Physical map of the nrdA-nrdB-ftsB-glpT region of the chromosomal DNA of Escherichia coli

309

Gene, 18 (1982) 309-318 Elsevier Biomedical Press

Physical map of the nrdA-nrdB-ftsB-glpT region of the chromosomal DNA of Escherichia coli (Recombinant

DNA;

restriction

map;

test; hybrid

complementation

plasmid;

DNA

cloning;

cell division

mutants)

Masao Yamada, Yutaka Takeda, Kazuo Okamoto and Yukinori Hirota National Institute of Genetics, Yata, I I1 1, Mishima, Shizuoka (Japan) (Received February Sth, 1982) (Accepted April 22nd, 1982)

SUMMARY

Seven pLC plasmids (pLC 3-46, 8-12, 8-24, 8-29, 14-12, 19-24 and 42-17) which complemented nrdA, nrdB, ftsB and/or glpT mutations of Escherichia coli were analyzed. A restriction map of each plasmid was constructed

and restriction

fragments

were subcloned

into pBR322.

A physical

map of approx.

a 15 X lo6

M, segment of the chromosomal DNA was deduced from the overlapping region of the pLC plasmids. pLC plasmids and newly constructed plasmids were examined for the ability to rescue the mutations. complementation

tests defined

n&A-nrdB-ftsB-glpT.

Functional

the location nrdAB

of the genes in the 15 X lo6 M, segment and ftsB genes

were located

in the following

The The order:

in the 3.1 X lo6 it4, EcoRI-PstI

fragment.

INTRODUCTION

the

To elucidate the molecular mechanism of bacterial cell division, we have isolated numerous cell division mutants and analyzed them geneti-

(1975)). Clarke and Carbon (1976) prepared a gene bank of E. coli in which random segments of chromosomal DNA of E. coli were ligated with a

same

gene

as pbpB

by

Spratt

and

Pardee

cally, physiologically and biochemically. Presently, at least 15 genes ( ftsA-ftsM, ftsQ and fts2) (Ricard and Hirota, 1973; Nishimura et al., 1977; Santos and DeAlmeida, 1975; Holland and Darby, 1976;

ColEl plasmid by the poly(dA-dT) screened the bank and found hybrid

Begg et al., 1980; Lutkenhaus et al., 1980) are thought to be involved in the division process of E. coli. However, the molecular nature of the gene products is not known except for ftsI, the structural gene of penicillin-binding. protein 3 ( ftsI is

and characterized penicillin-binding protein 3 from the cells carrying the hybrid plasmid, which contains the ftsI gene (Tamura et al., 1980). In this communication, we report the analysis of the DNAs of the pLC plasmids complementing the ftsB mutation, and the subcloning of restriction fragments into pBR322. These studies revealed the gene organization around the ftsB gene in the chromosomal DNA of E. coli.

Abbreviations: Amp, ampicillin; Tc, tetracycline. 0378-I 119/82/0000-0000/$02.75

0 1982 Elsevier Biomedical Press

method. plasmids

We that

suppressed each of ftsB, ftsE, f&I and ftsM mutations (Nishimura et al., 1977). We have purified

310

MATERIALSANDMETHODS

Fragments

(a) Bacterial strains and plasmids

To detect

with M,-values

lo6 could not be detected

All of the bacterial were derivatives

strains

used in this study

of E. coli K- 12. Their

and references

are as follows:

MFT84

small

fragments,

of less than about 0.2 ,x under

analyzed on a 5% polyacrylamide buffer. HindIII-digested h DNA

genotypes

M, standard

(F- , ftsB

1980).

these conditions.

the digests

(Allet and Bukhari,

were also

gel in the same was used as an 1975; Davis et al..

thr leu trp his thyA argH thi lacy malA xyl mtl mel tonA

supE

str; Ricard

and

Hirota,

(F- , nrdA thr leu thyA deoBCD str; Wechsler

and Gross,

1977), El01

thi lac Y tonA supE

xyl mtl str; Fuchs and Neuhard, thi; Cozzarelli

et al.,

1973) Lin6 (HfrC,

1968). The

plasmids

used are pBR322 (Bolivar et al., 1977) pLC plasmids (Clarke and Carbon, 1976) and newly constructed ones. They are summarized the RESULTS section.

