Deletions in the tetracycline resistance determinant reduce the thermosensitivity of A trfA(Ts) derivative of plasmid RP1 in Pseudomonas aeruginosa

Ann. Inst. Pasteur/Microbiol. 1987, 138, 151-164

(~) ELSEVIER

Paris

1987

DELETIONS IN THE TETRACYCLINE RESISTANCE DETERMINANT REDUCE THE THERMOSENSITIVITY OF A trf4(Ts) DERIVATIVE OF PLASMID RP1 IN P S E U D O M O N A S AEI~UGINOSA

by M. Rella (1) (*), J.M. Watson (1) (**), C.M. Thomas (2) and D. Haas (1) (1) Mikrobiologisches Institut, Eidgen6ssi~'che Technische Hochschule, CH-8092 Zfirich (Switzerland), and (2) Department of Genetics, University of Birmingham, Birmingham B15 2TT (UK)

SUM.~ARY A derivative of the broad-host-range plasmid RP1, pME301, was temperature-sensitive (Ts) at 43°C for maintenance in Pseudomonas aeruginosa, P. mendocina, Klebsiella aerogenes and Escherichia coli. In E. coli, the Ts defect of pME301 could be complemented in trans by th,: cloned trfA gene, which is known to be essential for RP1 replication in E. coli and P. aeruginosa. Because pME301 expressed a Ts phenotype in P. mendocina and K. aerogenes, we assume that the trfA function is also vital in these organisms. When plasmid-encoded carbenicillin resistance (on transposon Tn801) was selected at non-permissive temperatures in P. aeruginosa strain PAO carrying pME301, we obtained either Tn801 insertions into the chromosome or pME301 derivatives having a deletion (or point mutation) in their tet genes, which determine resistance to tetracycline and are not transposable. From cloning experiments, we infer that the tet gene product(s) destabilize the pME301 replicon in P. aeruginosa at 40-43°C. KEY-WORDS"Pseudomonas aeruginosa, R plasmid, Tetracycline; trfA(Ts) mutant, Resistance, RP1.

Submitted January 9, 1987, accepted March 11, 1987. Please send correspondence to Dieter Haas. (*) Present address: Ciba-Geigy AG, Agro Division, CH-4057 Basel (Switzerland). (**) Present address : CSIRO, Division of Plant Industry, GPO Box 1600, Canberra City, ACT 2601 (Australia).

152

M. RELLA A N D COLL.

INTRODUCTION The broad-host-range plasmid RP 1 carries determinants for resistance to kanamycin, tetracycline and ampicillin/carbenicillin [3, 8]. RP 1 is very similar or identical to a number of IncP-1 piasmids, including RK2, RP4, R68 and R18 [5, 35]. The constitutive penicillin resistance is due to a periplasmatic [3-1actamase, which is encoded by the bla gene in transposon TnS01 (= Tnl) [7]. Inducible tetracycline (Tc) resistance is due to a membrane protein specified by the tetA gene, which is controlled negatively by the tetR product; the RP 1 tet genes are not transposable [4, 43]. The replication and maintenance func:ions of RP1 are of particular interest because they function in almost all Cram-negative bacteria [29, 30, 40] in spite of considerable expression barriers that exist for genes of these bacteria in heterologous hosts [9]. Dissection of the RK2 replication and maintenance functions has revealed three separate, important regions: the origin of vegetative replication (oriV) and the trf4 and trfB regions coding for transacting replication functions and their c~ntrol elements [30, 31, 32, 39, 40]. For RK2 replication in Escherichia coli, Pseudomonas putida and P. aeruginosa, part of the trfA region (designated trfA*) and oriVare sufficient, although these mini-repticons are not entirely stable; in other bacteria, additional functions are required for maintenance [29, 30]. Temperature-sensitive (Ts) plasmid mutants are particularly useful for the analysis of essential replication factors and as tools in bacterial genetics. Several Ts mutants of IncP-1 plasmids have been isolated in E. coli [6, 13, 27] and in P. aeruginosa [15, 44]. Certain ef those plasmid mutants which cannot be maintained in E. coli at 42°C appear to be only weakly Ts (pMR5 [27] ; C. Thomas and C.A. Smith, unpublished results) or not at all Ys (pTHI0 [41]) in P. aeruginosa. The Ts defects of pMR5 arid another similar RP 1 (Ts) plasmid are complemented in trans by the cloned trfA gene in E. coil [36]. No complementation tests have been reported for those IncP-1 plasmids which are Ts in P. aeruginosa. We have therefore chosen to characterize pME301, an RP1 mutant which fails to replicate at 43°C in P. aeruginosa strain PAO

[44]. In previous work, it has been shown that suitable Ts derivatives of RP! can be integrated into the P. aeruginosa chromosome via Tn8Ol-mediated replicative transposition when carbenicillin (Cb) resistance is selected at non-permissive temperature [11, 15]o The presence of the plasmid in the

Cb Cm Km NA

= carbenicillin. = chloramphenicol. = kanamycin. = n u t r i e n t agar.

