FEMS Microbiology Letters 115 (1994) 191-196 © 1994 Federation of European Microbiological Societies 0378-1097/94/$07.00 Published by Elsevier

191

FEMSLE 05768

Transposon Tn5 mutagenesis fluorescens to isolate mutants in antibacterial activity

of Pseudomonas deficient

Narayanan Rajendran a,d, Dieter Jahn b,c, Kunthala Jayaraman a and Mohamed A. Marahiel .,1

a Biochemie/Fachbereich Chemie, Philipps-Universitiit Marburg, D-35032 Marburg, FRG, b Laboratorium fiir Mikrobiologie im Fachbereich Biologie, Phih'pps-Universitiit Marburg, Karl-von-Frisch-Strasse, D-35043 Marburg/Lahn, FRG, c Max-Planck-Institut fiir Terrestische Mikrobiologie, Karl-von-Frisch-Strasse, D-35043 Marburg/Lahn, FRG, and d Centre for Biotechnology, Anna University, Madras 600 025, India

(Received 23 September 1993; revision received and accepted 29 October 1993)

Abstract: Pseudomonas fluorescens was subjected to insertion mutagenesis studies using the transposon Tn5-GM to generate mutants deficient in antibacterial activity minus mutants. The transposon located on the temperature-sensitive plasmid pCHR84 was conjugally transferred into the non-pathogenic pseudomonad using the triparental mating procedure. Random integration of Tn5-GM into the chromosome of P. fluorescens was achieved by heat treatment of the transformed cells at 42°C. Approximately 2% of transconjugants revealed an auxotrophic phenotype indicating efficient integration of the employed transposon into the chromosome of P. fluorescens. One transposon insertion mutant was obtained showing an antibacterial activity minus phenotype. This mutant (MM-7) was found to be defective in the production of an unidentified antibacterial compound against B. subtilis. These results introduce Tn5 transposon mutagenesis as a new useful tool for the molecular analysis of P. fluorescens.

Key words: Pseudomonas fluorescens; Transposon; Tn5-GM; Antibacterial deficient mutant

Introduction

Pseudomonas f luorescens is a non-pa thogenic ,

Gram-nega t ive , rod shaped, polar f lagel la ted soil

bac te r ium [1]. In agr icul ture it plays a benef ic ia l

role in the p ro tec t ion of crop plants against dele-

* Corresponding author. Tel: (06421) 285722; Fax: (06421) 282191.

terious pathogenic microorganisms, particularly fungi [2]. A major attraction of the application of P. fluorescens in the medical field is its antibacte- rial ability. The organism produces a number of antibioticaUy active substances even against highly antibiotic-resistant strains of the human pathogen Pseudomonas aeruginosa [3].

In the rh izosphere , P. f luorescens is able to adapt to var ious ex t reme soil env i ronmenta l con- ditions. They usually live in the hosti le rhizo-

sphere env i ronment , in which the p lant p roduces

SSDI 0378-1097(93)E0460-T

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various toxic factors such as phenoilics and acti- vated oxygen-containing radicals. Moreover, sev- eral predators such as bacteriophages, bdellovib- rios and protozoa are present [2,3]. In order to survive in this unfavourable environment, P. fluo- rescens synthesises various antibiotics. They in- clude phenayine 1-carboxylic acid [4], pyrolnitrin, pyoluteorin [5], sperabillin [6] and an unidentified antibiotic [7]. Among these, sperabillin has spe- cial interest due to its economic uses. Sperabillin, a pseudo-peptide antibiotic is composed of 2,4- hexadienoic acid, 3,6-diamino-5-hydroxy hey- anoic(-heptanoic) acid and 2-amino ethanomidine in binding with amide bonds [8].

In recent years there has been a number of reports indicating the potential use of fluorescent Pseudomonads as a solution to the overuse of potentially harmful pesticides and fungicides [9,10]. The essential factors for biocontrol are the colonization of the rhizosphere and the produc- tion of antibiotics [11].

To understand the genetic basis of the antago- nism of P. fluorescens, a mutagenesis technique, using the transposon Tn5-GM, was carried out to isolate mutants deficent in antibacterial acitivity. This paper deals for the first time with the use of the transposon T n 5 - G M as a mutagen for P. fluorescens.

