FEMS Microbiology Letter~ 72 (1990) 177-182 177 Published by Elsevier

FEMSLE 04195

Syringomicin production is stimulated by cysteine in Pseudomonas syringae pv. syringae

Xavier Garr iga, Maria Cas taho and Jordi Barbd

Department of Genetics and Mwrob:oloKv. Faculty of Sczence~ A ulonomott~ Umt~rsitv o/ Barcelona. Bellalerro~ Bareelon~ Spain

Received 16 May 1990 Revision received 20 June 1990

A~:cepled 25 June 19qO

Key words: Syringomyciu production; Pseudomonas svringae pv. svringae; Cysteine mutant strain" Wild type strain

1. SUMMARY 2. INTRODUCTION

The influence of cysteine and serine in the production of syriugomycin by P£eudomonas syringae pv. syringae has been studied. Both amino acids increased toxin synthesis in wild-type strains, although cysteine has a higher stimulatory effect than seriue. To corroborate the role of cysteine in the production of syringomycin, a Cys- mutant of P. syringae pv. syringae was isolated by transposi- tioual mutagenesis with Tn5; this Cys- mutant did not produce syringomycin. Nevertheless, and after the addition of high concentrations of cy- steine, the cys: :Tn5 mutant recovered its ability to produce syringomycio. On the other hand, the addition of serine did not return the production of syringomycin to the cys::Tn5 strain; all these data indicate that cysteine modulates the synthesis of syringomycin in P. syrmgae pv. syringae posi- tively.

Correspondence to: J. Barb~, Dpt. Genetics and Microbiology. Faculty of Sciences. Autonomous Univ,:rsity of Barcelona. Benat©rra. 08193-Barcelona, Spain.

Many plant pathogenic strains of Pseudomonas syringae pv. s)'r:ngae produce a low-molecular- mass peptide toxin, known as syringomycin, which appears to play a role in the development of diseases caused by this organism [1]. This toxin is a broad-spectrum biocide affecting a variety of plant, fungal and bacterial cells, but sensitivity can vary widely [2]. in plants, it may induce necrosis, chlorosis and water-soaking of tissue [3]. Syringomycin production contributes significantly to virulence in P. ~yringae pv. syringae [4] and is associated with isolates from a wide variety of hosts [5]. On the other hand, a structurally related phytotoxin syringotoxin - has been shown to be produced only by citrus strains [6].

Syringomycin contains a positively-charged peptide moiety carrying arginine, phenylalanine, serine and diaminobutyric acid in a 1 : 1 : 2 : 2 molar ratio [7], although its precise structure is unknown. The biosynthetic pathway to syrin- gomycin has not been elucidated, despite this compound resembling many bacterial secondary metabolites in the mode in which its synthesis is regulated [8], and by its structural similarity to the

0378-1097/90/$03.50 © 1990 Federation of European Microbiological So~i¢Ues

178

peptide class of antibiotics [9]. It has been sug- gested that the biosynthesis of syringomyein is probably directed by polyenzymes which would be very close to those that are involved in the synthe- sis of peptide antibiotics [9]. Furthermore, assem- bly of syringnmycin would proceed via nonribo- somal synthesis on multienzyme protein surfaces, designated as peptide synthetases [9].

Syringomycin synthesis seems to be regulated by nutritional and environmental factors in a manner similar to that of other secondary metabolites produced by Pseudomonas aeruginosa and related bacteria [8]. Thus, it has been proved that syringomycin production is stimulated by some factors such as glucose and casamino acids [5], whereas this production is depressed by others such as peptone, phosphate [2] and aeration [5]. Nevertheless, the effect of serine as well as cy- steine on the syringomycin biosynthesis has not been analyzed so far, to our knowledge, despite the fact that it is known that the synthesis of phaseolotoxin [10] is stimulated by cysteine and that Cys- mutants of P. syringae show a lower production of syringotoxin [11]. For this reason, we have studied the influence of cysteine and serine on the production of syringnmycin by P, syringae pv, syringae.

3. MATERIALS AND METHODS

3.1. Bacterial strains, plasmids and growth condi- tions

The Escherichia coil K-12 strain used in this work was SM10 (thi thr leu reeA Muc+ chro- mosomally integrated RP4-2-Tc :: Mu) containing plasmid pSUP1011 carrying Tn5 transposon and mob region of RP4 plasmid 112]. The P. syringae pv. syringae UAlI029 strain was isolated from a diseased pear flower (Pyrus communis L.) in Spain, Strain UAll030 was obtained by selection for spontaneous resistance to rifampin (1(30 pgJml), UAll030 was indistinguishable from UAI1029 in ability to produce syringomycin on potato dex- trose agar. Geotrichum candidam F-260 was used in bioassays for syringomycin production [5],

Strains of P, syringae pv. syringae were routinely cultured on King medium B (KB) [13]

supplemented with glucose at 0.2%, potato dex- trose broth (PDB) and potato dextrose agar (PDA) or Ng minimal medium at 30*C 114]. E. coil strains were grown at 37°C in LB [15] broth supplemented with kanamycin (50 pg /ml ) and chloramphenicol (12.5 /~g/ml). G. candidum was grown in nutrient broth-yeast extract at 30°C.

