Enhanced instability of incfii basic replicon by the polA mutation

Vol. 139, No. 3, 1986

September 30, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 918-925

ENHANCED INSTABILITY OF IncFll BASIC REPLICON

BY THE poIA MUTATION

Hisashi YOSHIMOTO l, Chihiro SASAKAWA 2, Hirofumi DANBARA2/* Toshio UMEMOTO l and Masanosuke YOSHIKAWA 2

lDepartment of Oral Microbiology, Kanagawa Dental College, 82 Inaoka-cho, Yokosuka, 238 Japan

21nst i tute of Medical Science, Universi ty of Tokyo, Shiroganedai, Minato-ku, Tokyo, 108 Japan

Received June i0, 1986

Summary: IncF l l plasmids consist ing of a basic repl icon and of an addit ional fragment(s) unrelated to plasmid maintenance were a l l less stable in polAl than in i t s wi ld type. Reversion to UV-resistance recovered s t a b i l i t y ~ vice versa. UV i r rad ia t ion promoted i n s t a b i l i t y in polAl ce l l s . Larger p-T~-smfaT-~qowed a greater i n s t a b i l i t y and a fewer numbero----6-f-copies in a same host. Surpr is ing ly , polAl ce l ls with Tn3 on the plasmid showed a higher ampi- c i l l i n resistance than---~-h'ewild type, apparently suggesting that the polAl mu- ta t ion increases the copy number. The size-dependency of instabi l i ty- -wTs- less marked in polAl than in i t s parent. Comparison of the generation times has suggested a ~ i m e n t a l ef fect exerted by a basic repl icon in polAl hosts. © 1986 Academic Press, Inc.

The IncFl l plasmid such as RI00, R6-5 and R1 is stable under normal

cu l ture condi t ions, although the copy number is low. Replication and

subsequent pa r t i t i on is properly contro l led for stable plasmid maintenance.

The essential genes and si tes of IncFl l plasmids locate on two contiguous Pstl

fragments of 1.2 and 1.6 Kb in size ( I , 2), which correspond to a genet ica l ly

defined RepA region (3). The rep l ica t ion o r ig in , oriV (4), the pos i t ive

rep l ica t ion i n i t i a t o r gene, repA (5, 6), and the genes and/or s i tes for copy

cont ro l , Inc/Cop (7: 8, 9) ex is t on th i s region in c lus ter . This is cal led

basic repl icon or RepA repl icon.

Basic repl icon of the IncFl l plasmid is considerably unstable even i f i t

rep l icates normally. This indicates that an addit ional region is required for

Present address; Department of Bacteriology, The Kitasato Institute, 5-9-I Shlrokane, Minato-ku, 108 Japan.

Abbreviations: Kb, kilobase(s); Ap, ampiciIlin; Su, sulfonamide; Sm, streptomycin; Cm, chloramphenicol; Kin, kanamycin; Nm, neomycin; Tc, tetracycl in.

0006-291 X/86 $1.50 Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved. 918

Vol. 139, No. 3, 1986 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

stable maintenance of the plasmid. Such a region has been iden t i f ied in RIO0

and R1 (I0, I I ) .

Previously we have observed that a basic repl icon designated as pLC64

(personal g i f t from D. Lane) consist ing of the EcoRI-B fragment, the basic

repl icon of RIO0-1, and of an ampic i l l in resistance fragment was shown to be

markedly unstable in polAl than in i t s wild type (unpublished data). This

communication describes the ef fects of the polA mutation on the plasmid

s t a b i l i t y in re la t ion to the molecular size and the copy number.

MATERIALS and METHODS

Bacterial strains and plasmids: They are listed in Table 1 together with their relevant phenotypes. Media and antibiotics: Bacto Penassay broth (Difco Laboratories) added with thymine at lO mcg per ml (abbreviated as PAB) and agar medium made by solidifying PAB with agar at 1.5 % (abbreviated as PA agar) were usually used for bacterial growth. L broth (12) with or without agar and EMB agar (Nissan) were used for transformation and for scoring resistance by the replica plating, respectively. Ap (50 mcg per ml), Km (50 mcg per ml), Cm (25 mcg per ml) and Tc (25 mcg per ml) were all purchased from Sigma and used at a concentration indicated in parenthesis. Isolation and manipulation of plasmid DNA: Plasmid DNA was purified as described (2) except that tr i ton X-lO0 (0.2%) instead of Sarcosyl was used. Restriction enzymes and DNA ligase were purchased from Takara Shuzo Co.. Transformation was done as described by Cohen et al. (13). Transposition of Tn3: Plasmid DNA from C600 containig both pSClOl::Tn3 and a target plasmid (Cm-or Km resistant) was purified, transformed to C600, and selected by resistance to both Cm or Km and Ap. Then Tc sensitive colonies

