Location of the Rz gene in bacteriophage lambda

Gme. 26 (1983)

159-163

Elsevier GENE

907

Location of the Rz gene in bacteriophage lambda (Lysis genes; plasmid vectors; DNA sequencing; complementation;

transposon)

Alina Tayior *, Michaei Benedik * and Allan Campbell ** Department of Biological Sciences, Stanford University, Stamford, CA 94305 (U.S.A.) (Received

August

(Accepted

September

Tel. (415) 497-1170

12th, 1983) 23rd, 19X3)

SUMMARY

We report the nucleotide sequence of the Tn903 insertion in phage Adk23 identifying the Rz gene. The insertion is located after bp 46008 in the A nucleotide sequence. This is 43 bp after the start of the Rz gene, which is 459 bp long. Complementation experiments verify that the lysis defect in the 2Rz: :Tn903 mutant lies on a restriction fragment spanning the lysis region.

INTRODUCTION

Bacteriophage 2 codes for at least three genes involved in lysis of the bacterial host to allow the release of progeny phage. Two of these genes, R and S, are well characterized genetically. The R gene encodes a murein transglycosylase (BielikowskaSzewczyk et al., 1981; Taylor et al., 1975) of M, about 18 000, both determined from gel electrophoresis and predicted by the DNA sequence. The S gene product may act on the cytoplasmic

* Present

addresses:

University

of Gdarisk,

(98) 31-00-72; Road,

(A.T.) Kladki

Department

(M.B.) DNAX Research

Palo Alto, CA 94306 (U.S.A.)

** To whom

correspondence

of

24, 80-822 Gdalisk Institute,

Biochemistry, (Poland)

Tel.

1450 Page Mill

Tel. (415) 856-8444.

and reprint

requests

should

be

addressed. Abbreviations: bromide; mid-carrier

Ap, ampicillin;

Kn, kanamycin;

by, base pairs;

Tc, tetracycline;

EtBr, ethidium

[ 1, indicates

state.

0378-I ll9~83~~03.00

% 1983 Elsevier

Science

Publishers

plas-

membrane (Mukherj~e and Mandal, 1976). It has a predicted M, of 11500 from the DNA sequence. The third lysis gene is the newly discovered Rz gene (Young et al., 1979). They describe a Tn903 insertion mutation in phage d whose phenotype is inability to lyse the host cell in the presence of high concentrations of Mg2+ . Phage carrying the R?::Tn903 insertion can complement R - mutants in mixed infection, indicating that Rz is a complementation group distinct from R. Rz mutations did not turn up in previous hunts for conditions lethals (Campbell, 1961) because they do not prevent plaque formation on ordinary (low Mg” -+) media. The complete nucleotide sequence of phage i was determined by Sanger et al. (1982). In their work, identi~cation of the observed open reading frames with previously characterized genes was based on evidence from various laboratories. This communication documents the published assignment for the Rz gene.

160

MATERIALS

extraction and CsCl equilibrium-density-gradient centrifugation in the presence of EtBr.

AND METHODS

(a) Bacterial strains, phage, and plasmids (c) Molecular techniques Escherichiu coli strains W3 110 (sup”) and W3 110 ~rpEs~111(suPF) were originally obtained from C. Yanofsky. ARam is the mutant originally called s&O (Campbell, 1961). Phages Adk23 and Apk26 are derivatives of lb519b5 15~1857~~~5~~7. ldk23 carries a Tn903 insertion in the Rz gene at position 0.951, and Apk26 has a Tn903 insertion after Rz at position 0.979. AS’dk6 is Ab519cI857nin5 Rz::Tn903, carrying the Tn903 insertion in Rz at position 0.952 (Young et al., 1979). These phages were obtained from R. Young. Plasmids pAT5 and pAT7 are both derivatives of pBR327, each carrying a Hind111 fragment from the Aposition 44 142 bp (Fig. 1) Hind111 site before& to the HindIII site in the Tn903 insertion of Apk26 and Adk23, respectively. Bacteria were routinely grown in tryptone broth supplemented with 0.2% maltose.

Restriction digests, ligations and electrophoresis were performed according to standard techniques. Fragment purification and nucleotide sequencing were as described by Maxam and Gilbert (1980).