R Tc Ts S

= = = =

resistant. tetracycline. temperature-sensitive, sensitive,

P L A S M I D RP1, P. AERU(31NOSA A N D T E T R A C Y C L I N E

153

c h r o m o s o m e results in Hfr d o n o r strains [11, 15], which can be stabilized by mutations in the trfA locus, in the Tn801 resolvase gene and in the host recA gene [25]. In the absence of such mutations, the Ts l~,lasmid has a strong tendency to excise; after plasmid excision, a single Tn801 copy i~ found in the c h r o m o s o m e [12, 24, 4~!o D u n n g studies o f P. aeruginosa strains carr~,Sng pME3Ol, we found that selection for Cb resistance at 43°C did not always result in clones having chromosomal Tn801 or pME301 insertions; we alse recovered pME301 derivatives with deletions in the tet region. Here we present evidence that the tet genes modulate the thermosensitivity of pME301 in P. aeruginosa and we examine the host range of the Ts phenotype in different bacterial species.

MATERIALS AND METHODS Bacterial strains. ~ Cloning experiments were carried out in E. coli K12 strains ED8654 (metB suFE supF hsdR hsdM +) [4] or MV 10( = C600 trpE; thr-1 leu-6 thi-1 lacYi supE44 tonA21 trpES) [38]. Temperature sensitivity tests were done in P. aeruginosa strains PAO8 (met-28 ilv-202 str-1) or PAO25 (argFlO leu-lO) [10] and in E. coli K12 JC3272 (his lys trp lac A X74 gal str) [1]. Other bacterial species are listed in table III. Media and growth conditions. ~ Nutrient yeast broth and nutrient agar (NA) [33] were used for all bacteria. RPI and pME301 were transferred from E. coli ED8654 to various bacteria by conjugation at 30°C. Exponential cultures (0.2 ml) of the donor and recipient strains were mixed, concentrated 10 × and incubated on a NA plate for 4 h. The mating mixture was purified on selective minimal medium [33] containing 1000 ~g Cb/ml for P. mendocina, 500 l~g Cb/ml for P. aeruginosa, 250 tzg Cb/ml for Acinetobacier calcoaceticus, 150 ~tg Cb/ml for P. stutzeri or 50 ~tg Km/ml for Klebsiella aerogenes. The permissive temperature for pME301 was 30°C in all bacteria; to test the plasmid's Ts phenotype, the highest possible growth temperature was chosen (table III). Antibiotic concentrations usea for E. coli have been given previously [32, 37]; Km concentrations for other species are listed in table III. P. aeruginosa strains PAO8 and PAO25 carrying pME301 were grown on NA + Cb (500 ~tg/ml), NA + Tc (125 ~g/ml) or NA + Km (300 izg/mi) and the same antibiotic concentrations were used in nutrient yeast broth. CbRTs + derivatives of pME301 (see tt Results>>) were selected on NA + Cb at 43°C; those clones which lost their resistance to Tc or Tc +Km were purified .~everal times on selective medium. Plasmid construction, m A list of the plasmids used is given in table I. Plasmid DNA was extracted from E. coli or P. aeruginosa PAO as previously described [18, 36]. The conditions for restriction enzyme digestions, ligation, agarose gel electrophoresis and transformation were the same as those used in earlier studies [18, 36]. Plasmid pCT450 was derived from pCT87, a P15A replicon carrying Cm R as well as trfA and Tc R of RK2 [36], by ToA insertion between trfA and tetA followed by HindlII deletion of the majority of Tn5 and the fragment containing trfA. Thus, pCT450 consists of the P15A replicon pDS3 (2.5 Kb; table I) joined to a 5.0-Kb HindlII fragment carrying the RK2 tetA and tetR genes (fig. 2). Estimation o f plasmid copy numbers. ~ [3-Lactamase assays.were performed to estimate plasmid copy numbers [18]. Strain PAO1012, which cames a single bin gene (i.e., a Tn801 insertion) in the chromosome, was used as a standard [19].

154

M. R E L L A A N D C O L L .

TABLE I. -- Plasmids used. Plasmid RP1 pME206 pME301 pME305 pME144, pME145, pME146, pME147, pME449 pME490

Relevant properties Cb,Tc,Km Tra IncP-1 Cb,TcS,Km Tra IncP-1 (deletion of Tc) Cb,Tc,Km Tra Rep(Ts) IncP-1 (Ts maintenancein P. aeruginosa and E. colt) Cb,Tc,KmS Tra Reg(Ts) IncP-1 Cb,TcS Km Tra Rep(Ts) Ira.P-1 (Ts mainte, ance in & coli; maintenance in P. aeruginosa stabilized by mutatioa in Tc) Cb,Tc,KmS Tra PeptTs) IncP-1 (Ts maintenance in E. coil)

pME491

Cb,Tc,KmS Tra IncP-1

pCT87 pCT88 pCT450

Tc,Cm trfA + P15A replicon Tc,Cm trfA + P15A replicon Tc,Cm PI5A replicon

pDS3 pME285 pME324

Cm P15A replicon Hg,Km pVS1 replicon Km,Cm trfA + pVS1-PI5A cointegrate

Construction or reference

[si Haeil partial digestion of RP1, ligatio~ ( t ~ study, fig. 2). Nitrosoguani(Jneoinducedmutant derivative of RP1 [44].