Table I. Bacterial and plasmid strains used

Bacteria

Strain code Relevant genetic characters Source

C600 thi thr leu Our stock

YC256 ~ Our stock

WA5023 as YC256 but poiAl H. Ikeda

Plasmid

Plasmid code a Relevant genetic chracters Source

R6-5 IncFll, Su Sm Cm Km Nm (drd) Our stock

RIO0-1 IncFll, Su Sm Cm Tc Hg (drd) Our stock

pSClOl::Tn3 Tc, Tn3(Ap) S.N. Cohen

pSCl02 derivative of R6-5, Km Su K.N. Timmis

pKT071 derivative of pSCl02, Km K.N. Timmis

pMYll20 derivative of RlO0-1, Cm Our laboratory

pTHlO thermosensitive derivative of RP4 T. lino Kin, Tn3 (Ap)

aWith regard to the other plasmids see Fig. I.

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Vol, 139, No. 3, 1986 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

were isolated. For transposit ion onto the chromosome, a rep l ica t ion thermosensitive der ivat ive of RP4 (designated as pTHIO) (14) was used. Plasmid s t a b i l i t y tes ts : Overnight PAB cultures with appropriate an t ib io t i cs were di luted 50-fold with PAB with the same an t ib io t i c , incubated with shaking for 3 hrs to minimize plasmid-free ce l l s , and followed by 5-fold d i lu t ion with PAB containig the same an t ib io t i c for 1 more hr of incubation. These cultures were di luted I0 times with an t ib io t i c - f ree PAB and incubated at 37 C for 12 hrs with shaking. This subculturing was repeated and then the cultures were spread on PA agar to score plasmid loss by the repl ica plat ing. UV sensit iv i ty tests and UV-promoted plasmid loss: Cells with pKT071 at exponential growth phase in PAB with Km were spread on PA agar and irradiated with UV from a 15 W UV lamp covered by a carton with a 5 x 5 cm hole. During irradiation the plates were magnetically turned at 25 cm below the lamp. Colonies formed were either directly scored for Km resistance by the replica-plating or the whole cells of each colony were transferred to saline, fu l ly suspended and then spread on PA agar, the colonies on which were scored for Km resistance s imi lar ly . Single ce l l resistance to Ap: A f ixed volume of a d i lu t ion of ce l ls with a Tn3-bearing plasmid was spread on PA agar containing varying concentrations of Ap~ After 24 hr incubation the number of colonies was counted.

RESULTS

Instabi l i ty of various basic replicons in polAl and i ts parent host:

Stabil i ty was compared among plasmids of various molecular sizes with a basic

replicon either from pSCl02, a derivative of R6-5, or from pMYll20, a

derivative of RIO0-1. Their molecular sizes are different because of

additional Pstl fragments such as Km resistance fragment(s) of pSCl02 (P-l),

Cm resistance fragment(s) from plasmid S-a (P-Cm) and/or other fragment(s) of

pSCl02 origin (Fig. l ) . All plasmids tested are less stable in poIAl than in

i ts wild type cells. The larger in the molecular size the less stable is the

plasmid in a same stra in. Derivation of the basic replicon and i t s

or ientat ion re la t i ve to the additional fragments do not af fect the s t a b i l i t y .

UV-resistant revertants isolated spontaneously or selected by resistance to

ethylmethanesulfonate maintained pKT071 as stably as the wild type, YC256.

Inversely, a bacterial der ivat ive stably maintainig pKT071 isolated af ter

repeated growth in ant ib io t ic -conta in ing PAB was UV-resistant. The extent of

i n s t a b i l i t y is shown in Fig. 2 re la t i ve to the molecular sizes. A l inear

corre lat ion is observed between the s tab i l i t y and molecular sizes on the

semilogarithmic scale. Size-dependency of the s t a b i l i t y is less marked in the

polAl mutant than in the wild type. The two l ines for the mutant and the wild

type on Fig. 2 look to cross on the abscissa by extrapolat ion, indicat ing that

a basic replicon of 40 Kb or larger cannot be maintained by th is basic

repl icon i r respect ive of the polAl or the wild type backgrounD. However, i t

should be noted that a t ransfer -defect ive der ivat ive of RIO0-1 has never

produced a plasmid-free cel ls even in polAl mutant.