3.2. Transposon mutagenesis and auxotroph identifi- cation

Tn5 transposon, which codes for kanamycin resistance, was transferred into P. syringae pv. syringae by using the plasmid vector system of pSUPI011 [12]. The E. coli donor strain SMI0 (pSUP1011) and the rifampin-resislance P. syrin- gae were mated on membrane filters on KB agar for 6 h at 30°C as described previously [12]. After ii',cubation, kanamycin-resistant P. syringae trans- conjugants were selected on KB agar plus rifam- pin and kanamycin. These transconjugants were transferred to Ng minimal medium plus kanamy- cin, and auxotrophs were identified after 36 h of incubation, Lea- , Pro-, Lys- and Cys- mutants were characterized and isolated by the nutritional pool system of Davis et al. 116].

3.3. Syringomycin production test The method used to test production of syrin-

gomycin was the bioassay of Gross and DeVay [5]. P, syringae strains were grown overnight on KB medium, and 10/xl of a cell suspension containing 3 × 10 q c fu /ml were then inoculated on PDA plates supplemented with casa amino acids at 0,5% and incubated for 4 days at 23*C. After- wards, the plates were lightly oversprayed with a spore suspension of G, eandfdum, and zones of fungal growth inhibition were measured and re- corded after 16 h, When required, either cysteine or serine was added to the casa amino acids-supp- lemented PDA plates at the desired concentra- tions.

3,4. DNA techniques Plasmid and genomic DNA isolations, restric-

tion endonuclease digestion, southern transfer of DNA fragments and DNA-DNA hybridization were carried out as described peviously [17].

4. R E S U L T S

Fig. I shows that cysteine and sefine increased the production of syringomycin by P. syringae pv. syringae U A I I 0 3 0 , although the st imulatory effect of the cysteine was higher than originated by the serine. Likewise, the synthesis of syringomycin was stimulated by cysteine at lower concentrations that those of sefine (Fig. 1). However, there was a

l inear correlation between toxin-synthesis stimula- tion and amino acid concentration up to about 20 /xg/ml of either cysteine or serine (Fig. I).

A Cys- mutant of P. syringae U A I I 0 3 0 strain was isolated by transpositional mutagenesis with the Tn5 transposon to corroborate the effect of cysteine on the syringomycin production. Follow- ing this. the ability to produce syringomycin of this Cys - mutant was tested. Data obtained (Fig. 2) showed that the e y s : : T n 5 mutant was unable to synthesize this toxin, A Southern blot study was performed to analyze the possibility that there were double Tn5 insertions in the genome of the Cys- mutant. The genomic D N A of the Cys- mutant was digested with EcoRI because Tn5 does not contain a restriction site for this enzyme, and a H i n d l l l fragment of 3.3 kb of Tn5 contain- ing the central region of this transposon, as well as about 400 bp of each one of the two copies of the

"5

6 2'0' io' 6o' do ~oo Amlnoaod ConcentrQt~n ( H g l m t )

Fig, I, E f f~ t uf cysteine (n) and serine (a,) on the production of syrin~om~n by wild-type cells of P. syrmgae pv, s)Tmgae UAII030. ]'he siz~ of the growth inhibition zone of G. (~nd¢- dum for each concentrmion of either cysteine or senne is plotted against the amino acid concentration. All values are the averages of four independent euhures, with error bats repre-

s~nting standard deviations.

179

Fig. 2. G. candJdum bioassay for testing Ihe production of ~yringomycin by the x~ild-type strain (A.C) and the cys::Tn5 mutant (B,D) of P. svringae pv. srringae UAII030 in the absence {A,B) and in the presence IC.D) of eystein© at 75 /t g/ml in ¢asa amino acids-supplemented PDA plates. Bacterial cultures were grown on PDA for 4 days at 23°C and after- wards ovcrspraycd with the fungus to observe growth inhibi-

tion zones.

