FEMS Microbiology Letters 135 (1996) 179-l 85 ELSEVIER

Transposons Tn916 and Tn 925 can transfer from Enterococcus faecalis to Leuconostoc oenos

Manuel ZGiiga * , Isabel Pardo, Sergi Ferrer

Departament de Microbiologia. UrGersitat de Val.?ncia. Far&tat de Biologia. E-46100 Burjassot-Valhcia, Spain

Received 6 November 1995; accepted 8 November 1995

Abstract

The streptococcal transposons Tn916 and Tn925 were transferred to several strains of Leuconostoc (Ln.) oerlos using the filter mating method. The insertion of both transposons into the chromosome occurred at different sites. Transconjugants of Ln. oenos carrying Tn916 could serve as donors in mating experiments with Luctococcus luctis LM2301. Further analysis of L. luctis LM2301 transconjugants showed that the insertion of the transposon Tn916 into the chromosome was site-specific. These studies establish a basis for the initiation of genetic studies in this Leuconostoc species since there are no

efficient conjugal or transformation systems previously described for this microorganism.

Kexwwk: Eubacteria; Firmicutes; Letrconostoc oenos; Tn 916; Tn Y25; Conjugal transfer

1. Introduction

Leuconostoc fLn.) oenos is a lactic acid bacterium present in musts and wines and is often considered to

be the species responsible for malolactic fermenta- tion [I]. This is a secondary fermentation in which

t,-malic acid is converted to L-lactic acid and C02. This process reduces wine acidity which, in cold wine regions, is deemed essential because of the high malic acid content of the grapes, and in warm wine regions because of the enhanced microbiological sta- bility conferred upon the wine. Ln. oenos is the most

frequently used species in commercial starter cul- tures. Despite this, little is known about the genetics

* Corresponding author. Present address: I.A.T.A.-C.S.I.C.,

Poligono de la Coma s/n, P. Box 73, Burjassot, 46100.Valencia.

Spain. Tel: + 34 (6) 3900022: Fax: + 34 (6) 3636301.

Federation of European Microbiological Societies

SSDI 0378 1097(95)00446-7

of Ln. oenos. The presence of plasmid DNA has been reported in some strains of 01. MVIOS [2.3]. These studies have shown that Z.n. OPIIOS contains

little genetic information on extrachromosomal ele- ments. Other species of Leuconostoc have received more attention and the presence of plasmid DNAs

has been reported in many strains [4]. Moreover,

conjugal transfer [5,6] and electroporation [7.8] pro-

cedures have been described. We have concentrated our attention on the study

of the transfer of conjugative transposons to LII.

oenos. Tn916 191 and Tn9.25 [IO] belong to a group of transposons identified in some species of the genera Enterococcus and Streptococcus. These trans- posons are between IS and 17 kilobases in size, encode tetracycline resistance (Tet’) and are capable of promoting their own intercellular transfer to a broad host range [ 1 II. In addition. Tn Y 16 and Tn Y?5 share a high degree of sequence homology [I?].

180 h4. Z&&a et al./ FEMS Microbiology Letters 135 (19%) 174-185

There is strong evidence that suggests that these transposons move by an excision-insertion mecha-

nism [13]. Analysis of regenerated target DNA and the joint regions have shown that the insertion of the

transposon does not cause duplication of the target

sequence [14]. The choice of target site for integra- tion is not random: the target sequences contain a

conserved element that consists of a T-rich region

separated by about six bases from an A-rich region [15,16]. This is a major disadvantage for the use of

these transposons for mutagenesis. In this report, a procedure for the conjugal trans-

fer of Tn916 and Tn925 into Ln. oenos is described. This method will facilitate genetic studies on Ln.

oenos, since there are no transformation systems

described for this species.

2. Materials and methods

2. I. Bacterial strains and growth conditions

The bacterial strains used in this study are listed

in Table 1. E. fuecalis strains were routinely grown in brain-heart infusion broth at 37°C. Luctococcus

lactis was grown at 30°C in Ml7 medium 1201 in which glucose (5 g 1-l) replaced lactose. E. coli

was grown in LB broth at 37°C in a rotary shaker.