KmS derivative of pME301 [26]. Spontaneous Ts+ Tcs derivatives of pME301 obtained in P. aeruginosa (this study, fig. 2). 5-Kb HindlII Tc fragment of pCT450 cloned into HindIII site of pME145 (this study, fig. 2). 5-Kb HindlII Tc fragment of pCT450 cloned into HindlII site of pME206 (this study).

[361 [36] pCT87::Tn5 partially digested with HindlII removing most of Tn5 and trfA (this study, fig. 2). D. Stalker [36]. [17] Cointegrate between pCT88 and pME285 constructed in vitro by SalI digestion an~/~ation (this study).

RESULTS Stability of RP1 in P. aeruginosa at 43°C. An overnight culture of PAO8(RP1) grown at 30°C was diluted to 104 cells/ml in prewarmed nutrient yeast broth and incubated at 43 °C with vigourous shaking. After a short lag phase, PAO8(RP1) grew exponentially with a doubling time of about 30 min (fig. 1). Viable counts on NA, NA + Cb and NA + K m at 30°C were the same, and all colonies obtained on these media were also Tc R when replicated onto NA + Tc and grown at 30°C. Thus, RP1 was entirely stable without antibiotic selection in strain PAO at 43°C. Towards tile end of growth of PAO8(RP1), the efficiency of plating on N A + Tc (25 or 125 ~tg/ml) dropped somewhat (fig. 1), but this phenotypic

PLASMID RP1, P. A E R U G I N O S A A N D TETRACYCLINE

155

1~0 *-

i

109I

W .0

108

-

_

107 106 105 /

,iOt,

~ 0

2

~

6

8

. 10

12

1~

.3

-......L..-18

Time (h)

FIG. !. - - Growth

of PAOS(RP1) and PAO8(pME301) at 43°C.

Overnight cultures o f these strair,~ were grown at 30°C with antibiotic (Cb,Tc,Km) selection, diluted into fresh nutrient yeast broth without antibiotics and incubated at 43°C with vigourous shaking. Samples were plated on NA (ll), NA + Cb (O) and NA + Tc (&) and

incubated at 30°C for viable count determination. . . . . PAO8(RP1); - - = PAO8(pME301). For PAO8(RPI), the viable counts on NA, NA +Km and NA + Cb were the same; for PAO8(pME301) NA + Km gave tiae same results as NA + Cb (not shown).

loss of Tc resistance was entirely reversible at 30°C. The transient Tc sensitivity might be explained by a negative gene dosage effect [23] if RP 1 copy numbers were strongly elevated at 43 °C. However, there were 2-3 RP 1 copies at 43°C and 3-4 copies at 30°C, as estimated by ~-lactamase assays. Thus, it appears that prolonged growth o f P. aeruginosa at 43 °C can interfere with the expression o f Tc resistance, but this p h e n o m e n o n was not further investigated. No such effect was seen in E. coli carrying RP1 (data not shown).

Segregation of pME301 in P. aeruginosa at 43°C. A similar experiment was performed with PAO8(pME301) in order to follow the less of the RPI(Ts) plasmid at 43°C. During a long lag phase of 11 h, the number of antibiotic-resistant cells increased only very slightly; then

156

~ . R E L L A A N D COLL

growth resumed, with progressive loss of the plasmid resistance markers (fig. 1). After 10-16 generations at 43°C, 90-99 % of the cells had eliminated pME301 (fig. 1). Apparently, some plasmid replication was still possible at 43°C. Phenotypic loss of Tc resistance was not observed for pME301 (fig. I). Complementation of pME301 by trfA in trans.

Plas:nid pME301 could be transfer-red to E. coli by conjugatio~a and established in this host at 30°C. At 43°C, the pIasmid was Ts, as in P. aerugivosa (cf. table III). A complementation test previously developed for Ts derivatives of RP1 [36] was used to see whether the Ts phenotype of pME301 was due to a mutation in the trfA replication control gene. The trfA gene is close to tetA (fig. 2) [40]. The E. coli strain JC3272(pME301) was made competent and transformed with either pCT88 (P15A replicon, Cm R, trfA* = trfA + kilD- [30, 36]) or with the control pDS3 (P15A replicon, Cm R [36]). The selective medium contained Tc (25 t~g/ml)+ Km (50 ~ g / m l ) + Cm (25 ~g/ml). At 30°C, both pCT88 and pDS3 gave l 0 4 transformants/~g DNA. By contrast, at 43°C, pDS3 gave only 10 transformants/~g DNA, whereas pCT88 produced 104 transformants/~tg DNA. Thus, the Ts defect of pME301 could be complemented in trans by the cloned trfA region carried by pCT88. A complementation test was also attempted in P. aeruginosa. Plasmid pCT88 was cloned into the low copy number Pseudomonas vector pME285 [17]. The resulting co-integrate pME324 could be introduced into P. aeruginosa. However, we were unable to establish both pME324 and pME305 (= pME301AKm R) in P. aeruginosa, even at 30°C. Hence we could not test complementation in this bacterium.