in Table III in

(b) Preparation of plasmid DNA Plasmid DNA was prepared by a cleared lysate method (Vapnek and Rupp, 1971) and purified by ethidium bromide-CsCl equilibrium centrifugation (Radloff et al., 1967). (c) Endonuclease

method for construction of the

restriction map

1971) LD195 (Fe-, nrdB

leu his argG metB deoA cdd dcd lac Y or Z gal malA glpT

(d) End-assignment

digestion

A restriction

map

structed

primarily

(Takeda

et al.,

of plasmid

DNA

by an end-assignment 1980). If the restriction

was conmethod enzyme

which produces

the end of each fragment

is known,

the construction We found that

of a restriction map is simplified. the ends of restriction fragments

are easily assigned to a restriction enzyme when a comparison is made of the fragments produced in single, double and triple digests. The principal theory of this method is as follows. Assume A, B and C as restriction enzymes. The DNA is singly digested with each of A, B and C, doubly digested with A + B, B + C and C + A and triply digested with A + B + C. Then the restriction fragments in the above 7 different digestion mixtures are separated by gel electrophoresis. Fragments in the triple digest with A + B + C must appear in at

in 30 pl reaction mixtures containing about 0.7 pg DNA, l-2 units of enzyme, 900 nmol Tris . HCl,

least one of the other 6 digests, because the fragments in the triple digest are classified into at most 6 classes with respect to the nature of the ends

pH 7.4 and 300 nmol MgCl, at 37°C for 2 h. For the enzymes requiring a high salt concentration,

(A-A, B-B, C-C, A-B, B-C and C-A). If a homo-end fragment (the fragment cut by the same enzyme at

such as EcoRI, Sal1 and XhoI, NaCl was added in the reaction mixture to give a final concentration of 150 mM. Double and triple digestions were

both ends) exists in a triple digest, the fragment must appear not only in a single digest but also in double digests. For example, if an A-A fragment appears in the triple digest with A + B + C, the

Restriction

digestion

of DNAs

was carried

out

carried out by simultaneous addition of the enzymes as far as the enzymes can work at the same salt concentration. The reaction conditions employed were not necessarily optimal, but were convenient for double and triple digestions. Practically any enzyme tested was active under the above conditions. The restriction digest was separated by electrophoresis through 0.7% agarose gel in 89 mM Tris-89 mM borate-2.5 mM EDTA buffer, pH 8.3 (Davis et al., 1980) at 8 V/cm for 3 h. In some instances, electrophoresis was continued after the elution of marker dye from the gel to allow for a more complete separation of the larger fragments.

same fragment will also appear in A, A + B and C + A digests, because there are neither B nor C sites in the A-A fragment. If a hetero-end fragment (i.e. A-B) is obtained in the triple digest, it also appears in the double digest with A + B, but not in any other digests. We show an example of this method for the construction of restriction map of pLC 8-24. DNA of pLC 8-24 was digested with 7 different combinations of PstI (P), Hind111 (H) and KpnI (K). The electrophoretic profile is shown in Fig. 1, and end assignment of each restriction fragment to a

311

of I-IV-II-III-(I). fragments alone,

The order of adjacent

could not be determined

because

of whether

this method

the homo-end fragments

was based on the fact

or not there is another

in the fragments.

Using

fragments

homo-end

by this method restriction

the fourth enzyme enables

site

cutting

us to arrange

the

unambiguously.

