Cloning vectors, derived from a naturally occurring ... - Science Direct

Gene, 87 (1990) 145-149 Elsevier

145

GENE 03417

Cloning vectors, derived from a naturally occurring plasmid of Pseudomonas samstanoi, specifically tailored for genetic manipulations in Pseudomonas (Recombinant DNA; plasmid replication; shuttle vectors; Gram-negative bacteria; indoleacetic acid production;/aa~H operon; auxins)

C. Nieto, E. Femindez-Tresguerres, N. Sinchez, M. Vicente and R. Diaz Centre de Invesn'gaciones Biol6gicas (C.S.L C.), 28006 Madrid (Spain)

Receivedby S.R. Kuslmer:18 April 1989 Revised: 25 June 1989 Accepted: 8 August 1989

SUMMARY

A minimal replicon of 1.8 kb isolated from a 10-kb plasmid of Pseudomonas samstanoi, pPS 10, has been used to obtain a collection of small vectors specific for Pseudomonas (P. samstanoi, P. aeruginosa and P. putida). In addition, shuttle vectors that can be established both in Pseudomonas and Escherichia cell have been constructed by adding a pMB9 replicon. The vectors permit cloning of DNA fragments generated by a variety of restriction enzymes using different antibiotic resistance markers for selection and offer the possibility to screen recombinants by insertional inactivation, i ~s cloning system can be used to establish recombinant plasmids in Pseudomonas either at low or high copy number, pPSI0 derivatives are compatible with other Pseudomonas vectors derived from broad-host-range replicons of the incompatibility groups P 1, P4/Q and W. Introduction and expression ofthe iaaMH operon in a P. samstanoi mutant deficient in the preduction ofindoleacetic acid has been achieved using one of these vectors.

INTRODUCTION

The genus Pseudomonas includes a range of soil bacteria with wide metabolic versatility, together with other species

Correspondence to: Dr. R. Dlaz, Centre de lnvestigaciones Biol6gicas, Vel~zquez 144, 28006 Madrid (Spain)Tel. 3412611800, Ext. 278; Fax 34-1-2627518. Abbreviations: A, absorbance; Ap, ampicillin; bp, base pair(s); Cb, carbenicillin; IAA, indoleacetic acid; iaaMH, operon involved in the conversion of tryptophan to IAA; iaaH, gene encoding indoleacetamide hydrolase; iaaM, gene encoding tryptophan 2-monooxygenase; kb, kilobase(s) or 1000 bp; Km, kanamycin; p, plasmid; P., Pseudomonas; Pollk, Klenow (large) fragment ofE. coli DNA polymerase I; R, resistance; Sin, streptomycin; Sp, spectinomycin; Su, sulfonamide; Tc, tetracycline; [ ], denotes plasmid-carrier state. 0378-1119/90/$03.50 © 1990 Elsevier Science Publishers B.V.(Biomedical Division)

pathogenic for animals and plants (Clarke and Richmond, 1975). The ecological and biotechnological relevance of the Pseudomonads has led to the development of vectors for the genetic manipulation of several Pseudomonas species. Most vectors used for these manipulations derive from broad host-range plasmids belonging to incompatibility groups P-I (RK2, RPI, RP4, R68, RI8), P-4/Q (RSFI010, R300B, R1162), and W (pSa) (Bagdasarian and Timmis, 1982; Franklin, 1985; Schmidhauser et al., 1988). These vectors can be established and maintained in Pseudomonas as well as in other Gram- bacteria including E. coil. Pseudomonas syringae pv. savastanoi (P. savastanoi), is a phytopathogenic bacterium able to induce tumors (knots) in the branches of plants such as the olive tree, oleander and other related species. The formation of tumors by P. savastanoi is related to its ability to produce and secrete IAA, an auxin that induces the proliferation of plant cells

146 TABLE I Bacterial strains and plasmids Relevant characteristics

Source/reference

Strains HBI01 PAOI024 PAO1162 KT2440 PS3026a PS93a VlP-I ~ PS2009b

E. coil K-12;hsdS20 (r~, m~ ), recA 13, ara-14, proA2, lacYl,galK2, rpsL, xyl-5 mtl.l,supE44 P. aeruginosa; rrno- , rood+ P. aeruginosa; rmo-, mod +, leu-38 P. putida; rmo - , rood+ P. savastanoi; iaa + P. savastanoi; iaa- [pPSl0] P. samstanoi; iaa + P. samstanoi; iaa + [pIAA]

Boyer and Roulland-Dussoix (1969) K. NordstrOm (Uppsala) Dunn and HoHoway(1971) Bagdasarian and Timmis (1982) E. Alonso (Madrid) C.E.C.T. (Valencia) L. Caflas (Madrid) Kosuge et al. (1985)

