 1 proteins functionally interact during phage T4 infection. The groEL gene has been shown to map at 94 min on the revised E. coli map and to be the second gene of an operon, the order being promoter-pE&pEL (Geo~~poulos and Eisen, 1974; Tilly et al., 1981; Hemmingsen et al., 1988). None of the groES’ mutations block T4 morphogenesis,

2O suggesting that the host GroES protein is dispensable for correct T4 head assembly (Tilly and Georgopoulos, 1982). The groE operon has been shown to be heat-inducible, under the control ofthe E o "32 RNA polymerase holoenzyme (Neidhardt etal., 1981; Tilly, 1982; Tilly etal., 1983; Cowing etal., 1985). Both the groES and groEL genes have been shown to be indispensable for E. coli growth (Fayet et al., 1989). In addition, the GroES and GroEL proteins have been shown to be essential for correct ~. prohead and T5 tail assembly (Georgopoulos et al., 1973; Sternberg, 1973; Zweig and Cummings, 1973). The isolation ofintergenic suppressors suggested that the GroES and GroEL proteins functionally interact in E. coli (TiUy and Georgopoulos, 1982). Such an interaction has indeed been demonstrated with the two purified GroE proteins (Chandrasekhar et el., 1986). Because both the GroES and Gp31 proteins apparently interact with GroEL, we wished to determine whether the two former proteins exhibit structural similarities and identities at the aa sequence level. Since the aa sequence of the GroES protein is known (Hemmingsen etal., 1988), we proceeded to sequence the .~1 gene of T4 in order to compare the two proteins. Near the completion of our work, Drs. R. Nivinskas and L.W. Black independently established the gene 31 nt sequence (Nivinskas and Black, 1988). The sequence of their 656-bp fragment is in perfect agreement with the corresponding portion of our sequence.

31), T4amBL292 (in gene 55) and T4amBl7 (in gene 2:1) were obtained from Dr. W. Wood (Epstein etal., 1963; Georgopoulos etal., 1972). (h) Media and bacterial and phage manipulations The LB medium was used for bacterial growth, phage growth and phage genetic crosses (Georgopoulos et al., 1972; 1973; Georgopoulos and Hohn, 1978; Tilly et al., 1981). L-agar is LB medium supplemented with 1% Bacto agar (Difco). (c) Isolation of Tr + bacterial revertants Tr + revertants of the groES7, groE$42, and groES97 mutants were isolated by simply spreading bacterial cultures on L-agar plates and incubating at 43 °C for two days. The colonies that grew at 43°C (at a frequency of 10-s-10-7) were screened for their ability to propagate various phage T4 derivatives.

(d) Complementation and marker rescue tests Bacterial hosts were grown in LB medium to 3 × 10s cells/ml and infected with various phage derivatives alone at an moi of 5 phage per bacterium, or in combination at an moi of 5 of each phage per bacterium. The infected cultures were shaken at 370C for I h, treated with chloroform and assayed for plaque-forming ability on various bacterial hosts.

MATERIALSAND METHODS (a) Bacterial and phage strains

RESULTS AND DISCUSSION

All ofthe groE- mutants in our collection were originally derived from £. coil B 178 (Georgopoulos etal., 1972; Tilly et el., 1981). The groES7 and groE$97 strains each carry a mutation in the ~oES gene that results in inability to assemble properly the ~. prohead, as well as inability to form colonies at 430C (Georgopoulos st el., 1973; Georgopoulos and Eisen, 1974; Tilly etel., 1981). The groEL44, gwEL515, gwEL673 and groEL764 strains each carry a mutation in the groEL gene that also results in inability to assemble properly the ~ prohead (Georgopoulos etal., 1972; 1973; Georgopoulos and Eisen, 1974). The groEL44 mutation interferes with proper wt T4 head assembly, while the gwELSlS, gwEL673 and gruEL764 mutations interfere with the head ~ssembly of certain T48 mutants which have mutations that map in gene 31 (Georgopouios et el., 1972). The T4s mutants are phage derivatives able to grow on certain groEL- mutant hosts (see RESULTSAND DISCUSSION,section It, for details). The T4amN54, amNG71 and ts70 mutants (ell assigned to gene