(e) Subcloning of restriction fragments into pBR322 Restriction phoretically

fragments from

centrated under reduced centrifugal gel filtration Rad) as a separation with

a restriction

phosphatase, tion

fragment

were

the gel. The

eluted eluate

electrowas con-

pressure, and desalted by using BioGel P-60 (Bio-

gel. DNA of pBR322 was cut enzyme,

and ligated

treated

with

with the isolated

as described

(Ullrich

alkaline restric-

et al., 1977;

Davis et al., 1980). (f) Transformation

and test of phenotype

Cells were treated with 0.1 M CaCl, and transformed with a purified plasmid DNA (Lederberg and Cohen, The following the phenotype. n&A: Inability

1974). conditions

of cells to grow

L-agar (Lennox, 1955). nrdB: Inhibition of growth Fig. 1. Agarose gel electrophoresis of endonuclease-digested

0.25, 0.50 or 1.0 mg/ml

pLC 8-24 DNA. Lanes: (A) X DNA digested with Hind111 for

medium

M, standards. (B) Single digest with PsrI. (C) Single digest with HindIII. (D) Single digest with KpnI. (E) Double digest with PstI and HindIII. (F) Double digest with Hind111 and KpnI. (G) Double digest with KpnI and P&I. (H) Triple digest with PstI, Hind111 and Kpnl.

restriction

enzyme

is shown

in Table I. The frag-

ments that appeared in the double digests but not in the triple digest, were cut by the third enzyme as shown in Table IIA. The fragments were connected with each other to form the four partial arrangements shown in Table IIB. In this case, it was impossible to distinguish between two P-K fragments with almost the same size and between three small P-H fragments. Therefore, two possible arrangements remained. The order of four partial arrangements as I-III-II-IV-(I) was better fit for the result of single digestion rather than the order

were used

at 42°C

on

by hydroxyurea

at

in Davis-glucose

(Davis and Mingioli,

to detect

minimal

1950) supplemented

with required amino acids and 20 pg/ml thymine. frsB: Inability of cells to grow at 42°C on L-agar without NaCl. Mutation of ftsB in MFT84 was salt-repairable (Ricard and Hirota, 1973). glpT: Inability of cells to utilize 0.4% (Y-Lglycerol phosphate as a sole carbon source in Davis-minimal medium with supplements. Amp’ and Tc’: Resistance to 20 pg/ml ampicillin and 6 pg/ml tetracycline in L-agar, respectively. Colicin El immunity: The cells which showed resistance to colicin El and sensitivity to colicin E2 and E3 were regarded as the cells carrying ColEl plasmid. After transformation, L-broth at 30°C for 1 h the introduced genes, selective plates after an

the cells were cultured in to allow the expression of and then spread on the appropriate dilution.

312

TABLE Mr-value

I and end assignment

The approximate

Mr-value

ends of the restriction

of the restriction

fragments

(X 106) of each restriction

fragments

obtained

in single, double

fragment

by the double

and triple digests of pLC 8-24

which appeared

digestion

in the electrophoresis

and by the triple digestion

Hind111 (H) and KpnI (K). Since PstI and Hind111 cut the DNA at 6 and 4 sites, respectively, fragments

in P + H and P + H + K digests which were too small to be detected

included

in this table though

Enzyme Number

of cutting

sites

they were not detected P 6

and their M,-values

H

K

P+H

4

5

6+4=

10

on the agarose

were not determined

in Fig. 1 was determined.

were assigned

there must be additional

gel electrophoresis

The

to each of PsrI (P). employed.

three P-H They were

(ND).

H+K

P+K

P+H+K

4+5=9

5+6=-11

6+4+5=

P-3.97-P

P-3.95-P

15

9.95 7.60

K-7.53-K

4.50 P-4.50-H 3.95

P-3.97-P 3.13 3.4

2.80 P-2.60-K 2.58

P-2.55-P K-2,34-K

2.37

1.60

H-1.57-H 1.60

P-2.18-K

P-2.18-K

P-2.14-K

P-2.14-K

H-1.54-H K-

1.54-K

H-1.54-H K-1.54-K

K-1.54-K

P-0.64-P

P-0.64-P

P-0.99-H H-0.85-K 0.66

P-0.64-P

H-0.82-K

0.58 P-0.55-K H-0.48-K

H-0.48-K

0.44

P-0.38-H H-0.36-K P-0.33-K

P-0.32-K

P-0.26-K H-0.24-K

P-ND-H

(g) Ewmes The restriction enzymes BamHI, SgfII, EcoRI, HindIII, KpnI, PstI and Sal1 were products of Takara Co. (Kyoto, Japan). The enzymes Sac1 and Sac11 were purchased from New England Biolabs

