Lactococcus lactis subsp. lactis MG1363 can act as a conjugative donor of chromosomal markers. This ... The first example of a limitation on the promiscuity of ...... 1963. Preparation of transforming deoxyribonucleic acid by phenol treatment.
Vol. 174, No. 18
JOURNAL OF BACTERIOLOGY, Sept. 1992, p. 5840-5847
0021-9193/92/185840-08$02.00/0 Copyright ©) 1992, American Society for Microbiology
Transfer of Tn916 between Lactococcus lactis subsp. lactis Strains Is Nontranspositional: Evidence for a Chromosomal Fertility Function in Strain MG1363 FRANCOISE BRINGEL,"12 GAYLENE L. VAN ALSTINE,1 AND JUNE R. SCOTl* Department of Microbiology and Immunology, Emory University Health Sciences Center, Atlanta, Georgia 30322,1 and Groupement de Recherche du Centre National de la Recherche Scientifique, URA#1481 GEM, Institut Le Bel, 4, rue Blaise Pascal, 67070 Strasbourg Cedex, France2 Received 8 November 1991/Accepted 12 June 1992
Lactococcus lactis subsp. lactis MG1363 can act as a conjugative donor of chromosomal markers. This requires a chromosomally located fertility function that we designate the lactococcal fertility factor (Laff). Using inter- and intrastrain crosses, we identified other L. lactis strains (LM0230 and MMS373) that appear to lack Laff. The selectable marker in our crosses was Tcr, carried by Tn916, a transposon present on the chromosome. The transfer of Tcr was not due to Tn916-encoded conjugative functions, because (i) L. lactis cannot act as a donor in Tn916-promoted conjugation (F. Bringel, G. L. Van Alstine, and J. R. Scott, Mol. Microbiol. 5:2983-2993, 1992) and (ii) transfer occurred when the Tcr marker was present in a Tn916 derivative containing a mutation, tra-641, that prevents Tn916-directed conjugation in any host. In addition, we isolated a strain in which Tn916 appears to be linked to Laff; this strain should be useful for further analysis of this fertility factor. In this strain, Tn916 is on the same 600-kb SmaI fragment as Clu, a fertility factor previously shown to promote lactose plasmid transfer in L. lactis. Thus, it is possible that Clu and Laff are identical.
Conjugative transposons, such as Tn916 (14, 19), are elements that transpose during conjugation from a donor cell harboring the element to a recipient cell. The mechanisms of conjugative transposition were reviewed recently (6, 30, 39). The tetM gene (4) was found to be present on all conjugative transposons (the smallest of which is 16 kb); in the larger conjugative transposons, other antibiotic resistance genes such as ermnAM or aphA-3 are found as well (39). Dissemination of antibiotic resistance markers by these transposons is of great concern, especially when pathogens are involved. Conjugative transposons have a broad host range, since they are able to transpose in gram-negative bacteria and can conjugatively transpose with frequencies of 10-4 to 10-9 among almost all species of gram-positive bacteria that have been investigated. The first example of a limitation on the promiscuity of conjugative transposons is presented by Lactococcus lactis subsp. lactis (herein referred to as L. lactis) MG1363. In this gram-positive organism, Tn916 and Tn919 do not excise (3). Although it can act as a recipient for conjugative transposition from another genus, L. lactis MG1363 cannot donate conjugative transposons in plate matings with Bacillus subtilis, Enterococcus faecalis, or Streptococcus pyogenes. In intrageneric matings between L. lactis MG1363 derivatives, transconjugants are formed, but the transposons are present in the same location in the transconjugant chromosome as in the donor genome, indicating that no transposition has occured. Furthermore, the formation of transconjugants between L. lactis strains is independent of the presence of integrase, a transposon function required for conjugative transposition, confirming the absence of transposition of Tn916 in these matings. Bringel et al. (3) proposed that in matings between strains derived from L. lactis MG1363, *
Corresponding author. 5840
conjugative transposons are inherited by a mechanism that involves homologous recombination and not transposition. Since transfer of conjugative transposons occurs between L. lactis strains in the absence of transposition (3), the question of whether the mating event is promoted by the transposon arises. To study this, we introduced Tn916 tra-641, a conjugative transposon defective for conjugation into L. lactis MG1363. In this paper, we show that MG1363::Tn9l6 tra-641 acted as a donor in matings with L. lactis MG1363 derivatives, demonstrating that the formation of transconjugants does not depend on Tn916. Instead, it appears that fertility functions are present in L. lactis MG1363. Moreover, we found two other closely related plasmid-free L. lactis strains that do not appear to have fertility functions. In one strain, Tn916 appears to be located on the L. lactis chromosome close to the fertility factor. Data from Southern blot analyses and pulsed-field gel electrophoresis (PFGE) are consistent with the possibility that this fertility factor, which promotes transfer of chromosomal markers, is the Clu element previously shown (16) to promote high frequency conjugational transfer of lactose plasmids among L. lactis strains.