IS50 of Tn5 118], was used as a D N A probe. Results obtained indicated that there was only a single Tn5 insertion in the Cys - mutant located in an g.2 kb E c o R l fragment (Fig. 3). Furthermore, the T o x - behavior of the Cys- mutant cannot be explained by a second mutat ion produced by a ISS0 from Tn5 that, independently of the transpo- son, was inserted in the genome, because, as cited above, the D N A probe used in the Southe.'m. b!ot

180

contained two fragments of the IS50 sequence [18]. Afterwards, the effect of cysteine and serine addition over the production of syringomycin by the Cys- mutant was studied. Fig. 4 shows that concentrat~.ons of cysteine higher than 15 ,~g/ml increased toxin synthesis in the Cys mutant, which produced practically the same amount of toxin as the wild-type strain in the same condi- tions (Fig. 1). On the other hand, serine did not produce any effect on the syringomycin synthesis in the Cys- mutant (Fig. 4), as expected if cy- steine was really the stimulating factor of toxin production, because serlne is the precursor of cy- steine synthesis. Moreover, the eysteine-mediated induction of syringomycin production cannot be attributed to an unspecific effect of this amino acid upon the growth of the Cys- mutant since

A B C

8 .2 . .~

3.3.,.--

Fig, 3, Southern blot analysis of cys::Tn5 muto,:a of P, o'rin. gae pv, syrmgae UAII030. Total genomie DNA was cleaved with EcoRI. The DNA fragments were th¢~ separarcd by agaros¢ gel ¢lcctrophorcsis, transferred to nit/o0¢llnlose tncm- brant~ and assayed for byblidizatton witl- the 3,3-Hindll[ fragmtmt of "ins ~ the probe, (A) DNA .~rom the ¢ys::TnS mutant; (B) DNA from the wild-type strain as negative con- trot; (C) 3.3-1tindlll fragment of Tn5 as positive control, Size

standards in kb arc indicated on the left,

g 6

"6 ~2

"6 o

~3 0 20 40 60 Bo ~00 Arninouc,d ConcentrotK~n (pglrnl)

Fig. 4. Effect of cysteine (Ill and serine (,x) on the production of synngomycin by the c~'5::Tn5 mutant of P. syringae pv. syrmgae UA11030. Tn¢ size of the growth inhibition zone of 6:. candidum for each concentration of either cystcinc or ~rinn is plotted against the amino acid concentration. All values are the averages or four independent cultures, with error bars repre-

senting standard deviations.

the addition of cysteine did not modify the dou- bling time of the Cys- mutant growing in the medium used in the syringomycin-production test (data not shown). This is because it contains a sufficient quantity of amino acids to support a normal growth of any auxotrophic mutant. Like- wise, the addition of other amino acids - such as leucine, proline or lysine - did not affect sydn- gomycin production of either wild type or Cys- strains (data not shown). Finally, and in agree- ment with the above, auxotrophic mutants for amino acids such as leucine, proline or lysine showed the same pattern of syringomycin synthe- sis as the wild type strain (data not shown).

5. DISCUSSION

Results presented in this paper show that cy- steine and serinc stimulate the production of syringomycln in wild-type strains of P. syringae

pv. syringae, whereas in Cys- mutants only cy- steine has a stimulatory effect. Nevertheless, cy- steine is not present in the molecule of syrin- gomycin, but serine is [7], The positive effect of cysteine and serine on syringomycin production could be attributed to: (i) an increase of the intracellular pool of serin¢ which could be derived

for the toxin synthesis, (if) a regulatory e"fect of the serine, and (iii) a regulatory effect of cysteine. Our data showing that serine does not increase syrlngomycin production in the cys:: Tn5 mutant clearly indicate that possibilities (i) and (if) are not responsible for the eysteine- and seriae-mediated stimulation of syringomyein production. Thus, cy- steine must directly increase syrlngomycin synthe- sis. In bacterial cells, cysteine is mainly produced from serine by the action of cystelne synthetase. in this respect, the effect of sefine in the syrin- gomycin production may be due to the increase of the inlracelhilar pool of cysteine originated by its addi t ion rather than to the act ion of itself. The increase of syringomycin production by cyst¢in¢ could be explained by (i) cysteine being a donor of some group to lhe toxin or by (ii) cysteine being a positive effector of some of the enzymes which are involved in the production of' syringomycin. It has been shown that the syringomycin-production pat- tern is typical of so-called secondary mctabolites because it normally occurs during postexponential growth [8,19]. Cysteine could therefore be the sig- nal that triggers the incorporat ion of serine to the synthesis of syringomycin when there is a high conocntration of this last amino acid. However, further work is necessary to clarify tiffs aspect.

Finally, it is also worth noting that production of other phytopathogen toxins from several Pseu- domonas such as syringotoxin and phas¢olotoxln is also related to cystelne [tO, I l l , despite this amino acid not being present in either of these two toxins [7,20], This fact suggests that perhaps a similar cysteine-degendent regulatory pathway of toxin synthesis in the phytopathogen strains of the Pset~lomonas genus may exist.

A C K N O W L E D G E M E N T S

Wc wonld like to thank M,A. Calve, J. Engwall and E. Momesinos for their generous gifts of several strains, and J .M. Cuartero for drawing the figures. We acknowledge the help of the Comisi6

ln terdepar tamenta l de Reccrea i Innovacifi tec- nol6glea of the General i ta t de Catalunya for a grant to carry out this work. X.G. was supported by a predoctora] fellowship from the Autonomous University of Barcelona, and M.C. by a predoc- total fellowship from the Spanish Ministry of Education and Science.

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