H-0.23-K

H-0.22-H

0.23

P-0.22-K

P-0.22-K

P-O. 17-K

P-O. 17-K P-ND-H

P-ND-H

P-ND-H

P-ND-H

P-ND-H

Inc. (Beverly, MA, USA), and Xhol from Bethesda Research Laboratories Inc. (Bethesda, MD, USA). T4 ligase was a generous gift from Dr. Y. Sadaie of our Institute, and bacterial alkaline phosphatase was purchased from Worthington Biochemical Corp. (Freehold, NJ, USA).

313

TABLE II Arrangement of the fragments in the triple digest a (A) The fragments in the double digests were cut by the third enzyme as follows. P-0.38-H = P-O.17-K-0.23-H P-0.79-H= P-0.22-K-0.82-H P-2.55-P=P-(2.14 or 2.18)-K-0.32-P P-4.50-P=P-(2.14 or 2.18)-K-1.54-K-0.48-H H-0.22-H = H-ND-P-ND-H H-0.36-K= H-ND-P-0.32-K K-2.34-K=K-0.22-P-(2.14 or 2.18)-K K-7.$3-K=K-0.17-P-(0.64 or 3.95)-P-(3.95 or 0.64)-P-(2.14 or 2.18)-K P-0.26-K= P-ND-H-0.23-K P-0.55-K= P-ND-H-0.48-K P-2.60-K= P-ND-H-1.54-H-0.82-K (B) Four I. II. III. IV.

partial arrangements of the fragments in the triple digest. P-ND-H-O.23-K~.l7-P~0.~ or 3.95)-P-(3.95 or 0.64)-P-(2.14 or 2.18)-K P-ND-H-1.54-H-0.82-K-0.22-P-(2.14 or 2.18)-K P-(2.14 or 2.18)-K-1.54-K-0.48-H-ND-P-ND-H P-(2.14 or 2.18)-K-0.32-P-ND-H

BMr-value of restriction fragments and the restriction enzyme that cut at the ends are expressed in the same manner as in Table I.

RESULTS

and Carbon (1976).

(a) Mapping of f&B

using the purified plasmid DNAs, we reconfirmed our previous finding that pLC 3-46, 19-24 and

gene by Pl transduction

By the transformation

assay

42-17 could rescue the ftsB mutation ~is~~a Ricard and Hirota (1973) showed by a mating experiment that ffsB is located near the his gene.

et al., 1977). These results also agreed with Weiner’s

Plasmids complementing f&B, such as pLC 3-46, 19-24 and 42-17, could also complement g@T at

complement the glpT mutation (cited in Clarke and Carbon, 1976). We examined whether or not

48 min in a linkage map of E: coli (Bachmann and

these plasmids could complement other genes near ftsB and glp7: Thermoresistant colonies of El01 and hydroxyurea resistant colonies of LD195 could be obtained by transformation with the DNA of pLC 19-24 followed by selecting the phenotypes directly. When LDl95 cells were transformed with the DNAs of pLC 3-46 or 42-17, almost no colonies appeared on hydroxyurea plates by the di-

Low, 1980). Therefore, we reevaluated the location site

of frsB.