Materials and Methods

Media, strains and plasmids The type strain of P. fluorescens, possessing

antibacterial activity, used throughout this study was described previously [12]. The employed transposon T n 5 - G M resides on the broad-host range plasmid R388ts carrying a thermosensitive origin of replication, designated as pCHR84. The vector contained a gentamycin-resistant marker on the transposon and a t r imethoprim marker on the thermosensitive plasmid part. This transpo- son delivery system was obtained from Dr. C. Sasakawa, Institute of Medical Science, Univer- sity of Tokyo, through Dr. J. Reiser, ETH, Switzerland [13].

Strains, plasmids and their genotypes are listed in Table 1. The donor E. coli MC1061 harboring

Table 1

Bacterial strains and plasmids

Strain or plasmid Genotype or description Refer- ence

Pseudomonas fluorescens Parent recipient 12

Bacillus subtilis JH642 trpC2 pheAl 23 MI100 trpC2 pheAl Sigb::cat 23

Escherichia coli H B 1 0 1 SupE44hsdS20(r B- m B- )

recA13 ara-14 ProA21acY1 gal K2 rps L

20 xyl.5rntl-1 14 MC1061 pla.pCHR84,388::Tn 5-GM 13

Plasmids pKT240 repR300B APt kl mr(12"9k°) 25 pCH R71 re 13. R388ts Tn 5.Tpr 13 pCHR81 reP.R388ts, TnS"TPrKmr 13 pCHR82 reP.R388ts,Tn5-Tcl'TprTcr 13 pCHR84 rep. R388ts,Tn5'Tpr Gmr 13

pCHR84 (R388ts : :TN5-GM)and E. coli HB101 carrying the mobilizing plasmid pKT240 were maintained on Luria-Bertani (LB) agar [14], con- taining antibiotic gentamycin (12.5 /zg /ml) or kanamycin (50 ~ g / m l ) respectively to which they provide resistance. The recipient P. fluorescens strain was maintained on LB agar. Medium selec- tion for transconjugants of P. fluorescens contain- ing the plasmid R388ts: :Tn5-GM was carried out on Pseudomonas Isolation Agar (PIA, Difco Lab) containing gentamycin (100 /zg/ml) . Auxotrophs were screened on M9 Minimal medium [14] con- taining gentamycin (100/zg /ml) .

Chromosomal DNA and plasmid preparations The two commonly used protocols for bacterial

genomic D N A extraction using enzyme detergent lysis and an organic solvent t reatment [15] and using Guanidine Isothiocyanate with CsCI [16] are powerful but rather complex and time-con- suming. Therefore, a simple and rapid method for the extraction of genomic D N A from Gram- negative bacteria, recently published [17] was em- ployed for the first time for P. fluorescens, in this study.

For this purpose a saturated culture of 1.5 ml was harvested by centrifugation for 30 min at

12 000 rpm. The cell pellet was resuspended and lysed in 200 jzl of lysis buffer (40 mM Tris-acetate (pH 7.8), 20 mM sodium-acetate, 1 mM EDTA, I%SDS) by vigorous pipetting. In order to re- move most proteins and cell debris, 66 tzl of 5 M NaC1 solution was added and mixed well. The viscous mixture was then centrifuged at 12000 rpm for 10 min at 4°C. After transferring the clear supernatant into a new vial, an equal vol- ume of chloroform was added and gently mixed. The milky solution was centrifuged at 12 000 rpm for 3 min. The supernatant was mixed with 2 vol of chilled 100% EtOH and centrifuged at 12 000 rpm. The pellet was washed with 70% E tOH and air-dried.

Plasmids were prepared according to manufac- turer protocols, using the Qiagen plasmid prepa- ration Kit (Qiagen, USA).

Triparental mating procedures Triparental broth matings (TBM) were per-

formed by using a modification of the original technique described earlier [18,19]. A 2 ml cul- ture of E. coli carrying pCHR84 and a 2 ml culture of E. coli harboring the mobilizing plas- mid pKT240 were grown separately at 30°C and 37°C, respectively, for 16 h at 280 rpm in liquid LB with appropriate antibiotics. A 3 ml culture of the recipient P. fluorescens was grown at 30°C, at 300 rpm in LB broth containing rifampicin (34 /zg/ml) for 18 h. All the three cultures were pooled and incubation continued at 30°C for 24 h. In order to induce transposition of the trans- duced transposon residing on the pCHR84 vec- tor, grown cultures were transferred to 42°C for 48 h at 300 rpm. Since the parental P. fluorescens strain was sensitive to gentamycin, transconju- gants were purified on selective PIA plates with gentamycin. Purified single colonies were used for further studies.