RESULTS

(a) Plasmid construction Derivatives of plasmid pBR327 carrying lysis genes from Apk26 and ldk23 were constructed (Fig. I). The transposon Tn903 has a Hind111 site within the KnR gene. Restriction fragments spanning the region from the Hind111 site (44 142 bp) before the pk promoter to the Hind111 site in Tn903 were inserted into the Hind111 site of pBR327, and ApRTcS colonies were screened for the presence of the correct fragment. Plasmid pAT5 carries the Sam7, R and Rz genes from Apk26, and pAT7 carries Sam7, R and part of Rz from hdk23.

(b) Preparation of DNA Phage DNA was prepared from CsCl-banded phage by phenol extraction followed by ethanol precipitation. Plasmid DNA was prepared by the method of Guerry et al. (1973) followed by phenol

HI11 I

RI PR’

Fig.

r

I

+

I

II II

s

b

R

1. Lysis region of phage I, showing gene order and relevant restriction

carrying

tge Hind111 fragment

the Hind111 site before site before

from this region cloned in the unique HindHI

V

HI11 I

Rz

sites. Plasmids

pAT7 and pAT5 are derivatives

site in the Tc gene. In pAT7 the fragment

pk to the I-lirrdIII site in the Tn903 insertion of ldk23. In pAT5 the fragment

pk to the Tn903 insertion

beyond

Apk26.

vcos

1

extends

of pBR327 extends

from

from the same HindIll

161

determined (Sanger et al., 1982), and the sequence of the ends of Tn903 is available (Grindley and Joyce, 1980). Comparing restriction digests of pAT7 with those of pAT5 and pBR327, we identified a unique Hue11 fragment of about 250 bp that spanned the

(b) Nucleotide sequence determination We determined the nucleotide sequence across the junction of the Tn903 insertion in ldk23 (pAT7). The complete nucleotide sequence of 2 has since been 10