Enhancement of instabi l i ty of pKT071 in poIAl cells by UV irradiation: The

poIAl mutant is in fact more sensitive to UV than the wild type, whereas no

920

VoI. 139, No. 3, 1986 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Stb

E Ep m

p P

DES i GNAT I ON STRUCTURE BASIC REPLICON

P P5 PH P

#593 . . . . ' ( 6 . 3 ) PSCI02 | | ! | P-4 Ix-(, lx-Cm

P PS P P

' " ' I (6 s) PMY1120 PMY1122 . , (e- 4)(P- 6) P- c=

P PS P P H P

PYH347 ", ,~ 'I' I (6.7) PSCI02 P-4 P-6 P-Cm

P-8

PYH351 A (6.7) PSCI02 P- 4 P-Cm 8

~ " ( 1 4 . s ) p S C l 0 2 PKT071 ' ,s | ! i , P-4 P-6 P- 1 (Kin)

PYH333 , ~ ; (14. s) PMY1120 C P- 4)CP- 6) P- 1C~)

P Ps i PYH341 ' ~ 'P s ~ (14.S) PMYI120

i J

(P- 4)C~ 6) P- z O(m)

. . . . [ s i PYH329 " ~ ' , , ~ (17 9) PSCI02 | ! •

p-4 ~ 6 P-Cm P- 1 (](m)

. . . . . i s [ pYH344 " " ; ! ' ~ ( 1 7 . 9 ) pMYII20 '(P- 4)(P- 6) P-Cm P- 1(Km)

, PLs ~ ~ ~ PH ~ (17.9) pSCI02 PYH326 " " ! l • P- 4 P- ~ P- I (IOn) P- Cm

PYH357 '. ~s ; ~ ~ ,,, , , I ' ~ pSCI02 I~4 P-6 P- 1 (Kin) P-Cm P-Cm P-3 (24.6)

Km

P p

P DERIVATION OF

Fi 9. I Der iva t ions and const ruc t ion of plasmids wi th d i f f e r e n t s izes. Pst l -d igested pSC]02 ( d e r i v a t i v e of R6-5) or pMYII20 ( d e r i v a t i v e of RIO0-1) DNA was mixed wi th Ps t l -d iges ted Cm fragment (P-Cm) from S-a or P-I from pSCI02 and l i ga ted wi th T4 DNA l igase . Pst l -d isgested pSCI02 DNA was a lso s e l f - l i g a t e d . C600 was transformed wi th these DNA by se lec t ing wi th res istance to Km, Cm or both. The basic r ep l i con -ca r r y i ng r e s t r i c t i o n fragments (P-4 + P-6 of pSCI02 or the corresponding fragments of pMYll20, ind icated as (P-4)+(P-6)) are shown by heavy l i nes . The hor i zon ta l arrows ind ica te the o r i e n t a t i o n of the fragment(s) r e l a t i v e to the basic rep l i con . The sizes of plasmids are expressed in parenthesis by k i lobase (Kb). Abbrev iat ions fo r r e s t r i c t i o n enzymes are P; Ps t l , S; Sa l l , E; EcoRl, H; H i n d l l l .

9 2 1


500

o_ 100

50 2

m 10

~ s

1

® Fig. 2

\

4o

~_ 30

"\ "\ ~: 20 x xx~\ • i0

i i l ~\ o i0 20 30 40 SIZE OF PLASMID IN KILOBASE

A B

50 100 50 100

% PLASMID-BEARING CELLS IN COLONIES

Relationship between instabil i ty and size of plasmids. Instabil i ty was expressed by the number of generations required for 50 % plasmid loss, 0 and • are the averages for YC256 (polA ÷) or WA5023 (poIAl) with al l plasmids of the respective sizes ind lc~d in Fig. I. ~ r standard deviations are shown with vertical solid lines. Broken lines are extra- polations from the results actually obtained.

o • 0 Promotion of plasmid loss by UV irradiation. The whole cells of each colonies derived from UV-irradiated (A) or unirradiated (B) polAl cells

with pKT07I were spread on agar plates and grown. The colonies ut-h-u-s--formed were scored for plasmid carriage. Vertical arrows and horizontal open boxes show the average and standard deviations, respectively.