Bioassay studies The mutated cells carrying the transposon on

their chromosome were tested for antibacterial activity as described by Kamdar [20], with out- lined modifications. The LB agar plates contain- ing a lawn of indicator cells of B. subtilis were precultured for 6 hours at 37°C. The colonies to

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be tested were spotted onto sterile Whattman paper discs along with control colonies of the parent P. fluorescens. Plates were incubated at 30°C for 18 h and transferred to 37°C for 16 h. These plates were then overlaid with a thin layer of bacto-agar containing the indicator strain in order to ensure that the observed inhibition zones around parental colonies were due to the antibac- terial ability. Control plates were also used sepa- rately with and without mutant strains.

Results and Discussion

Several transposon delivery systems were tested in this studies (listed in Table 1) for the mutagen- esis of P. fluorescens. The most effective and workable transposon donor was pCHR84, carry- ing a thermosensitive R388 replicon and gen- tamycin and trimethoprim markers. We used this Tn5-GM transposon system throughout this study to mutagenize P. fluorescens.

Transfer of transposon vector pCHR84from E. coli to P. fluorescens by the triparental broth mating procedure

The donor plasmid containing Tn5-GM trans- poson was temperature-sensitive [13] and not self-transmissible at 30°C from E. coli to P. fluo- rescens without a helper strain. In order to mobi- lize the transposon plasmid from E. coli to Pseu- domonas an E. coli harboring the mobilizing plasmid pKT240 was used. During triparental mating, this mixture was kept at 30°C to enable the Tn5-plasmid to enter the recipient cells.

We screened the transconjugants from the donor and mobilizer by using PIA plates with gentamycin on which only transformed P. fluo- rescens is capable of growing. The selective agar medium reduces the bacterial competition and increases the recovery of the transconjugants and the gentamycin-resistance determinant of Tn5- GM was a useful selective marker in Pseu- domonas. After conjugation with P. fluorescens as the recipient, Tn5-GM transfer from E. coli donor was observed at a frequency of approximately 7 × 10 -8 per donor. Earlier study using Tn5-259 gave the transconjugants frequency of approxi-

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mately 10 -6 [21]. Using the described method 4790 transconjugants of P. fluorescens carrying pCHR84 were recovered.

Transposon mutagenesis using a Tn5 derivative, yieM 2% auxotrophy

After the entry into the cell, the transposon was integrated into the chromosomal DNA of P. fluorescens at 42°C only and not at 30°C or 37°C. Triparental mixture kept at 42°C instead of 30°C gave a negative result. This was presumably be- cause the thermosensitive plasmid pCHR84 may already have lost its transposon into the donor strain of E. coli itself rather than integration into the chromosomal DNA of P. fluorescens. The obtained transconjugants were replicated after heat treatment at 42°C for transposon mobiliza- tion onto M-9 minimal medium agar plates con- taining gentamycin and incubated at 30°C for 48 h. Colonies that failed to grow on minimal agar plates were considered putative auxotrophs. Among 4790 transconjugants, 95 colonies were identified as auxotrophs. This relatively high number of auxotrophs indicated the sucessful in- tegration mutagenesis via the Tn5 transposon and makes the employed system a useful tool for the molecular analysis of P. fluorescens. Never- theless, since the auxotrophs were not further characterized, possible hot spot of integration cannot be excluded.

Isolation of a mutant (MM-7) of P. fluorescens, deficient in antibacterial activity by transposon mu- tagenesis

A number of reports [22-24] were available on the isolation of mutant bacterial cells that were impaired in the synthesis or secretion, or releas- ing of extracellular and cell-bound secondary products. In order to obtain a P. fluorescens mutant losing its antibacterial ability, we screened the obtained transconjugants for their ability to restrict the growth of B. subtilis. The employed method is outlined in the Materials and Methods section. One antibacterial deficient mutant, des- ignated as MM-7, was isolated using the growth inhibition petri plate assay technique. The physi- cal presence of Tn5-GM intergration into the

genomic DNA of this mutant was confirmed by Southern hybridization (data not shown).

Initial characterization of the MM-7 mutant For the initial characterization of the obtained

mutant, culture cells of mutants MM-7, and par- ent P. fluorescens were directly spotted (20 ml per Whattman paper disc) on the lawn of the indicator strain B. subtilis. The plate was incu- bated at 30°C. The zone of inhibition of B. sub- tilis growth was observed only around P. fluo- rescens and not around mutant MM-7, even after 48 h (Fig. 1). All other transconjugants tested on the bacterial lawn produced a zone of inhibition on the B. subtilis lawn roughly equivalent to those of the parental P. fluorescens.