Isolation of Ts '~ derivatives of PAO(pME301) strains.

The efficiency of plating of PAO8(pME301) pregrown at 30°C was ca. 10-6 when cells were plated on NA + Cb at 43°C (table II). Most Cb R colonies were also resistant to Tc and Km, b:~t this phenotype was not stable at "i3°C during purification. This can be explained in two ways. Either some plasmid replication continued at 43°C because of leakiness of the Ts defect, or pME301 might unstably integrate into the chromosome and replicate under chromosomal control for some time. In ca. 1 °7o of the Cb R colonies growing at 43°C, transposition of Tn801 into the chromosome had occorred. Such Tn801 insertions have been described elsewhere [12]. Unexpectedly, some stable Cb R Ts + clones of PAO(pME301) strains were Km R but TcS; they could be isolated at 40 or 43°C. ii: is difficult to estimate the frequency at which they arose (~< 10-s), but they could be isolated readily after D-cycloserine enrichment for Tc s cells by the method described previously [11]. Five isolates wexe tested; they all contained a transfer-proficient

P L A S M I D RP1, P. AERUGINOSA A N D T E T R A C Y C L I N E

RP~

(0}

{1301~3)

i o

I!

1%/*) (~0)

,

"';'-..

~

I

(600)

I,

30

I

~

~

"

157

~

"'x

, "

"

1127) ¢13.01

I

12.0 pM~. 1 / ' ~ ' ~

(15.t,)

Ill

13.0

•q=== oriV

-,

15.0

erR = ;

16.0

~

~_ . . . . . . . .

I

>'

,

I

,

,

I

kilD

t r f A (Ts)'

'

__~

,

<.._.~l I

I

= 0.35 kb

~E~06

180kb

tr fk(Ts)

/~ : 0.55 kb

p~9

~70

fr'^'n-82',~3~

fetA

~- . . . . . . . . . . . . . .=. . .2.6-2.1~ . . . . . . kb

r-

"" -..

I

lt,,.O

Rept,catio.

p~6/' pilE1/,5

(lt,31 (lt,5)flt, t 5)

t

1<. . . . . . . . . . .

tr fA (Ts)

,

~

i

,

,

A = 1.6 kb

trfA* =

p~ ~90 0

10 /~Tc5 20

=

pET/+50 F--T 0

.~C,

Tc ~

-

~,~ 2

7erR "-tetA

50

=

I

, t.

I

60kb

=

, 6

I 7.5 kb

onR

FIG. 2. - - Restriction maps o f RP1 and its derivatives. The maps are drawn according to the R P 4 / R K 2 maps o f Lanka et al. [21] and Thomas et al. [40]. The tet deletion plasmids pME 144, pME 145, pME 146 and pME449 were aerived from pME301 as described in the text; pME206 is a derivative o f RP1 obtained after partial digestion with HaeII. Brackets indicate maximal extent o f a deletion. Arrows denote the open reading frames o f the kilD~ trfA(P3s2), tetR and tetA genes; points indicate the transcription starts. The 5.0-Kb HindIII fragment o f pCT450 carrying the RK2 Tc R genes was cloned into the HindIII site o f pME145 to produce pME490.

plasmid. Four plasmids (pME144, pME145, pME146 and pME~49) contained a deletion in the tet region (fig. 2). One plasmid (pME147) had the same restriction pattern as pME301 and may carry a point mutation or a very small ( < 0.1-Kb) deletion. Deletion formation in the Tc resistance genes was also observed in a recombination-deficient PAO strain (data not shown). The

158

M. R E L L A A N D COLL.

TAaLE II. -- Stability of RP1 and its derivatives in P. aeruginosa PAO8. Plasmid in strain PAO8

RP1 pME301 pME145 pME490 pME206 pME49I

Plasmid stability coefficient 30°C liquid culture grown withantibiotic selecfic,~a,plated at 30°C on NA with:

30°Cliquid culture 30°C liquid culture 43°Cliquid culture gxownwith antibiotic grown without grown without selection,plated at antibiotics 0), plated antibiotics~2),plated 43°Con NA with: at 30°C on NA with: at 30°C on NA with:

Cb

Km

Tc

Cb

0 0 0 0 0 0

0 0 0 S 0 S

0 0 S 0 S 0

Km

Tc

0 0 0 -6.0-5.0 -5.0 -2.0 -2.5 S -6.0 S -5.0 0 0 S 0 S 0

Cb

Km

Tc

0 0 0 0 0 0

0 0 0 S 0 S

0 0 S 0 S 0

Cb

Km

Tc

0 0 -2.3 -3.0-5.0-5.0 -1.5-1.6 S -2.5 S -3.0 0 0 S 0 S -1.3

The p!asmid stability coefficient is defined as log ~ viable count on NA + antibiotic "~ viable count on NA J Therefore, a value of 0 denotes stability and increasing negative values correspond to increasing instability. The viable counts of all liquid cultures grown at 30°C or at 43°C were very similar on NA (1-4 × 109 ceils/ml), aAntibiotic selection~> means that all antibiotics to which the plasmids specify resistances were added to nutrient yeast broth. (1) Growth in liquid culture for about 20 generat'ons. (2) Growth in liquid cv'mre for about 40 generations. S = sensitive.