Ln. oenos was grown in ML0 medium 1211 at 30°C. Antibiotics were added to the following final concen-

trations: tetracycline 5 pg ml-l, vancomycin 50 pg ml - ’ . Solid media contained 2% (w/v) agar.

2.2. Mating procedure

Unless otherwise noted, filter matings between E.

faecatis and Ln. oenos were carried out as follows: 9 ml of medium were inoculated with 1 ml of an

overnight donor culture or a 48 h recipient culture.

Both were grown to an OD at 600 nm of 0.3. The cultures were centrifuged at 5000 X g at 4°C for 10 min, washed with distilled water and finally resus- pended in 5 ml of distilled water. 1 ml of donor and

5 ml of recipient cells were mixed and collected on a membrane filter (0.45 pm pore size). The filters

were then placed on ML0 agar plates (pH 6.0) and incubated overnight at 30°C under a CO, atmo-

sphere. Each filter was washed with 1 ml of distilled water and appropriate serial dilutions of the cell

suspension were made. Donor viable cell counts were performed on BH plates containing 5 pg ml - ’ of tetracycline and incubated at 37°C. Recipient vi-

able cell counts were performed on ML0 plates containing 50 pg ml-l of vancomycin at 30°C and transconjugant viable cell counts on ML0 plates with both antibiotics present. Plates for counting

donor and recipient were incubated aerobically and those for counting transconjugants were incubated under a CO, atmosphere. Presumptive transconju-

gants were identified as Ln. oenos taking into ac-

Table 1

Bacterial strains and plasmids used in this study

Species Strain Relevant chromosomal markers Plasmid content References or remarks

Enterococcus faecalis

Escherichia coli

Lactococcus lactis

Leuconostoc oenos

Ceil 10

CG180

OGlSSp

CG120

LM2301

840

841

866

872

873

874 874- 1

riffus tet

rrjj’us {Tn 916)

str spc (Tn 925)

F recA1, end Al, gq’rA96,

thi, hsdR17, supFA4

Lac-, Str’

Van’

Van’

Van’

Van’

Van’

Van’

Van’, {Tn 916)

None

pAMl80

(pAMSI::Tn916)

pCF- 10

PAM120

None

None

None

None

None

None None

None

Gawron-Burke and Clewell [ 171

Gawron-Burke and Clewell[17]

Christie et al. [ 121

Gawron-Burke and Clewell [ 131

McKay et al. [18]

Pardo [19]

Pardo [19]

Pardo [ 191

Pardo [ 191

Pardo [ 191

Pardo [ 191 This work

M. Z&&a et al. / FEMS Microbiology Letters 135 ( 1996) 179- 185 181

count their heterolactic degradation of fructose as tested on M5 medium plates [22].

Matings between Ln. oenos and L. lactis LM2301 were essentially performed as described above with the following modifications. Donor cells were grown in ML0 broth supplemented with 5 kg ml-l of tetracycline and recipient cells in modified Ml7 broth (see above). For matings the donor:recipient ratio was 1: 1. Donor viable cell counts were per- formed on ML0 agar plates containing 5 pg ml-’ of tetracycline and 50 pg ml-’ of vancomycin. Recipient viable cell counts were made on Ml7 agar plates since Ln. oenos is unable to grow on this medium. Transconjugant cell counts were made on M 17 agar plates containing 5 ,ug ml-’ of tetracy- cline. The identity of the transconjugants was checked using the API SOCHS system.