(a) Isolation of phage 1'48 mutants Phage T4 mutants, called e, can be readily isolated on groE£- mutant bacteria simply based on their ability to form plaques on these restrictive hosts (Georgopoulos et el., 1972; Takano and Kakefuda, 1972; Coppo et el., 1973; Revel etel., 1980). The T4al mutant was thus isolated on groEL44 mutant bacteria and shown to map in gene 31 (Georgopoulos etel., 1972). Simultaneous to acquiring this new phenotype, the T4sl mutant was shown to have lost the ability to propagate on groEL515, 673 and 764 mutant bacteria (Table I). Both phenotypes were shown to be consequences of the same mutation since the majority ofthe T4el 'revertants', isolated as plaque formers on groELSl$ bacteria, were shown to simultaneously lose their ability to propagate on gro£L44 bacteria (data not shown). In an earlier study, we showed that, although all known groES- mutant hosts propagate phage T4 normally, certain

21 TABLE I P l a q u e - f o r m i n g ability o f T48 m u t a n t s o n various

Eschertchla cob"groE- hosts

~roE$ 6 !9

E. coli h o s t s a B I 7 8 g r o E ÷

groEL44

8roEL59

groEL 140

~oEL 764

groEL673

8roELSl5 Phage T4 mutants b

30 °

37 °

42°C

30 °

37 °

42°C

30 °

37 °

42°C

30 °

37 °

420C

30 °

37 °

42°C

30 °

37 °

42°C

+

+

30 °

37 °

+

+

+

-

T4D o wt

+c

+

+

+

+

+

±

_

_

+

+

+

+

+

+

+

3181

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

.

31871 !

+

+

+

+

+

+

-

-

-

+

+

-

+

±

-

+

31e714

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

.

318421 !

+

+

+

+

+

+

-

-

-

±

-

-

±

-

-

+

+

+

-

-

31e9723

+

+

+

+

+

+

-

-

-

+

±

-

+

±

-

+

+

+

±

-

31e9725

+

+

4-

+

+

+

.

+

±

-

-

-

.

.

.

.

.

.

.

.

.

. +

.

.

.

.

.

+ .

42°C

a T h e bacterial hosts are d e s c r i b e d in G e o r g o p o u l o s et al. (1973), G e o r g o p o u l o s a n d Eisen (1974) a n d Tilly e t a l . (1981). b P h a g e T 4 D o is the w t p a r e n t for all 31e m u t a n t s isolated ( a t frequencies o f 1 0 - s - 1 0 - 7), e x c e p t e9725, w h o s e p a r e n t is p h a g e T 4 D o el. Phage m u t a n t s e71 ! a n d 8714 w e r e isolated as p l a q u e f o r m e r s o f T 4 D o o n bacterial m u t a n t g r o E $ 7 T r ÷ i ( G e o r g o p o a l o s and Eisen, 1974). Phage m u t a n t ~4211 w a s isolated as a p l a q u e f o r m e r o f T 4 D o o n bacterial m u t a n t g r o E S 4 2 T r ÷ 1 ( G e o r g o p o u l o s a n d Eisen, 1974). Phage m u t a n t s 89723 a n d 89725 were isolated as p l a q u e f o r m e r s o f T 4 D o a n d T 4 D o e l , respectively, on bacterial m u t a n t

gwE$9Trr ÷2 ( G e o r g o p o u l o s a n d Eisen, 1974).