The mutant MM-7, capable of growing on min- imal medium, was found to be defective in the production of an unidentified antibacterial com- pound (Table 2). According to Hida [8] and Katayama [6], sperabillin (a new antibiotic ob- tained from P. fluorescens) showed antibacterial activity against B. subtilis. In order to determine the deficiency of antibacterial activity in mutant MM-7, whether it is due to the loss of synthesis of an antibiotic compound or due to some other secondary metabolites, another growth inhibition study was carried out. The cell pellets of the mutant MM-7 and control parental strain P. fluo- rescens (dissolved in minimum volume of sterile double distilled water) as well as the supernatant of the same cell culture were used in this assay.

i Fig. 1. Growth inhibition of B. subtilis using Petri plate assay techniques. 20 /xl of grown cell cultures of parent Pseu- domonas fluorescens (A) and mutant MM-7 (B) were spotted on sterile Whattman paper on the lawn with the indicator strain of B. subtilis. The plate was incubated for 24 h, at 30°C.

Table 2

Properties of the mutant and parental strains

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Phenotypes Mutant Other Trans- Parental strains MM-7 conjugants

Recipient Donor Mobilizer

Inhibition i D LP + + + - - Markers 2 a) Rifampicin R R R S S b) Kanamycin S S S S R c) Gentamycin R R S R S Growth parameters A: Temperature a) at 30°C + + + + + + + + + + + + + b) at 37°C + + + + + + + + + + + B: Optimum pH 7-8 7-8 7-8 6-8 5-8 C: Type of medium a) PIA + + + + + + + + + - - b)LB + + + + + + + + + + + + + + + c )2×YT + + + + + + + + + + + + + +

1 Antibacterial activities of mutant MM-7, other transconjugants and the parent Pseudomonas fluorescens, were tested on the lawn of indicator strain of Bacillus subtilis (a) JH642 and (b) MH1.

2 Antibiotics used in this study, for the selection of phenotypes of transconjugants, parental strains P. fluorescens and E. coli containing donor and mobilizer plasmids, in which their growth was restricted or not. D, Deficient; LP, Like Parent P. fluorescens; R, Resistance; S, Sensitive; + + +, good/high; + +, little/low; +, poor; - , nil.

T h i s tes t was c a r r i e d o u t as d o n e b e f o r e wi th

c u l t u r e cells. In b o t h cases , m u t a n t M M - 7 d id n o t s h o w any a n t i b a c t e r i a l activity. T h i s sugges t s t ha t

t h e de f i c i ency o f a n t i b a c t e r i a l ac t iv i ty in M M - 7 is

d u e to an i m p a i r e d syn thes i s o f an u n i d e n t i f i e d

c o m p o u n d wi th a n t i b a c t e r i a l activity.

T h e s e resu l t s r evea l t he p o t e n t i a l use o f T n 5 -

G M as a m u t a g e n in P. f luorescens. T h e o b t a i n e d

m u t a n t will be f u r t h e r c h a r a c t e r i z e d fo r t h e phys-

io log ica l ro le (s ) in a n t i b a c t e r i a l ac t iv i ty aga ins t B.

subtilis s t ra in . C o m p l e m e n t a t i o n e x p e r i m e n t s

s h o u l d p r o v i d e access to t h e i nvo lved genes . F u r -

t h e r s tud ie s o n the c h a r a c t e r i z a t i o n o f t h e m u t a n t a r e c u r r e n t l y u n d e r w a y in o u r l abora to ry .

Acknowledgements

W e t h a n k Dr . C. Sasakawa , I n s t i t u t e o f M e d i - cal Sc i ence , U n i v e r s i t y o f Tokyo , a n d Dr . J. R e i s -

er , E T H , S w i t z e r l a n d fo r T n 5 - p l a s m i d . N . R . g r a t e fu l l y a c k n o w l e d g e s t h e U G C ( U n i v e r s i t y

G r a n t s C o m m i s s i o n , Ind i a ) a n d D A A D ( D e u t -

s che r A k a d e m i s c h e r A u s t a u s c h d i e n s t , F R G ) fo r a fe l lowship . Th i s w o r k is pa r t i a l ly s u p p o r t e d by

D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t , and F o n d d e r C h e m i s c h e n Indus t r i e , F R G .

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