2.3. DNA isolation

Plasmid DNA from Ln. oenos was isolated fol- lowing the alkaline lysis method [23]. Chromosomal DNA from Ln. oenos was isolated as follows. An overnight culture was diluted lOO-fold in 10 ml of ML0 medium and grown at 30°C until an OD at 600 nm of 0.6-0.8 was reached. The culture was cen- trifuged (5000 X g 10 min at 4°C) and washed once with sterile distilled water. The pellet was resus- pended in 0.5 ml of lysis solution (25 mM Tris . HCl, pH 8.0, 50 mM EDTA, 50 mM glucose, 5 mg ml-’ lysozime) and incubated for 15 min at 37°C. After addition of 20 ~1 20 mg ml-’ proteinase K and 25 ~1 10% (w/v) SDS, the incubation was continued for 1 h. The lysate was extracted twice with phenol- chloroform-isoamyl alcohol (25:24: 1) and once with chloroform-isoamyl alcohol (24: 1). 50 ~1 3M sodium acetate (pH 5.2) were added and the DNA was precipitated by adding 2 volumes of 100% (v/v) ethanol (- 20°C). The pellet was washed once with 70% (v/v> ethanol, vacuum dried and dissolved in 100 ~1 10 mM Tris . HCl (pH SO>, 1 mM EDTA supplemented with 20 pg ml-’ boiled RNaseA. The solution was incubated for 30 min at 37°C and stored at 4°C. Total DNA from E. faecalis and L. lactis

was isolated following the same procedure except that the incubation in lysis solution was prolonged for 30 min.

2.4. Southern blot analysis

Alkaline transfer of DNA in agarose gels to GenScreenPlus’” membranes (DuPont) was done as

described by Chomczynski and Qasba [24]. pAM 120 plasmid HindIII-digested DNA was labelled with digoxigenin-dUTP by using the Non-ratioactive

DNA Labelling Kit (Boehringer). Hybridization, washing and staining were done according to the instructions of the supplier.

3. Results and discussion

3.1. Transfer of Tn916 and Tn925 to Ln. oenos

Initially, assays were performed in order to deter- mine if Tn916 could be introduced into Lra. oenos.

For this purpose, E. faecalis CG180 was used as a

donor strain in filter matings with several strains of tn. oenos. Donor viable cell counts ranged from 5 X 10’ to 1 X lo9 cfu ml- ’ and recipient viable

cell counts from 1 X lo9 to 5 X lo9 cfu ml-’ after mating. Tetracycline-resistant variants were recov- ered after matings with all the strains tested, while

no spontaneous resistance mutants were observed in control assays with recipient cells alone. SCmilarly,

no vancomycin resistant mutants were obtained with donor cells. Transfer frequencies of Tet’ resistance are listed in Table 2 and ranged between 1 x lop6

and 1 X lo-*. High variability (greater than one order of magnitude in some cases) from one experi-

Table 2

Frequencies of conjugal transfer of Tn916 from E. faecalis CC1 80 or CC1 10 to several strains of Ln. 0eno.y

Mating Frequency per

donor cell *

CG180x840 2.3 x 1O-8

CG180x841 9.7x lo-”

CG180X866 2.2 x lo+

CGI 10X866 4.8X lo+

CGlSOX872 ND b

CG180~873 ND

CG180x 874 ND

CGl10X874 1.6~ lo-’

’ Average based on three replicates.

b Not determined.

Frequency per

recipient cell a

2.4X lo-’

1.1 x10-x

9.5x lo-”

1.9x 10-h

1.9x 10-x

1.2x lO-h

1.3x lomh

ND

182 M. ZLiiga et al. / FEMS Microbiology Letters 135 f I9961 17% 185

ment to another was observed. This variability may

be due in part to the viable counting method itself, since both E. faecalis and Ln. oenos grow as chains.

In addition, both microorganisms need complex me- dia to grow, making it difficult to reproduce exactly the experimental conditions from one replicate to another.

Analysis of Tet-resistant strains showed that they

did not contain any plasmid DNA. This result sug- gested that either pAM81 was not involved in the

conjugative transfer or that it might have been un- able to replicate in Ln. oenos. In order to demon-

strate that the conjugal transfer was enabled by the

transposon, we used E. faecalis CGl 10 (a plasmid- free strain carrying several copies of Tn916 in the

chromosome) as a donor in matings with 866 and 874 strains of Ln. oenos. E. faecalis CG180 was

used as a positive control in this experiment. The results are shown in Table 2. CGI 10 was able to act as donor strain for the transfer of Tet-resistance to both strains of Ln. oenos, confirming that Tn916

can promote its own transfer to this host without the involvement of a conjugative plasmid.