© T h e ( + ) s y m b o l indicates a n c o p o f approx. 1.0 a n d g o o d p l a q u e size. T h e ( ± ) s y m b o l indicates an c o p o f 0. i-1.0, a n d small plaque size. T h e ( - ) s y m b o l indicates an c o p o f < 1 0 - 4 a n d n o visible p l a q u e f o r m a t i o n (except for o c c a s i o n a l 8 mutants).

of their 'pseudorevertants', mapping in the groEL gene, did block T4 growth at the level of Gp31 action (Georgopoulos and Eisen, 1974; "lilly and Georgopoulos, 1982). The plating efficiencies of key examples of these various T4s mutants are summarized in Table I. The results obtained reveal a wide spectrum of phage growth properties on the various gro£- strains tested (Table I). However, it is noted that T48711 and T4s9723 mutants, although originally isolated on different hosts, nevertheless exhibit identical plating properties when tested on various bacterial mutant hosts and at various temperatures (Table 1). (b) Identification of the cloned T4 gem 31

The original plasmid clone pKLI03-44 containing an intact phage "1"4gene 31 was obtained from Dr. H. Krisch, University of Geneva, Switzerland. The pKLI03-44 plasmid was prepared by digesting to completion T4 cytosine-containing DNA with EcoRl + Bg/ll. The fact that it carries an intact gene 31 was verified by both complementation and marker rescue tests. When plasmid pKLI03-44 was introduced into B178 sup ° bacteria by transformation, it was shown to restore the ability of T4amN54 and T4amNG71 phage to propagate at 37°C and T4tsA70 at 42°C (all of these mutations were previously shown to reside in gene 31). As a control, T4amBL292 (in gene 5~) and T4amB 17 (in gene 23) phage did not propagate on BI78[pKL103-44] bacteria (not shown). When the T4 mutants 3;~1,8711, 8714, 84211, 89723 and 89725 were propagated c,n BI78[pKLI03-44] bacteria,

31 + wt recombinant phage were found among the progeny at a frequency of approx. 10 - 3 (varying from 2 × 10- 4 to

5 × 10- 3): W!len the same mutant phage were propagated on B178 bacteria, no 'recombinant' phage above background (< 10 -5) were found. These results indicate that all of these T4s mutants map either in gene 31 or in its immediate vicinity. The preliminary conclusion that these T4s mutants map in gene 31 was reinforced by T4 genetic crosses. For example, T48711 complemented T4amBL292 (in gene 55) but not T4amN54 (in gene 31) for growth on groELl40 sup ° bacteria (not shown). This complementation test formally places the s711 mutation in gene 31. In further subcloning experiments, we found that the approx. 400-bp Hincll DIqA fragment, totally derived from T4 DNA, could marker rescue all known mutations in gene 31 including our various T4s mutations. This result strongly suggested, but did not prove, that gene 31 was contained in its entirety within this approx. 400-bp T4 DNA fragment.

(c) Sequencing gene 31 and its surrounding sequences Because of the uncertainty of whether gene 31 was completely contained within this 400-bp T4 DNA fragment, we proceeded to subclone and sequence the entire 1.l-kb EcoRI-Bglll T4 DNA fragment. Primarily, the sequencing technique used was that of Sanger etal. (1977). Both DNA strands were sequenced in their entirety using this technique. In addition, approx. 450 nt were sequenced from the EcoRI end using the technique of Maxam and Gilbert (1977). Fig. I shows the six possible ORFs predicted by the

22 1000

500

.::.,,h,.4-.' I1111 llllllllr

I

I

i

i

---"lllJ 3 il lilll!,.ll I I I,h I, J IIII III Ill II IIII 2 ,I, I, I,I ,I I i l I III ,°r3Y lill II -1 II III III, I , II II I III -2 I, i I I IIIII IIILU -3 I I II I'--"5 s''' I I i

500

1000

Fig. !. The ORFs predicted by the nt sccluence using the DNA Strider program (Marck, 1988). All six possible ORFs (in both directions) are shown. The full-length vertical bars indicate stop codons; the half-sized vertical bars indicate potential start (AUG) codons. The arrows indicate the direction and extent ofthe ORFs. The horizontal scale represents the nt sequence relative to that shown in Fig. 2. The nomenclature used in

naming the ORFs in the immediate vicinity of gene 31 follows the agreed 1"4 nomenclature (Kutter etal., 1989; see RESULTS AND DISCUSSION, sections e and d, for details).