temperature sensitivity of plasmid maintenance was greatly reduced in all five isolates. Table II shows that one typical representative, pME145, was 100-1,000 times more stable than pME301 at 43°C in strain P A O . Reversion of the Ts defect of pME301 was improbable because pME145 and the other four Tc s derivatives were still fully Ts in E. coli (data not sho~;n). Characterization of the tet deletion plasmid pMEI45. To test whether the tet gene products and, in particular, the tetA-encoded membrane protein [14], may interfere with pME301 at 43°C, we inserted the wild-type tet genes of RK2 into pME145 at a site remote from the trfA ... oriV region. A 5.0-Kb HindIII fragment carrying the tet genes was cloned into the unique HindIII site of pME145; this gave pME490 (Cb,KmS,Tc) (fig. 2). As a control, the same tet fragment was also inserted into pME206, an R P I derivative with a 1.6-Kb de!etion in tetA and tetR (fig. 2), producing pME491 (Cb,KmS,Tc). Table II shows that insertion o f the wild-type tet genes into pME145 restored the temperature sensitivity of the plasmid; pME490 and pME301 were very similar with respect to loss from P. aeruginosa at 43°C. The tet genes inserted into the HindIII site of pME206 did not

P L A S M I D RP1, P. A E R U G I N O S A A N D T E T R A C Y C L I N E

i59

p e r se destabilize plasmid maintenance at 43°C (pME491, see table II). From these observations, we conclude that the tet genes enhance the instability of pME301 at 43°C. Host

range

of pME301.

Ts maintenance of pME301 was observed in two P. aeruginosa strains, P. mendocina, Klebsiella aerogenes and in E. coli at incubation temperatures of 40-43°C (table III). At 30°C, the plasmid was stable without antibiotic selection in all strains (data not shown). In P. stutzeri ATCC11607, no loss of pME301 could be observed at 40°C; unfortunately, this strain could not grow at temperatures above 40°C. In another P. stutzeri strain, JM303 [34], pME301 was not lost at 4 2 ° C ; after transfer back to E. coil, pME301 was

TABLE lIT.

-

-

Stability of pME30!

Strain

i n d i f f e r e n t b a c t e r i a l s p e c i e s at 4 0 - 4 3 ° C .

Selective Km Plasmid stability coefficient concentration in Liquid culture grown at Liquid culture, plated NA (~tg/rnl) 30°C with antibiotic on NA+Km at 30°C, after growth in liquid selection, plated on NA+Km at: without selection (*) at: 40°C

P. aeruginosa PAOI(RP1) PAOI(pME301) PACI(RP1) PACI(pME30!) P. mendocina NCIB10541(RP1) NCIB10541(pME301) P. stutzeri ATCC11607(RPI) ATCC11607(pME301) A. calcoaceticus BD413(RPI) BD413(pME301) K. aerogenes MK53(RP1) MK53(pME301) E. coli ED8654(RP1) ED8654(pME301)

300 300 250 ! 50

43°C

40°C

43°C

0

0

0

0

- 0.3

- 6.0

- 1.6

- 3.6

0 -3.3

0 -3.0

0

10 i0

- 2.0

-

0 0.7

NG NG

25 20

0 0

NG NG

0 0

NG NG

25 25

0 0

NG NG

- 0.7 - 0.3

NG NG

0 < - 7

0 -4.0

NG NG

0 -6.3

0 -1.5

0 -6.0

100 50 25 25

0 0

The plasmid stability coefficient (defined in table II) was also tested on NA + Cb and NA + \ c , with similar results (not shown). (*) Growth in liquid culture for c a . 20 generations. NG = no growth.

160

M. R E L L A A N D COLL.

still Ts (G.J. Stewart, personal communication). Thus, it appears that the maintenance of pME301 is not Ts in P. stutzeri. Acinetobacter calcoaceticus (table III) and Rhizobium meliloti [16] both retained pME301 stably at 40°C but could not be tested at 43 °C for lack of growth.