The

mutation

of f&B

could

be

transduced out by selecting gIpT+ at 86% co-transducing frequency, and 79% of thermoresistant colonies of ftsB became glpT_ after transduction with PI phage grown in Lin6 (f&B’, gtpT_). These co-transducing frequencies were slightly higher than that between glpT and nrdA (78%). These results confirmed that ftsB is located very close to glp7: Further plasmids.

mapping

was done using hybrid

(b) Complement&ion test of the pLC plasmids Plasmid DNAs of pLC 3-46, 8-12, 8-24, 8-29, 14-12, 19-24 and 42-17 were prepared from the hybrid plasmid colony bank prepared by Clarke

finding that all of the seven pLC plasmids could

rect selection. However, once the transformants were selected by the phenotype of colicin El-immunity, they could grow on the hydroxyurea plates. This suggests that the nrdB gene of pLC 3-46 and 42-17 was weakly expressed compared with pLC 19-24. These results are summarized in Table III. None of the seven pLC plasmids could complement the menC mutation located 0.4 min apart from g@T.

314

TABLE

III

Characterization

of plasmids

pLC plasmids

were constructed

here was pBR322. the direction

Orientation

by Clarke and Carbon

is in the reverse

selective

plates

direction

at the frequency

in pBR322

of I. The results

of 104-106/pg

cells. + or 2, Transformants

appeared

of the cloned segment or the direction

appeared

Plasmid

DNA.

by complementation

The vector of the pMY plasmids

described

shown in Fig. 2 from left to right is in accordance

of replication

of complementation

plasmid

of ColEl.

were as follows.

The colony

Orientation

cells. Details of the character

II means

+ +, Transformants

size on the plate

on the selective plates but the number

DNA or the colony size was smaller than that of wild-type colonies

region of the E. colr chromosome

(1977) and their vector was ColEl.

I means that the direction

of tetracycline-ori-ampicillin

segment wild-type

the m&l-nrdB-ffsB-g&T

carrying

was indistinguishable

of the transformants

appeared

on the

from

that of

was less than

for each case are described

with

that the cloned

104/pg

in the text. ~, No

on the selective plates. Cloned

fragment

Orientation

pLC

19-24

random

segment

II

pLC

3-46

random

segment

I

pLC 42-17

random

segment

II

pLC

14-12

random

segment

I

pLC

8-12

random

segment

II

pLC

8-24

random

segment

II

pLC

8-29

random

segment

II

pMY308

EcoRI-Hind111

pMY324

EcoRI-Sal1

of pLC 19-24

of pLC 19-24

of BglII-BglII

I I

pMY336

deletion

pMY312

EcoRI-PstI

pMY322

deletion

of BgZII-BglII of pMY312

II

pMY318

deletion

of Kpn I-Kpn I of pMY308

I

pMY328

EcoRI-BamHI

of pLC 19-24

I

pMY331

EarnHI-EarnHI

of pLC 3-46

II

pMY338

PSf I-PsrI of pLC 3-46

of pMY308

of pLC 19-24

I

Complementation nrdA

nrdB

++ _

++ + +

-

_

++ f-t +

++ ++ ii ++ ++ _ _

++ i ++

II

ff.YB

&T

++ ++ ++ _

++ +t ++ ++ ++ ++ ++ ++ ++ ++

++ ++ ++ ++ ++

++

II

++ + -t

(c) Restriction sites in the nrdAB-f&B-glpT region of the chromosome of E. cofi

ftsB-g&T

The DNAs of the seven pLC plasmids were analyzed with 10 restriction enzymes to construct the restriction map. Three plasmids, pLC 8-12, 8-24 and 8-29, had an identical restriction map

(Fig. 2). Since the pLC plasmids were constructed by the poly(dA-dT) connection method, the transition point from chromosomal DNA to ColEl DNA was uncertain. The region covered by these pLC

including the connecting region of the cloned segment to ColEl vector DNA and also with respect to the orientation of the cloned segment to the vector. They also showed the same complementation pattern. Therefore, we refer to pLC 8-24 as a representative plasmid of this group. Plasmids pLC 19-24 and pLC 8-24 contained an overlapping region comprising common 4.2 X lo6 M, PstI-Hind111 and small HindIII-Hind111 fragments. All the restriction fragments derived from cloned segments of pLC 3-46, 42-17 and 14-12 coincided with those derived from either

plasmids extended about 15 X lo6 M, and contained a total of 31 restriction sites for ten different restriction enzymes. Although deletion, addition or even rearrangement may take place in the cloned DNA, the physical map shown in Fig. 2, especially its left half, was the most likely reflection of the chromosome of E. coli for the following reasons. (1) Functional genes existed on the cloned fragments in the order of mapping data by a Pl transduction (see below). (2) The chromosomal DNA isolated from 2 different strains was digested with EcoRI,