To verify the presence of Tn916 in the recipients,

chromosomal DNA was isolated from the parent

strains and several transconjugants picked randomly from matings among recipient strains 866 and 874, and donor strains CGl 10 and CG180. After digestion with HindIII, the DNAs were electrophoresed, blot-

ted and probed with digoxigenin-dUTP labelled pAM120 DNA. pAM120 contains the EcoRI

F::Tn916 fragment of pAM211 cloned in the E. coli

vector pGLlO1 [ 131. pAM120 has only a single target for HindIII, being located within the sequence of Tn916. Due to this, two chromosome-transposon junction fragments should be detected after digestion with HindIII, and hybridization with the probe. The results for strain 874 are shown in Fig. 1. Similar

results were obtained for strain 866. Only the transconjugants gave positive hybridization signals and all of them contained no more than two frag- ments that hybridized with the pAM120 probe. This indicated that all the transconjugants checked har- boured only one copy of Tn916. The sizes of the fragments generated after the digestion with Hind111 varied from one transconjugant to another indicating that Tn916 inserts at different sites on the recipient chromosomes. Moreover, this result discounted the

1 2 3 4 5 6 7 8 9 10 1112 13 14

1 2 3 4 5 6 7 8 91Olll21314

Fig 1. Agarose gel electrophoresis (upper panel) of Hind111

digested chromosomal DNA of Ln. oeno~ 874 transconjugants

obtained from matings with CC180 and Southern transfer analysis

(lower panel) with digoxigenin-dUTP labelled PAM 120. Lanes

I - 10, 874 transconjugants (lane 1, strain 874-l); 1 I, recipient

strain 874; 12, donor strain CG180; 13, pAMI not digested; 14,

A DNA digested with Hind111 (molecular mass marker).

M. ZGCga et al. / FEMS Microbiology Letters 135 (1996) 179-185 183

1 o-9

0 0.2 0.4 0.6 0.8

O.D. 600 nm

Fig. 2. Effect of recipient cell age on transfer efficiency of Tn916

to Ln. oenos 874. Empty symbols, frequency expressed as

transconjugants per donor. Filled symbols, frequency expressed as

transconjugants per recipient. 0, CGl 10 donor strain; I : 10 ratio.

A, CC3110 donor strain; 15 ratio. 0, CG180 donor strain; 1:5

ratio.

possibility that pAM180 could be present in the

transconjugants. The conjugal transfer of Tn925 to Ln. OEXOS was

also investigated. E. faecalis OGl SSp containing

pCF- 10 was chosen as a donor. Ln. oenos strains

12345678 12345678

866 and 874 were used as recipients. Tet’ transcon-

jugants of both recipient strains could be recovered at frequencies of 8.6 X lo-’ (transconjugants per

donor) for 866 and 5.2 X 10d8 for 874. Analysis of some randomly selected presumptive transconjugants

revealed that none of them contained plasmids. The

presence of Tn925 in the transconjugants was tested by Southern blotting. pAM120 was used also as a probe since Tn916 and Tn925 share extensive ho-

mology [lo]. Chromosomal DNA from the ttanscon-

jugants was digested with Hind111 which hasa single target within Tn925 [12]. The results are shown in

Fig. 3. It could be observed that one transconjugant

(Fig. 3, lane 1) harboured more than one coRy of the

transposon. In some of the other transconjugants examined additional bands hybridizing with the probe

also appeared but these were probably due to a

partial digestion of the chromosomal DNA.