DISCUSSION In the course of the experiments reported here, true reversion of the Ts mutation in pME301 was not observed. The plasmid had a Ts phenotype in P. aeruginosa and in enteric bacteria. Although the trfA complementation test could be done only in E. coli, we assume that the same defect in trfA is also responsible for the Ts phenotype in P. aeruginosa. This is consistent with the finding that trfA is necessary and sufficient for RK2 replication from oriV in both E. coli and P. aeruginosa [29, 30]. In P. mendocina and K. aerogenes, pME301 was Ts and therefore probably also under ttfA control. In all bacterial species which are able to maintain RP1 and have beep examined in detail, trfA is required for plasmid replication [28, 29, 30]. !-iowever, it has become evident that a particular trfA (Ts) defect need not have the same effect on plasmid maintenance in various bacterial hosts at a given temperature. We speculate that the cellular context, in particular the membrane, may critically affect trfA function. Kornacki and Firshein [20] have demonstrated RK2 replication in vitro by a DNA-membrane complex containing the trfA gene product. A membrane involvement in plasmid replication is further suggested by oar finding that the tet genes modulate the degree of temperature sensitivity of pME301, at least in P. aeruginosa. Other interpretations are also possible. It ha~: recently been shown that the tet gene of pBR322 affects plasmid DNA super coiling in topoisomerase I mutants of E. coli; it has been postulated that the transcription of tet influences pla~mid supercoiling [24a]. Analogous considerations may apply to the replication of pME301. RP1 and RK2 are very stable plasmids; they cannot be cured easily and, to ore' knowledge, spontaneous deletions in the Tc resistance genes have not been observed previously. In our present study, we noticed two modes of ~oss of Tc resistance from P. aeruginosa at 43°C: either the entire pME361 plasmid was eiiiminated or the tet genes of pME301 were inactivated by deletion or point mutation. There are precedents for our observations. The cloning vector pBR322 has an adverse affect on growth of E. coli in both batch and chemo.,~tat cultures. As a consequence, plasmid-free segregants appear within 20 gem-rations [22, 42]. The adverse effect disappears when the tet gene of pBR322 is inactivated by inserti~3n, and hence the plasmid is maintained much more stably [22, 42]. The pSC101 tet gene has also been found unsuitable for the const~'uction of IncQ vector plasmids because such vectors are not stably inherited in Pseudomona~ [2]. It is assumed that the tet proteins may cause membrane alterations that are deleterious to the host or to plasmid replication [2, 22].

P L A S M I D RP1, P. AERUGINOSA A N D T E T R A C Y C L I N E

161

While the mechanism that leads to the elimination of the tet genes of pME301 in P. aeruginosa is unknown, we have no indication for a multicopy effect [23] being involved. Considering the properties of pME490, we favour the hypothesis that the tet genes can exert their adverse effect in trans.

R SUM ; CARACTI~.RISATIOND'UN Dt~RIVI~TI-i[ERMOSENSIBLEtrfA(Ts) DU PLASMIDE RP 1 CHEZ PSEUDOMONASAERUGINOSA La r6plication du plasmide pME301, d6riv6 du plasmide h large spectre d'h6tes RP 1, est thermosensible (Ts) h 43 °C chez Pseudomonas aeruginosa, P. mendocina, Klebsiella aerogenes et Escherichia coli. Cette mutation Ts peut ~tre comp!~ment6e en trans chez E. coli, par un plasmide recombinant portant le g~ne trfA; la fonction de ce g~ne est essentielle pour |a r6piication de RP1 chez E. coli et P. aeruginosa. Puisque le ph6notype "l-s du pME301 s'exprime chez P. mendocina et K. aerogenes, la fonction trfA devrait 6galement ~tre vitale chez ces microorganismes. Lorsqu'une souche de P. aeruginosa PAO croTt ~ temp6rature non permissive~ la s61ection du marqueur plasmidique ae r6sJstance h la carb6nicilline (port6 par lc transposon TnS01) conduit h l'insertion du Tn801 dans le chromosome ou ~t l'obtention de d6riv6s du pME301 off les g6nes tet (d6terminants non transposables de la r6sistance h la t6tracycline) sont inactN6s ~ la suite de d616tions ou de mutations ponctuelles. A partir d'exp6riences de clonage, nous d6duisons que ie(s) produit(s) des g~nes tet d6stabilise(nt) le replicon pME301 chez P. aeruginosa, 40-43 oC. MOTS-CLI~S: Pseudomonas aeruginosa, Plasmide R, T6tracycline; Mutant trfA(Ts), R6sistance, RP1. ACKNOWLEDGEMENTS

We are grateful to Paola Moretti for preliminary experiments and the isolation of pME449, and to ThCmas Leisinger for support. We thank Beecham AG for providing carbenicillin. This work was supported by the SchweizerischeNationalfonds (project 3.620-0.84) and a UK Medical Research Council Project (grant No. G80/374/9CB). REFERENCES

[1] ACHTMAN,M., WILLETTS,N. & CLARK,A.J., Beginning a genetic analys~.s of conjugational transfer determined by the F factor in Escherichia coli by isolation and characterization of transfer-deficient mutants. J. Bact., 1971,106, 529-538. [2] BAGDASARIAN,M. 8/; TIMMIS, K.N., Host:vector systems for gene cloning in Pseudomonas, in < ",P. H . Hofschr~eider & W. Goebel) (pp. 47-67). Springer-Verlag, Berlin, 1982.