obvious e f fec t of carrying pKT071 in UV sens i t i v i t y is observed. I f the

survived colonies af ter UV i r rad ia t ion are examined for plasmid loss, only the

polAl mutant but not the wi ld type shows an enhanced plasmid loss by the same

doses of UV. The polAl mutants e i ther i r radiated or unirradiated were plated

and incubated at 37°C overnight. Then the whole ce l ls of each colony were

suspended in saline and spread on PA agar without Km. The colonies formed

were scored for Km resistance. The resul ts on I00 i n i t i a l colonies are shown

in Fig. 3. The percentage of plasmid-bearing ce l ls in the majori ty of

colonies formed af ter UV i r rad ia t ion is usually smaller than that without

i r rad ia t ion . I t should be noted that 99 out of I00 i r radiated colonies

contained plasmid-bearing ce l l s , indicat ing that at least these 99 colonies

emerged from plasmid-bearing ce l ls and that plasmid-bearing ce l ls were not

se lec t ive ly k i l l ed before f i r s t cel l d iv is ion on the agar plates. These

observations together with an obvious lack of di f ference in UV sens i t i v i t y

between polAl ce l l s with and without pKT071 have revealed that UV i r rad ia t ion

pos i t i ve ly enhances the emergence of plasmid-free cel ls in polAl ce l l s .

Selective overgrowth of plasmid-free cells as the main mechanism of apparent

i n s t a b i l i t y in the polA background: A tendency has sometimes observed that

plasmid loss in poIA cel ls is apparently accelerated during subculturing. The

growth rates of poIAl and i t s wild type with and without pKT071 were compared.

No or l i t t l e , i f any, difference is observed in the growth rates of the wild

9 2 2


i00 .--o o " ~ . A

50 )

0 LL./; , , X o 8 . . ~ - < , , X , . j j

IP.5 50 100 200 400 800 1600

i00 W~

-- ~ 50 (9.4~, ° ) . ~ . i )

2 0 , . . , " , . J ]2.5 50 100 200 40(] 800 1600

- I " 3 100 - ~ ~ ~-~-~

so "\:

0 L I I i i ,~ ~ , 1 0 1,06 3.12 6.25 12.5 25 50

AMPICILLIN 60NCENTRATION (#g/m0)

Fig. 4 Relationship between size and copy number of plasmids. Appropriate dilutions of overnight cultures of YC256 (A) or WA5023 (B) carrying a plasmid with Tn3 ( • ; #593::Tn3, <> ; pKTO71::Tn3 and O; pYH357::Tn3) were spread on agar c~taining variou-s concentrations-of Ap and grown. -The results are expressed by the number of colonies formed relative to those formed on agar with 12.5 mcg of Ap per ml. The f igure (C) is for YC256 ( A ) and WA5023 ( A ) with Tn3 on their chromosome and the results are expressed by the number of colonieS-formed relative to those formed without Ap. Note that the unit of abscissa in (C) is different from that in (A) or (B). The f igures in parenthesis on (A) and (B) are evaluated by multiplying the 50 % inhibitory concentrations of Ap (mg/ml) by the molecular size in Kb.

type with or without pKT071. In contrast, the poIAl cells with pKT071 showed

a generation time (25.7 min) ca. 1.5 min longer than the same cells without it

(24.2 min). This difference was much more marked in the presence of Km than

in its absence. These observations indicate that once plasmid-free cells

appeared, they overgrow the plasmid-bearing cells, leading to an apparent

instability.

Molecular sizes and the number of copies of various basic replicons: Relative

copy number of 3 representative plasmids, pYH357, pKT071 and #593 were

examined by transposing Tn3 on each plasmid and by comparing Ap resistance

(15). As in Fig. 4A and B, the larger is the size of plasmids, the lower is

the Ap resistance in the same host. Surprisingly, any of 3 plasmids tested

revealed a higher Ap resistance in poIAl than in its wild type. Since no

difference in Ap resistance was observed between these hosts when their

chromosome carries Tn3, the difference in Ap resistance between poIAl and its

wild type may have to be ascribed to the difference in the copy number.