nt sequence. Noteworthy features of this sequence include (l) the existence of at least six ORFs coding for putative polypeptides larger than 9kDa; (2)both ends of the sequenced DNA appear to code for ORFs ofindeterminate length since they originate outside the sequenced DNA portions [i.e., ORF 31.4 and ORF 31.-1 (the nomenclature used is that of Kutter et al., 1989)], and (3)there are potentially three cases of ORFs that are both overlapping and oppositely oriented. 1"4 codon usage is specific in that the phage uses certain codons that are rarely used in strongly expressed genes in E. coil (Hahn et al., 1986; C. AIf-Steinberger, personal communication). A computer analysis was performed to compare the codon usage in gene L~; and ORF 3 !.1 with those used in 37 other T4 proteins. The results of the comparison suggest that both of these genes comply with the I'4 codon usage pattern (C, Alf-Steinberger, personal communication). It is possible, then, that both of these ORFs indeed encode bona fide 1"4 proteins. Of the various ORFs predicted by the nt sequence there are two candidates, gene 31 and ORF 31.-I, that may encode Gp31 partially or in its entirety. These are both located within the righthalf of the D N A segmentshown in Fig.1. In agreement with the genetic studies mentioned above, gene 31 and ORF 31.-1 sequences are found in the 396-bp Hincll fragment. To determine which of these two ORFs encodes Gp31, we sequenced various mutations. These included the amN54 and amNGTl mutations, which were originally used to de/me gene 31 (Epstein et al., 1963), and four of the six T46 mutations, whose properties are shown in Table I.

(d) Sequencingof gene 31 mutations Phage stocks of the various 'I'4 mutants to be sequenced were used as sources of DNA for sequencing. The phenol-extracted DNA was amplified using the PCR

technioue (Saiki et al., 1988). The two DNA oligos chosen for the amplification reaction were those at nt 581-602 (oligo l) and 936-961 (oligo 2). The results are shown in Fig. 2. Since the change to an amber (nonsense) codon in amNG71 DNA is not contained within ORF 3 l.- l, and the amN54 mutation that results from the CAG-, UAG change in gene 31 corresponds to a CTG ~ CTA missense codon change in ORF 31.-1, we conclude that ORF 31.-1 does not encode Gp31. The structural portion ofgene 31 is located between nt 129580 and 129247 on the T4 physical map (Kutter et al., 1989). The locations of the amNGTl and amN54 mutations, near the N-te_rmi_naland C-terminal-coding portions ofgene 31, respectively, are consistent with (/)the previous observation that amN54, but not amNG71, allows the formation of a long polypeptide fragment (Castillo and Black, 1978), and (ii) the fact that these two codons are the only codons in gene 31 which can be altered by a predominantly transition-promoting mutagen to amber codons. It is interesting to note that all gene 3Is mutations that were sequenced are clustered in the middle third of the coding portion of gene 31 (Fig. 2). In addition, some of the aa substitutions result in very subtle changes, e.g., the sl mutation, which completely restores the ability of "1'4 to propagate on gro£L44 mutant hosts (Table I) and results in complete inability to propagate on the ~oEL515, groEL673 and gro£L764 mutant hosts (Table I), substitutes lie for Leu at aa position 35 (Fig. 2). Since all T4e mutants were isolated spontaneously at a frequency of 10-s-10 - 7 it is not surprising that only a single mutational event was seen in each case (excluding the AGAA seen in two independent 8 isolates; Fig. 2).