CATAAGGCTG

TGCAAAATTC

50

40

30

20 ACAGCCTGAT

AAAGAAGCGG

GCGGAACGGT

ATC Ile

GTC Val

TGC Cys

GTC Val

CTG Leu

125 TCA Ser

TGG Trp

GCT Ala

GTT Val

AAT Asn

140 CAT His

TAC Tyr

CGT Arg

GAT Asp

AAC Asn

155 GCC Ala

ATT Ile

ACC Thr

TAC Tyr

AAA Lys

170 GCC Ala

CAG Gln

CGC Arg

GAC Asp

AAA Lys

185 AAT Asn

GCC Ala

AGA Arg

GAA Glu

CTG Leu

200 AAG Lys

CTG Leu

GCG Ala

AAC Asn

GCG Ala

215 GCA Ala

ATT Ile

ACT Thr

GAC Asp

ATG MET

230 CAG Gln

ATG MET

CGT Arg

CAG Gln

CGT Arg

245 GAT Asp

GTT Val

GCT Ala

GCG Ala

CTC Leu

260 GAT Asp

GCA Ala

AAA Lys

TAC Tyr

ACG Thr

275 AAG Lys

GAG Glu

TTA Leu

GCT Ala

GAT Asp

GCT Ala

AAA Lys

GCT Ala

GAA Glu

AAT Asn

305 GAT Asp

GCT Ala

CTG Leu

CGT Arg

GAT Asp

320 GAT Asp

GTT Val

GCC Ala

GCT Ala

GGT Gly

335 CGT Arg

CGT Arg

CGG Arg

TTG Leu

CAC His

350 ATC Ile

AAA Lys

GCA Ala

GTC Val

TGT Cys

365 CAG Gln

TCA Ser

GTG Val

CGT Arg

GAA Glu

380 GCC Ala

ACC Thr

ACC Thr

GCC Ala

TCC Ser

GGC Gly

GTG Val

GAT Asp

AAT Asn

GCA Ala

410 GCC Ala

TCC Ser

CCC Pro

CGA Arg

CTG Leu

425 GCA Ala

GAC Asp

ACC Thr

GCT Ala

GAA Glu

440 CGG Arg

GAT Asp

TAT Tyr

TTC Phe

ACC Thr

455 CTC Leu

AGA Arg

GAG Glu

AGG Arg

CTG Leu

470 ATC Ile

ACT Thr

ATG MET

CAA Gln

AAA Lys

485 CAA Gln

CTG Leu

GAA Glu

GGA Gly

ACC Thr

500 CAG Gln

AAG Lys

TAT Tyr

ATT Ile

AAT Asn

515 GAG Glu

CAG Gln

TGC Cys

AGA Arg

TAG

537 AGTTGCCCAT

290

395

Fig. 2. Nucleotide the Tn903 insertion The overlined

sequence

of the I strand

of L&23 is marked.

TGA is the termination

GTT Val

ATC Ile

AGA Arg

ATC Ile

CTG Leu

-Tn903 w TGC ATC Cys Ile

A'L'tiAGC MET Ser

ATT Ile

TCC Ser

GATGT

80 ACC Thr

GCG Ala

95 GCT Ala

60 CAGAGAGATT

of the Rz gene (Sanger

The bp = 1 corresponds codon

of the R gene.

547 ATCGATGGGC

et al., 1982), with its translation

(Daniels

AAC

et al., 1983). The site of

to bp 45966 in the maps of Sanger et al. (1982) and Daniels et al. (1983).

162

Rz: :Tn903 insertion. and end labeled,

This fragment

and separated

was gel purified strands

W3110[pAT5]

are Rz’

recombinants. Also, fewer than lOO;, of the phage present in plaques of AS + dk6

were se-

quenced.

plated on W31 lO[pATS]

The Tn903 bp 46008 RZ.

insertion

in pAT7

is situated

(Fig. 2) 43 nucleotides

after

that

after the start of

they

have

attribute

W3 1 lO[pATS] tests

the

growth

than to recombinational

If a wild-type

phage gene cloned on a plasmid

at a sufficient

rate, phage mutated

is

transcription

in that

protein

gene may form plaques on a plasmid-bearing host under nonpermissive conditions. Table I shows that neither LRam60 nor i;S+ dk6 plates on the parent strain W3 110 (tested, in the case of AS + dk6, in the

The

is reduced when 100 mM MgSO, is present throughout the growth cycle. This reduction is partially reversed by the presence of PATS, but not pAT7 (not shown). Fewer than 1% of the phage particles liberated in such a single cycle on

rescue of Rz + Presumably of both R and Rz results from

initiated

nucleotide

sequence

Efficiency

genotype

of plating

of phage mutants

su/!J*

6 x 1O-5

7 x 10-h

W31 lO[pAT7]

sup+[R+Rz-]

I x 10-j

0.8

W3 1 lO[pATS]

sup+[R+Rz+]

(1)

0.9

C600

SLpE was

on tryptone

across

the

A::Tn903

agar,

(1)

supplemented

I lO[pAT7] proved to be Rr + revertants

“

Ram60

W3110

” Plating

by the Q

phage.

segment covered by PATS, and is caused by the Tn903 insertion in Rz (Young et al., 1979).

St&6

W3

at J& and extended

of the infecting

of R + and Rz + functions Relevant

Host

on

transglycosylase determined by the R gene, A-infected cells express another lysis-related activity (endopeptidase; Taylor, 1971) whose relationship, if any, to Rz is unknown. Our complementation data demonstrate that the lysis defect of Adk23 can be overcome by a restriction fragment carrying the intact lysis region and not by the shorter L DNA fragment in pAT7 (Fig. l), which expresses the R gene. This supports the conclusion that the only lysis defect in Adk23 lies in the DNA

These results are corroborated by one-step growth curves in liquid medium. The yield of AS’dk6

I

LS + dk6

in tram rather

the protein product of Rz nor its biochemical function has yet been identified. In addition to the

the test plates. Phage picked from such plaques plate with full efficiency on W3 110 and have lost the ability to generate kanamycin-resistant lysogens. The frequency of revertants was different for each of several Adk6 lysates tested, but the efficiencies of plating of any one lysate on W3 110 and W3 1 lO[pAT7] were always about equal.