3.2. effect of the growth stage on the tramfer fre-

quency of Tn916

The effect of the growth stage of the recipient cells was also studied. Recipient strain 874 and

Fig. 3. Agarose gel electrophoresis (left panel) of Hind111 digested chromosomal DNA of Ln. oeno~ 874 and 866 transconjugauts obtained

from matings with OGlSSp and Southern transfer analysis (right panel) with digoxigenin-dU’II’ labelled pAM120. Lanes l-5. 874

transconjugants; 6, 866 transconjugant; 7, pAM120 not digested; 8. h DNA digested with Hind111 (molecular mass marker).

184 M. Zhiiga et al./ FEMS Microbiology Letters 135 (1996,61 179-185

donor strains CGllO and CG180 were chosen for this experiment. Donor strains were grown to an OD of 0.3 at 600 nm and the same culture of each donor strain was used for all the matings. Several cultures of strain 874 grown to different optical densities were mated as described in Materials and methods. When CGllO was used as a donor, matings were performed at 15 and 1: 10 donor to recipient ratios in order to check the effect of the donor:recipient ratio on the transfer frequency. CG180 was mated at 1:5 donor:recipient ratio. The results are shown in Fig. 2. The age of the culture considerably affected the transfer frequency. Highest transfer frequencies were obtained with cultures of the recipient strain har- vested at an OD of 0.2 to 0.4. Higher optical densi- ties resulted in a great reduction of the frequency. A 1: 10 donor to recipient ratio resulted in higher trans- fer frequencies.

3.3. Transfer of Tn916 from Ln. oenos to L. lactis

LM2301

We also investigated the ability of Ln. oenos to act as a donor for the transfer of Tn916. For this experiment we used strain 874-l (see Table 1, and Fig. 1, lane 1) as the donor and L. lactis strain LM2301 as the recipient. Tn916 could transfer from Ln. oenos to L. Zactis LM2301. The conjugal trans- fer frequency observed was 6.8 X lop9 (transcon- jugants per donor). The presence of Tn916 in the transconjugants was confirmed by Southern analysis (not shown). This result indicated that Ln. oenos

could act as a donor in conjugation experiments. The same band pattern was observed in all the transcon- jugants tested suggesting that the insertion of Tn916 was site-specific. Since LM2301 is a plasmid-free strain, the pattern observed could not be explained by the insertion of the transposon in a resident plasmid. This phenomenon has been previously ob- served with other strains of L. lactis and is due to the fact that Tn916 cannot transpose to the chromo- some of these strains, but can be inserted by site- specific recombination [25].

3.4. Stability of the transposons

To study whether or not Tn916 and Tn925 were lost from the chromosome when tetracycline was

missing during growth, two randomly selected transconjugants harbouring Tn916 or Tn 925 were grown for several passages in ML0 medium without tetracycline, corresponding to approx. 100 genera- tions. After the culture of each passage had reached the early stationary phase, it was used as an inocu- lum (1: 100) for a further one. Samples were taken periodically and analysed for viable cell count and percentage of resistant cells. Segregation could not be detected, indicating that both transposons were stably maintained. We also investigated whether sec- ondary transposition events occurred after growth for several generations. To do this, five single colonies obtained from the last passage of each culture used to determine the segregation frequency were isolated and grown to stationary phase. Chromosomal DNA from these cultures was extracted and analysed by Southern blotting hybridization. All the isolates showed the same band pattern as the original cul- tures, indicating that the secondary transposition fre- quency must be low for both transposons.

The results of this study indicate the applicability of Tn916 and Tn925 as tools for the genetic manipu- lation of Ln. oenos. This is of special interest since there is no previously described way to introduce foreign DNA sequences into the chromosome of this microorganism.

Acknowledgements

We thank Dr. D.B. Clewell for kindly providing the strains harbouring Tn916 and pAM120, Dr. G.M. Dunny for providing the strain harbouring Tn92.5 and Dr. L.L. McKay for providing strain LM2301. We also thank Dr. A. Macabbe for critical reading of the manuscript. This work was supported by the Comisidn Interministerial de Ciencia y Tecnologia (ALI93-0246) and by a grant from the Generalitat Valenciana to M.Z.

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