(e) Relationship between Gp31 and GroES The sane 31 protein consists of 111 aa residues, with a putative pl of 4.88 and Mr of 12064. The E. coil GroES protein consists of 97 aa residues, with a putative pl of 4.71 and an Mr of 10373 (l-lemmingsen et al., 1988). Because both the Gp31 and GroES proteins appear to intimately interact with GroEL (Georgopoulos et al., 1972; Tilly and Georgopoulos, 1982, and this work), we compared their aa sequence and protein structures in a variety of ways to determine if they possess any common features. Hydropathic indexes, calculated according to Kyte and Doolittle (1982), indicated that neither protein is a membrane protein, although both were shown to be hyclrophobic overall. However, computer analysis of Gp31 and GroES sequences failed to reveal significant homology at the aa level (Heedleman and Wunsch, 1970; Feng et al., 1985) or similarity in secondary structures (Gamier et al., 1978; Hovotny and Auffray, 1984).

23 1-96

ATGAATT T C G T T T G G T A A / ~ C T C A C A G C A A T T A ~ . C I T T T A T T C T A A C C A G A A f ' A ~ T ~ A ~ A T ~ T A ~ ~ T

97-192

GGAAAcAGTATTATAAcAc/~A~GTT1~TATTcATAcACCATATA~T(~jkRATC~T~TTTAGAARA~TGt~-A~A~%TTAt~TT(y2~`TTA

193-288

RAGAACGI~7~ACAACl~ATATTARAACl~TAATA~GTARATARATC~AACIT~~~A~T~~

289-384

AAATTGTt'T.ATGGGTGTT 1TAAACGTAGTTT~%AAGTCIT~TGGTACTA~A~A~~A~~~A~

385-480

AT G C J ~ A C T G G C ~ A A T T G A ~ T t ~ A C l ~ G G ~ A A G ~ A T C T A C E C ~ ~ A ~ ~

481-576

AT T G A C A ~ T T G A T T T C C G T T A A T G G T A ~ G T T G A G C A T T T G C T T C A A ~ A T ~ T ~ T ~ ~ A ~ A ~ A ~ T

5??-653

TCATCTGTTAA~GGAAP,AACG ATG TCT GAA GTA CAP. CAG eTA CCA ATT CGT GCT GTC GGT GAA TAT GTT &TT ,> Met: Ser GIu Val Gin Gin Leu Pro Ile Arg AIa Val Gly Glu TyE Val Ile olJ.go 1

GAG~-A-A-x-t-Ax.-~"

5~ mnWG71

t

A£711,

-.

E9723

A tl(Xle)

& eD?2S(&op)

!

t

1-17

654-725

TTA GTT TCT GAA CCT (;CA CAA G ~ GGT CAT GAA GAA GTT ACA GAA TCA GGA ~ AT? AT(2 GGT AAA CGT GTT Leu Val SeE GIU Pro Ala Gin Ala GI¥ Asp Glu Glu Val ThE Glu SeE GI¥ Leu Tie Tie GI¥ Lys Arg Val

18-41

726-797

CAA GGT GAA GTT OCT GAA CI~ TGT GTA GTT CAC TOT GTC GGT COT GAT GTT COT ~ ~ T TTC T ~ GRA GTT Gin Gly Glu Val Pro GIu Leu Cys Val Val His Set Val GIy Pro Asp Val Pro Glu GIy Phe Ojs Glu Val

42-$5

798-869

GGT CAT TTG ACT TCT CTT CCA GTT GGT ~ ATT CGA AAT GTT CCG CAT CCT TTT GTA GCT CTG GGT ¢TT AAG GIy Asp Leu ThE Ser Leu Pro Val GIy Gln Ile Arg Asn Val Pro His Pro Phe Val AIa Leu Gly Leu Lys

6G-89

T a~154

t

870-935

C~G ~TA AAA GAA ATT AAA CAP, ~ TTC GTT ACC TGT CAC TAT AAA GCT ATT CCG TaT CTT TAT A~G Gin Pro Lys Glu Ile Lys Gin Lys Phe Val Thr Cys His Tyr Lys Ala Ile Pro Cys Leu Tyr LFS

936-1019

TGATATAAATAATAATATGAATTC~TGTCGG~ATAATAAGTTRAt~T~ACAATTCTA~~ A ~ A ~ ~ C A ~