162

M. R E L L A A N D COLL.

[3] BENNETT,P.M., GRINSTED,J. & RICHMOND,M.H., Transposition of TnA does not generate deletions. Mol. gen. Genetics, 1977, 154, 205-211. [4] BORCK,K., BEGGS,J.D., BRAMMAR,W.J., HOPKINS,A.S. & MURRAY,N.E., The constrt~ction in vitro of transducing derivatives of phage lambda. MoL gen. Genetics, 1976, 146, 199-207. [5] BtmrARDT,H.-J., RIESS,G. & PtrEHLER,A., Relationship of group P1 plasmids revealed by heteroduplex experiments: RP1, RP4, R68 and RK2 are identical. J. gen. MicrobioL, 1979, 114, 341-348. [6] DANILEVICH,!"~N., STEPANSHIN,Y.G., VOLOZHANTSEV,N.V. & GOLUB, EA., Transposon-mediated insertion of R factor into bacterial chromosome. MoL gen. Genetics, 1978, 161, 337-339. [7] DOUGAN,G., SAUL, i . , TWIGG, A., GILL, R. & SHERRATT,D., Polypeptides expressed in Escherichia coli K-12 minicells by transposition elements Tnl and Tn3. J. Bact., 1979, 138, 48-54. [8] GRINSTED,J., SAUNDERS,J.R., INGRAM,L.C., SYKES,R.B. & RICHMOND,M.H., Properties of an R factor which originated in Pseudomonas aeruginosa 1822. J. Bact., 1972, 110, 529-~37. [9] H.~.s, D., Genetic aspects of biodegradation by pseudomonads. Experientia (Basel) 1983, 39, 1199-1213. [10] HAAS, D. & HOLLOWAY,B.W., R factor variants with enhanced sex factor activity in Pseudomonas aeruginosa. Mol. gen. Genetics, 1976, 144, 243-251. [11] HAAS, D., WATSON,J., KRIEG, R. & LEISINGER,T., Isolation of an Hfr donor of Pseudomonas aeruginosa PAD by insertion of the plasmid RP 1 into the tryptophan synthase gene. MoL gen. Genetics, 1981, 182, 240-244. [12] HAAS,D., CRYZ, S.J. Jr, ITOH, Y., LEISINGER,T., LUETHI,E., MERCENIER,A., REIMMANN, C., RELLA, M., SOLDATI,L., WATSON, J.M. & WRETLIND,B., Some applications of transposon insertion mutagenesis in Pseudomonas, in <>(H. Heslot) (pp. 91-111). Soci6t6 Fran~aise de Microbioiogie, Paris, 1984. [13] HARAYAMA,S., TSUDA, i . & lING, T., High frequency mobilization of the chromosome of Escherichia coli by a mutant of plasmid RP4 temperaturesensitive for maintenance. MoL gen. Genetics, 1980, 180, 47-56. [14] HEDSTROM,R.C., CRtDER, B.P. & EAGON, R.G., Comparison of kinetics of active tetracycline uptake and active tetracycline efflux in sensitive and plasmid RP4-containing Pseudomonasputida. J. Bact., 1982, 152, 255-259. [15] HOLLOWAY,B.W., CROWTHER,C., DEAN, H., HAGEDORN,J., HOLMES, N. & MORGAN, A.F., Integration of plasmids into the Pseudomonas chromosome, in <~Drug resistance in bacteriz'> (S. Mitsuhashi) (pp. 231-242). Japan Scientific Societies Press, Tokyo, 1982. [16] HOOYKAAS, P.J.J., DEN DULK-RAS, H. & SCHILPEROORT, R.A., Phenotypic expression of mutations in a wide-host-range R plasmid in Escherichia coli and Rhizobium meliloti, d. Bact., 1982, 150, 395-397. [17] ITOH,Y. & HAAs, D., Cloning vectors derived from the Pseudomonas plasmid pVS1. Gene, 1985, 36, 27-36. [18] ITOH, Y., WATSON, J . i . , HAAS, D. & LEISINGER,T., Genetic and molecular characterization of the Pseudomonas plasmid pVS1. Plasmid, 1984, 11, 206-220. [19] JEENES, D.J., SOLDATI, L., BAUR, H., WATSON, J . i . , MERCENIER,A., REIMMANN,C., LEIS~GER,T. & HAAS, D., Expression of biosynthetic genes from Pseudomonas aeruginosa and Escherichia coli in the heterologous host. Mol. gen. Genetics, 1986, 203, 421-429. [20] KORNACKI,J.A. & FIRSHEIN, W., Replication of plasm~d RK2 in vitro by a DNA-membrane complex: evidence for initiation of replication and its coupling to transcription and translation. J. Bact., 1986, 167, 319-326. [21] LANKA,E., LoP,Z, R. & FtJERSTE,J.P., Molecular cloning and mapping of SphI restriction fragments of plasmid RP4. Plasmid, 1983, 10, 303-307.