complementation

of

cycles

We there-

junction in the Rz mutant Adk23 allows us to determine the exact site of the Tn903 insertion. The insertion occurs about 40 bp beyond the end of the R gene, within an open reading frame, which would give a protein of 18 kDa1. This open reading frame is that denoted as Rz by Sanger et al. (1982). Neither

which carries both R and Rz, both phages plate. The few plaques of&S + dk6 on W3 110 or W3 1 lO[pAT7] are wild-type revertants, some of which may arise on

Plasmid

growth

DISCUSSION

presence of 100 mM MgSO,). On W3llO[pAT7], which carries all of R and only part of Rz, ARam plates but l.S’dk6 does not. On W31 lO[pATS],

TABLE

despite the fact

was possible.

to complementation

the complementation expressed

several

during which recombination fore

(c) Complementation

are Rz’,

undergone

for iSi

dk6 assays

with

that had lost the TnY03 insertion

100 mM MgSO,.

Plaques

(see RESULTS.

section

of i.S’dk6 c).

on W31 IO and

Grindley,

ACKNOWLEDGEMENTS

N. and Joyce, C.: Gencttcs

of the kanamycin

We thank Dr. Ryland Young for phage stocks and helpful discussions,

Dr. Desmond

advice and materials,

Mascarenhas

and Rosemary

for

Redfield

and

Gary Foster for phage lysates. This work was supported by NIH Grants AI08573, 5701-GM158 and

Acad. Guerry,

isolation

analysts

TnYOS. Proc. Natl.

P. and Falkow.

of plasmid

S.: General

deoxyribonucleic

method

for the

acid. J. Bacterial.

1 I6

(1973) 1064-1066. Maxam,

A. and Gilbert,

base-specific

Press,

Sanger.

end-labeled

cleavages, Methods

New York,

P.K. and Mandal,

phage lambda 70

W.: Sequencing

chemical K. (Eds.),

Academic Mukherjee,

REFERENCES

and DNA sequence

transposon

Sci. USA 77 (1980) 7176-7180. P., LeBlanc,

Moldave,

5T32-GM07276.

resistance

in

DN.4 wuh

in Grossman.

1.. and

Enzymo!ogy,

Vol. 65.

1980, pp. 499-560.

R.K.: Role of S gene of bacterrnr-

in host lysis. Biochcm

Biophys. Res. Commun.

C1976) 302-309. F., Coulson.

Nucleotide

A., Hong,

sequence

G., Hill, D. and Peterson,

of bacteriophage

G.:

1 DNA. J. MO!. Biol.

162(1982)729-774. Biehkowska-Szewczyk, gene product glycosylase. Campbell.

K., Lipitiska,

of bacteriophage

lambda

Mol. Gen. Genet.

A.: Sensitive

mutants

B. and Taylor,

A.: The R

is the murein

trans-

1. Virology

14

D.L.,

Schroeder,

J.L.,

Coulson,

A.R., Hong,

G.-F.,

Blattner,

F.R.:

Hendrix,

R.W., Roberts,

Complete

R.A. (Eds.),

Lambda

Cold Spring

Harbor.

Szybalski,

W..

Sanger,

Hill, D.F., Peterson,

annotated J.W.,

Stahl.

II. Cold Spring

lambda

F.,

G.B. and

sequence,

in

F.W. and Weisberg, Harbor

NY. 1983, pp. 519-676.

activity of phage R-endoly sin Nature

A., Das, B. and Van Heijenoort,

peptidoglycan

fragments

lysin and containing

(1961) 22-32. Daniels,

A.: Endopcptidaae

New Biol. 234 (1971) 144-145. Taylor.

184 (1981) 11 l-1 14. of bacteriophage

Taylor,

J. Biochem. Young,

produced

J.: Bacterial-cell-wall

by phnge R or VII I

1,6-anhydro-?-acetylmurnmic

53 (1975) 47-54.

R.. Way, J.. Way, S., Yin. J. and Syvancn. of bacteriophage

mutagenesis

affecting

ceil lysis. J. Mol. Biol. 132 (1979) 307-322.

Laboratory, by W. Szybalski.

lambda.

M.. Trans-

position

Communicated

Endo-

acid. Eur.

A new gene