DISCUSSION

In this study i t has been made clear that the polAl mutation is one of

the factors to enhance apparent instability of the basic replicon of the

923


IncFll plasmid. At th is moment i t is not sure how the deficiency of DNA

polymerase I provokes such i n s t a b i l i t y . However, i t is sure that the polAl

mutation i t s e l f is responsible for th is phenomenon because ( i ) UV resistant

revertants selected by ethylmethanesulfonate carry the basic repl icon as

stably as the wild type, ( i i ) stable revertants are res istant to UV and ( i i i )

UV i r rad ia t ion enhances the apparent i n s t a b i l i t y without af fect ing UV

sens i t i v i t y of both plasmid-bearing and plasmid-free polAl ce l ls (Fig. 3).

In a same host a plasmid with a larger molecular size is less stable.

This presumably re f lec ts a smaller copy number due to a larger molecular size

as shown in Fig. 4A and B. I t should be noted that the two l ines in Fig. 2

showing the relat ionship between the molecular size of basic replicons and

the i r i n s t a b i l i t y in polAl and i t s wi ld type host cross at the point where the

number of generations required for 50 % loss of the replicon is 1 in both

hosts. This may suggest that there exists a l im i t in the molecular size for

th is basic replicon capable of maintainig and that th is l im i t is equal in both

hosts. I t also means that the slope of the l ine is more steep in the wild

type than in the mutant, suggesting that molecular size-dependency of the

i n s t a b i l i t y is more marked in the wild type than in the mutant. Another

in terest ing observation is that the tota l DNA content of basic replicon in a

ce l l , estimated by the product of the molecular size of a replicon and the

minimal inh ib i to ry concentration expressed by Tn3 on i t , is almost constant

for each host but larger in polAl mutant than in the wild type (Fig. 4).

Since Ap resistance expressed to the same level by the transposon on the

chromoseome in both hosts, i t is conceivable that the polAl mutation plays a

certain role in increasing the plasmid copy number.

With more copies a replicon has been considered more stable i f there is

no par t i t i on mechanism (16). Nevertheless the basic replicon in polAl mutant

is apparently less stable than that in the wild type mainly due to a

detrimental e f fec t of the repl icon in polA mutant and as a consequence

select ive overgrowth of plasmid-free ce l ls . This detrimental e f fect may also

be copy number-dependent as suggested by the d i f f e ren t slopes in Fig. 2 for

the mutant and the wild type host. As the detrimental e f fec t may be so

overwhelming that the ef fect of the polA mutation to increase copy number may

have been overcome.

REFERENCES

I . Kollek, R., Oertel, W. and Goebel, W. (1978) Molec. Gen. Genet. 162, 51-57.

2. Timmis, K. N., AndrOs, I . and Slocombe, P. M. (1978) Nature 273, 27-32. 3. Yoshikawa, M. (1974) J. Bacter iol . 118, 1123-1131. 4. Synenki, R. M., Nordheim, A. and Timmis, K. N. (1979) Molec. Gen. Genet.

168, 27-36.

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5. Andres, I . , Slocombe, P. M., Cabello, F., Timmis, J. K., Lurz, R., Burkardt, H. J. and Timmis, K. N. (1979) Molec. Gen. Genet. 168, 1-25.

6. Rosen, J., Ryder, T., Inokuchi, H., Ohtsubo, H. and Ohtsubo, E. (1980) Molec. Gen. Genet. 179, 527-537.

7. Danbara, H., Brady, G., Timmis, J. K. and Timmis, K. N. (1981) Proc. Natl. Acad. Sci. USA. 78, 4699-4703.

8. Light, J. and Molin, S. (1981) Mol. Gen. Genet. 184, 56-61. 9. Molin, S. and NordstrOm, K. (1980) J. Bacteriol. 141, 111-120.

I0. Miki, T., Easton, A. M. and Rownd, R. H. (1980) J. Bacteriol. 141, 87-99. I I . NordstrOm, K., Molin, S. and Aagaard-Hansen, H. (1980) Plasmid 4, 215-227. 12. Mi l ler , J. H. (1972) Experiments in molecular genetics, Cold Spring Harbor

Laboratory. 13. Cohen, S. N., Chang, A. C. Y. and Hsu, I. (1972) Proc. Natl. Acad. Sci.

USA. 69, 2110-2114. 14. Harayama, S., Tsuda, M. and l ino, T. (1980) Molec. Gen. Genet. 180, 47-56. 15. Uhlin, B. E. and NordstrOm, K. (1977) Plasmid I , I-7. 16. Meacock, P. A. and Cohen, S. N. (1980) Cell 20, 529-542.

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Enhanced instability of incfii basic replicon by the polA mutation