P L A S M I D RP1, P. A E R U G I N O S A A N D T E T R A C Y C L I N E

163

[22] LEE, S.W. & EDLIN, G., Expression of tetracycline resistance in pBR322 derivatives reduces the reproductive fitness of plasmid-containing Escherichia coli. Gene, 1985, 39, 173-180. [231 MOVED,H.S., NGUYEN,T.T. & BERT~ND, K.P., Multicopy TnlO tet plasmids confer sensitivity to induction of tet gene expression. J. Bact., 1983, 155, 549-556. [24] NICAS, T.I. & IGLEWSKI,B.H., Isolation and characterization of transposoninduced mutants of Pseudomonas aeruginosa deficient in production of exoenzyme S. Infect. Immun., 1984, 45, 470-474. [24a] PRUSS,G.J. & DRLICA,K., Topoisomerase I mutants: the gene on pBR322 that encodes resistance to tetracycline affects plasmid DNA supercoiling. Proc. nat. Acad. Sci. (Wash.), 1986, 83, 8952-8956. [25] REIMI~_NN,C. & HAAS, D.~, IS21 insertion in the trfA replication control gene of chromosomally integrated plasmid RP1 : a property of stable Pseudomonas aeruginosa Hfr strains. Mol. gen. Genetics, 1986, 203, 511-519. [26] RELLA, M., MERCENIER,A. & HAAS, D., Transposon insertion mutagenesis of Pseudomonas aeruginosa with a Tn5 derivative: application to physical mapping of the arc gene cluster. Gene, 1985, 33, 293-303. [27] ROBINSON,M.K., BENNEa% P.M., FALKOW,S. & DODD, H.M., Isolation of a temperature-sensitive derivative of RP1. Plasmid, 1980, 3, 343-347. [28] SATO, M., STASr~WlCZ, B.J., PANOPOULOS,N.J., PETERS, S. & HONMA, M., A host-dependent hybrid plasmid suitable as a suicidal carrier for transposable elements. Plasmid, 1981, 5, 325-331. [29] SCHMIDHAUSER, T.J., FILUTOWICZ, M. & HELINSKI, D.R., Replication of derivatives of the broad host range plasmid RK2 in two distantly related bacteria. Plasmid, 1983, 9, 325-330. [30] SCHMIDHAUSER,T.J. • HELINSKI, D.R., Regions of broad-host-range plasmid RK2 involved in replication and stable maintenance in nine species of Gramnegative bacteria. J. Bact., 1985, 164, 446-455. [31] SCHREINER,H.C., BECHHOFER,D.H., POHLMAN,R.F., YOUNG,C-, BORDEN,P.A. & FIGURSKI, D.H., Replication control in promiscucds plasmid RK2: kil and kor functions affect expression of the essential replication gene trfA. J. Bact., 1985, 163, 228-237. [32] SHINGLER,V. & THOMAS,C.M., Analysis of the trfA region of broad-host-range plasmid RK2 by transposon mutagenesis and identification of polypeptide products. J. tool Biol., 1984~ 175, 229-249. [~3] STANISICH,V. & HOLLOWAY, B.W., A mutant sex factor of Pseudomonas aeruginosa. Genet. Res., 1972, 19, 91-108. [34] STEWART,G.J., CARLSON,C.A. & INGRAHAM,J.L., Evidence for an active role of donor cells in natural transformation of Pseudomonas stutzori. J. Bact., 1983, 156, 30-35. [35] STOKES,H.W., MOORE,R.J. & KRISHNAPILLAI,V., Complementation analysis in Pseudomonas aeruginosa of the transfer genes of the wide host range R plasmid R18. Plasmid, 1981, 5, 202-212. [36] THOMAS, C.M., Complementation analysis of replication and maintenance functions of broad host range plasmids RK2 and RP1. Plasmid, 1981, 5, 277-291. [37] THOMAS,C.M., Genetic evidence for the direction of transcription of the trfA gene of broad host range plasmid RK2. J. gen. MicrobioL, 1984, 130, 1641-1650. [38] THOMAS, C.M., MEYER, R. & HELINSKI, D.R., Regions of broad-host-range plasmid RK2 which are essential for replication and main,tenance. J. Bact., 1980, 141, 213-222. [39] THOMAS, C.M. & SMITH, C.A., The trfB region of broad host range plasmid RK2: the nucleotide sequence reveals incC and key regulatoiy gene trfB/korA/korD as overlapping genes. Nucl. Acids Res., 1986, 14, 4453-4470.

164

M. R E L L A A N D C O L L .

[40] THOMAS,C.M., SMITH,C.A., SHINGLER,V., CROSS, M.A., HUSSAIN,A.A.K. & PINKNEY, M., Regulation of replication and maintenance functions of broad-host.range plasmid RK2, in ~ Pla~mids in bacteria~ (D.R. Helinski, S.N. Cohen, D.B. C!ewelI, D.A. Jackson & A. Hollaender) (pp. 261-276). Plenum Press, New Yori¢ 1985. [41] TSODA, M., HARAYAMA, S. & hNO, T., Tn501 insertion mutagenesis in Pseudomonas aeruginosa PAO. _VIOL gen. Genetics, 1984, 196, 494-500. [42] VERNET, T., MCDONALD, I.J., CAMERON, D.R. & VISENTIN, L.P., Stable maintenance in chemostat-grown Escherichia col;, of pBR322 and pACYC 184 by disruption of the tetracycline re.~istance gene. Bioscience Rep., 1985, 5, 29-37. [43] WATERS,S.H., ROGOWSKY,P., GRINSTED,J., ALTENBUCHNER,J. ~ SCHMITT,R., The tetracycline resistance determinants of RP1 and Tn1721 : nucleotide sequence analysis. NucL Acids Res., 1983, 11, 6089-6105. [44] WATSON, J., Replication mutants of the IncP-1 plasmid RP1. Experientia (Basel), 1980, 36, 1451.