Plasmids pBR322 pUC7 pUCl8 pKT231 pCP3 plAA pPSl0 pNSVI pCN38~ pCN39* pCN40~ pCN41© pCN50~ pCN51c pCN60© pCN61©

pMB9 replicon; ApR, TcR pMB9 replicon; ApR pMB9 replicon; ApR RSFI010 replicon; SmR, KinR pMB9 replicon; ApR Isolated from P. samstanoi; PS2009; iaaMH + Isolated from P. savastanoi PS93; cryptic pPSI0 and pMB9 replicons; ApR/CbR, KmR pPSl0 repficon; Km~o pPSl0 replicon; KmR pPSl0 replicon; SmR, KmR pPSI0 repficon; SmR pPS10 and pMB9 replicons; ApR/CbR, KmR, TcR pPSl0 and pMB9 replicons; KmR pPS10 and pMB9 replicons; ApR/CbR, KmR, Smx pPSl0 and pMB9 replicons; ApR/CbR, SmR

Bolivar et al. (1977) Vieira and Messing (1982) Norrander et al. (1983) Bagdasarian et al. (1981) Yamada et al. (1985) (section d) Kosuge et al. (1985) Present work (section a) Present work (section d) Present work (section a) Present work (section b) Present work (section b) Present work (section b) Present work (section ¢) Present work (section ¢) Present work (section e) Present work (section ¢)

a Strains isolated from olive-tree tumors. b Strain isolated from an oleander tumor. c Further information in Fig. 1.

(Comai and Kosuge, 1980; Kosuge et al., 1985). The production of IAA depends on the expression of the i a a H and i a a M genes. These genes are organized in an operon and are plasmid-borne in strains isolated from oleander tumors but seem to be located in the chromosome in isolates from the olive tree (Kosuge et al., 1985; Palm and Kosuge, 1985). The aim of this study was to construct a series of cloning vectors based on the functions of a replicon present in pPS 10, a plasmid naturally found in a strain of/'. s a m s t a n o i isolated from an olive three gall. The vectors described here, while specifically suited for the genetic manipulation of P. samstanoi, can also be established in strains of P. aeruginosa and P. putida. Plasmids containing the pPS l0 and pMB9 replicons, which can be established in E. coil as well as in Pseudomonas, have also been constructed. We report the introduction and expression of genes that code for IAA in an iaa strain ofP. savastanoiusing one of the new vectors.

EXPERIMENTAL AND DISCUSSION

(a) Isolation and preliminary characterization of a minireplicon, pCN38, from a naturally occurring plasmid of Pseudomonas samstanoi

A 10-kb cryptic plasmid, pPS 10, present in P. s a m s t a n o i PS93 (Table I), was digested with P s t l and the two ensuing fragments, 7.6 and 2.4 kb in size, were ligated in vitro to a 4.2-kb P s t l fragment containing a K m R gene, from plasmid pKT231. To avoid incompatibility problems that could interfere with the establishment or maintenance of the recombinants, the ligation mixture was used to transform PS3026, a strain of P. samstanoi different from PS93, the isolate originally containing the p P S l 0 replicon. The obtained Km R transformants contained a plasmid of I 1.8 kb that resulted from the ligation of the 7.6-kb P s t l fragment of p P S l 0 and the 4.2-kb P s t l fragment of pKT231. One of these recombinants was shortened by deletion of non-essential regions: the smallest derivative obtained was a 4. l-kb minireplicon denominated pCN38

147

A

B

pCN38 Pv

Pv

~_T.~ E K

K

pCN39 pCN50 ~

Pv

E

pCN51

K pCN40

pCN41 K

¢'~

pCN60

-

pCN61

Fig. 1. Physical maps ofplasmid vectors containingthe replication functions ofpPS 10. (A) Specific vectors for Pseudomonasderived from pCN38, pCN39 contains part ofthe multicloning site ofpUC7 inserted at the BamHl site ofpCN38 and was obtained in three steps: first a 0.9-kb Pstl fragment from an RI mim'plasmid was introduced at the Pstl site of the pUC7 vector, then the 0.9-kb BamHl fragment of this recombinant was cloned at the BamHl site of pCN38 and finally the internal 0.9-kb Pstl fragment of this recombinant was deleted, pCH40 was constructed substituting the original Pmll-Pstl fragment of pCN39 by the 1.9-kb Pmll-Pstl fragment of pKT231 that includes the SmR gene. To obtain pCN41, pCN40 was digested with BamHl + Hindlll, then the 5' -cohesive ends were ligated alter being made blunt using Pollk. Transformation of Pseudomonaswas as described by Comai and Kosuge (1980). A preincubation of P.putida cells at 42°C for 30 min before starting the competence treatment, increased substantially the yield of transformants (F. Rojo, pets. comman.). (B) Shuttle vectors containing the pPSI0 and pMB9 replicons. To construct pCNS0, the pCN38 and pBR322 plasmids were linearized and then joined at their Pmll sites, pCN51 is a smaller derivative ofpCNS0 that was originated by a 2.6-kb spontaneous deletion. To construct pCN60, the pUCI8 and pCN40 replicons were iinearized and joined at their Kpnl sites. PCN61 derives from pCN60 by an in vitro deletion ofa 0.6-kb Hindlll fragment. The arrows indicate the length, location, and direction oftranscription ofthe antibiotic-resistance genes. Blackened segment, DIqA derived from pPS 10; open and blackened circles, origin ofrepfication ofpPS 10 and pMB9 replicons, respectively. Restriction enzymes: B, BamHl; H, Hindlll; K, Kpnl; P, Pstl; Pv, PmlI; S, Sinai; Sa, Sail; X, Xbal. tc R = TcR.