19-24 or 8-24. A physical map of the n&A region of E. coli chromosome was deduced from the restriction maps of these plasmids

pLC

315

wrA

n
aroc

47.52

1A

0

-%%Y

r

l 9g1

0

II OEco RI

.

b

0

5

0 pLC pLC pLC pLC pLC pMY pMY

19-24 3-46 42-17 14-12 B-24 308 324

c: pMY pMY pMY pMY pMY

;:: 322 318 328 331 338

Fig. 2. Physical investigation. complementation indicate construct

15 . 10

2.0Kb Mdal

-.

map of the n&4-nrdB-f&B-glpT Restriction

sites are drawn

test but their extremities

the deleted

region

BamHI, HindIII, Southern blotting

are uncertain.

fragments

Although

the plasmid

whose molecular

fragment of pLC agreed well with the

on the physical

map

in Fig. 2

(data not shown). (d) Subcloning of the restriction complementation tests

fragments

as possible.

Solid lines indicate

PstI and KPnI, and analyzed by technique (Southern, 1979) using

the 3.1X lo6 M, EcoRI-PstI 19-24 as a probe. The profile based

region of E. co/i chromosome

to scale as closely

in the plasmid.

this map, small homo-end

prediction

10

. 5

rSac i1 *Sal I xXho I

and

To determine the precise location of ftsB, restriction fragments of the pLC plasmids were subcloned on pBR322. The newly constructed plasmids were tested to determine whether or not they rescued the mutation of nrdA, nrdB, ftsB and glpT. Properties of the plasmids are summarized in Table III, and the region of the chromosomal DNA carried by each plasmid is shown in Fig. 2.

DNAs

as deduced The location the covering

from the structure of the genes region

were extensively

of plasmids

is indicated

of each plasmid

analyzed

used in this based

on the

and double

with restriction

lines

enzymes

to

weights are less than 0.1 X lo6 M,, if present,

might have been missed.

The largest fragment

7 X lo6 M,) in the

(about

double digest of pLC 19-24 with the combination of EcoRI and Hind111 was subcloned into pBR322 to form pMY308. This plasmid was Amp’ and TcS and could complement nrdA, nrdB, ftsB and glpT. Then a series of plasmids were constructed in which the right terminus of the cloned fragment was shortened successively while the left terminus was kept at the EcoRI site. Plasmid pMY312 having a 3.1 X lo6 M, EcoRI-PstI fragment of pLC 19-24, could complement nrdA, nrdB and ftsB but not glpT, while pMY328 having a 1.10 X lo6 M, EcoRI-BamHI fragment of pLC 19-24 could complement none of the mutations tested. Plasmid pMY318 was constructed by KpnI-digestion of pMY308 followed by ligation. In this plasmid, two KpnI-Hind111 segments were retained as chromosomal DNA. Among the mutations tested this

316

plasmid

could complement

The deletion BgUI fragment the activity El01

only n&l.

in pMY324

and pMY312

and pMY322,

colonies

appeared.

with

We examined obtained

The

inoculated

on L-agar, and incubated

or

Small

at 42°C

drug-resistant

by an indirect

transformants

thermoresistant and the number

fragment

contains

were

at either 30°C colonies

were

two reasons.

nrdA affected reversion sistance

frequency became

14-12, because corrected plasmid

(1) The left-hand

nrdB activity

the ftsB

by and

of MFT84

higher the

the

must be located

within

cells to thermore-

mutation

between

chromosome.