(Fig. IA) that conferred resistance to Km and retained a 1.8-kb region of pPSI0 that should contain the plasmidreplication functions. Plasmid pCN38 can be established by transformation in restriction-deficient strains ofP. aeruginosa(PAO1024 and PAOl162) and P.putida (KT2440) but not in a similar strain of E. coil (HBI01). pCN38 was found to be compatible with broad-best-range plasmids of the incompatibility groups PI, P4/Q and W, and was maintained at a copy number of 15-20 copies per genome equivalent in P. savastanoi (data not shown).

(b) Pseudomonas-specmc vectors based on_ the pCN38 replieon Plasmid pCN38 can be used as cloning vector as it contains a selectable marker, Km R, and three potential cloning sites that can be used to insert fragments without inactivating either the antibiotic resistance or the replication

functions (Fig. IA). To increase the versatility of the cloning system, the following derivatives of pCN38 were constructed (Fig. IA): pCN39, containing part of the multicloning site of pUC7; pCN41 carrying SmR, instead of KmR. as the selectable marker, and pCN40 containing both KmR and SmR as selectable markers, allowing the use of insertional inactivation.

(c) Psadomonas and Esckerickia coil shuttle vectors containing the pPSIO and pMB9 replicons Plasmids that could replicate both in Pseudomonas and E. coil were constructed (Fig. IB). Two of them, pCN50 and pCN51, were derived from the pCN38 and pBR322 vectors, pCN50 contains three selectable markers (ApR, KmR and TcR). Insertional inactivation can be used to screen for recombinants at some unique cloning sites ofthis vector, pCN51, a smaller derivative of pCN50, contains KmR as the only selectable marker. Similar shuttle vectors,

148

peN60 and peN61 based on the pUCl8 and peN40 vectors, were also constructed, peN60 contains three selectable markers (ApR, KmR and SmR) and makes it possible to use insertional inactivation to screen for recombinants containing inserts at some of the cloning sites, pCN61 was derived from pCN60 and contains two of its three antibioticresistance markers (ApR and SmR). The deletion ofa 0.6-kb H/ndIIl fragment of pCNt0 that originated pCN61, removed 0.2 kb to the left of the KpnI site in the pPS10 repficon. The copy number of pCN61 in Pseudomonas is two to three per genome equivalent (data not shown). The stability of pCN61, compared to that of the parental miniplasmid pCN40, was reduced. Plasmid stability was measured as the % of SmR colonies after propagation ofthe cells in solid medium and in the absence of selection, using the procedure described by NordstrOm (1980). After 90 generations of growth without selection, over 99% of the population derived from pCN40-containing cells were SmR, whereas the proportion was only 80~o for the population derived from pCN61-containing cells. It is concluded that a 0.2-kb fragment of the pPS 10 replicon located to the left of the Kpnl site, is not strictly required for replication of the miniderivatives but increases the stability of the shuttle vectors. Therefore, pCN61 allows to establish the recombinants in Pseudomonas at a relative low copy number and also, if required, to cure the host from these recombinants. The shuttle vectors contain in addition the mobilizable site of pMB9 and can therefore be mobilized if suitable functions are provided in trans.

(d) Use of the pPSl0-derived vectors: establishment of genes needed for the production of IAA in a strain of Pseudomonas samstanoi pPC3 is a recombinant plasmid containing the iaaMH operon, that derives from the E. coii vector pBR328 (Yamada et al., 1985). The natural promoter of the iaaMH operon is not active in E. coli, but strains of E. coil containing pCP3 express these genes and produce IAA, probably due to read-through transcription, from the cat promoter of the vector (Yamada et al., 1985; Palm et al., 1989). To be able to establish the iaaMH operon in P. savastanoi we constructed pNSVI, a plasmid in which pCN38 and pCP3 were fused at their PvuII sites. This fusion created a discontinuity between the cat promoter and the iaaMH operon and resulted in an laa- phenotype in E. coli (Table II). To avoid DNA degradation by restriction systems, we first introduced pNSVI by transformation into P. aeruginosa pAO1024, a R - M + strain. Plasmid pNSV1 DNA was isolated from this background and transformed into an l a a - strain of P. savastanoi (PS93), selecting for KmR transformants. A transformant of PS93 containing pNSVI, recovered the ability to produce IAA (Table If).

TABLE II Levels of IAA production in strains of Escherichia coil and Pseudomonas savastanoi

Host a

Plasmid a

IAA productionb

E. coil K-12 HBI01 E. coil K-12 HBI01 E. coil K-12 HBI01 P. savastanoi VIP-1 P. savastanoi PS93 P. savastanoi PS93 P. savastanoi PS2009

-pCP3 pNSVI m m pNSVI pIAA