Gene

the overlapping

is nrdA-nrdB-ftsB-glpT.

pLC

was probably the glpT

region

pLC 14-12 and pLC 42-17. We conclude order

of

(2) The

in the cells carrying

recombination host

region

but not ftsB.

of

the gene

The possible

loca-

tion of the genes is shown in Fig. 2.

of the colonies

was 6.5% of that at 30°C. This suggests BglII-BglII

of

the growth prop-

selection.

formed

the DNAs

very small thermoresistant

erty of the transformants

42’C.

reduced

of nrdA but not nrdB nor ftsB. When

cells were transformed

pMY336

following

of a 0.26 X IO6 M, of the BglII-

that the

a part of nrdA gene

and that the chromosomal segment without the BglII fragment still has a weak activity of n&A.

DISCUSSION

These results indicate the gene order in the 7 X lo6 M, EcoRI-Hind111 fragment as EcoRInrdA-(nrdB-ftsB)-glpT-HindIII. A functional nrdA

We deduced a physical map of approx. 15 X lo6 M, segment of E. coli chromosomal DNA including the location of nrdA, nrdB, ftsB and glpT based

gene is situated in the 1.96 X lo6 M, EcoRI-KpnI fragment and its right terminus extended into the

on the restriction map plasmids and subcloning

BgfII-BglII fragment. 3-46 covered almost

pBR322. Genes nrdA and nrdB are structural genes coding for the Bl and B2 proteins of ribonucleotide reductase, respectively (Fuchs and Neuhard, 1973). A mutation in nrdA in El01 cells caused tempera-

Chromosomal DNA in pLC the entire EcoRI-KpnI seg-

ment, because the size of the fragment from the KpnI to the P.rtI site in ColEl was 1.70 X lo6 M,. However, pLC 3-46 could not complement This suggests that the region very close

nrdA. to the

EcoRI site in the EcoRI-KpnI fragment was essential for the functional n&4. The region close to the EcoRI site also affected the nrdB activity. Whenever the plasmid had the chromosomal region starting at the BumHI site to the right, such as pLC 42-17, 3-46 and pMY331, they complemented nrdB by an indirect selection. On the other hand, whenever the chromosomal region in plasmids started at the EcoRI site to the right, the plasmids complemented nrdB by a direct selection as well as by an indirect selection. The nrdB gene cloned on pLC 42-17 and 3-46 may have some defects which reduce its activity. Another possibility is that nrdA and nrdB constitute an operon with nrd4B polarity and that the main promoter sequence is located near the EcoRI site. Further experiments will be necessary to clarify this matter. Both nrdB and ftsB genes were located in a 2.00 X lo6 M, BamHI-PstI segment in pMY312. Although we did not construct the plasmid carrying ftsB without nrdB, we suppose that ftsB is located at the right-hand side of nrdB for the

of seven kinds of pLC of the fragments into

ture-sensitive growth of the cells DNA synthesis stopped immediately restrictive temperature. ribonucleotide reductase the free radical

because their upon shift to

Hydroxyurea inhibits in vitro by inactivating

in the protein

B2. Mutation

of

nrdB in LD195 cells decreased the activity of B2 resulting in hypersensitivity to hydroxyurea (Fuchs and Karlstrom, 1973). Mutation of ftsB in MFT84 cells caused cells. When shifted

temperatue sensitive growth of the the temperature of the culture was

up to restrictive

temperature,

the cells of

MFT84 formed multi-nucleated, long filamentous cells, because cell division was inhibited but DNA, RNA and protein syntheses continued (Ricard and Hirota, 1973). Sensitivity of MFT84 cells to hydroxyurea was almost the same as its parental strain PA3092. Therefore, ftsB must be a different cistron from nrdA or nrdB. We demonstrated that a 3.10 X lo6 M, EcoRIPstI fragment contained the nrdA, nrdB and ftsB genes. The M,-values of Bl and B2 proteins of ribonucleotide reductase were previously reported as 160000 and 78000, respectively. Each of them

317

consists

of

Therefore, 80000

two

almost

identical of nrdA

the products

and

39000

(Thelander,

of 3.10 X lo6 M, double-stranded ftsB must

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