MATERIALS AND METHODS Media. L. lactis was grown on GM17, which is M17 medium (38) supplemented with 0.5% glucose, at 30°C without agitation; B. subtilis was grown on LB medium (29) at 37°C with agitation; and S. pyogenes was grown on Todd-Hewitt yeast extract medium (11) at 37°C without aeration. The antibiotic concentrations were as follows: erythromycin, 5 ,ug/ml; fusidic acid, 25 p,g/ml; rifampin, 25 p,g/ml; streptomycin, 1,000 ,g/ml; and tetracycline, 10 ,ug/ ml. Kanamycin was used at 300 ,g/ml for S. pyogenes. Plasmids and bacteria. Plasmid pAM120 contains the wildtype conjugative transposon Tn916 (20), and plasmid
VOL. 174, 1992
LACTOCOCCAL FERTILITY FUNCTION
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TABLE 1. Bacterial strains Relevant phenotypea
Strain
Bacillus subtilis BB1421 CKS102 FB2002 MEN205 W168S
Lactococcus lactis subsp. lactis BL112
Parent(s)
Remarks, source, or reference
This work 31 W168 transformed with This work pAM641
Emr Tcr Tc' Tcr
Tn9l6AE, Tn916 tra-641 Tn916 Tn916 tra-641
Emr Smr
Tn916AE
Laff+ Smr Tcr
Tn916 (4 in sites A, B, C, CKS102 x MG1363Sm 3 and D) Tn916 (2) This work CKS102 x MMS373 Tn916 (2) This work CKS102 x MMS373 Tn916 (2) CKS102 x LMO23OFus This work Tn916 CKS102 x LMO23OFus This work Tn916 tra-641 BB1421 x MG1363Sm This work Tn916 tra-641 BB1421 x MG1363Sm This work Tn916 tra-641 BB1421 x MG1363Sm This work Tn916 tra-641 BB1421 x MG1363Sm This work 3 Tn919 (2) GF590 x MG1363Sm 3 MG1363 3 Tn916C BL112 x FMCB1 Tn919 (2 as in EFL201) EFL201 x LMO23OFus This work Tn916 (in site A) BL112 x LMO23OFus This work Tn916 (in site B) BL112 x LMO23OFus This work Tn916 (in site A) BL112 x MMS373 This work Tn916 (in site A) BL112 x MMS373 This work Tn9J6C BL1924 x FMCB1 This work C2 Plasmid-free strain (10) LM0230 3 LM0230 Smr spontaneous derivative, this work 712 Plasmid-free and prophagefree strain (15) MG1363 3 ML3 Rec- mutant and plasmidfree strain (35) MMS373 Fusr spontaneous derivative, this work
LM0230 LMO23OFus LMO23OSm
Emr Laff- Rec- Tcr Emr Laff- Rec- Tcr Fusr Laff- Tcr Fusr Laff- Tcr Ems Laff+ Smr Tcr Ems Laff Smr TCr Ems Laff+ Smr Tcr Em SLaffSmr Tcr Laff+ Smr Tcr Fusr Laff+ Rif' Fusr Laff+ Rif Tcr Fusr Laff- Tcr Fusr Laff+ Tc' Fusr Laff- Tcr Emr Laff- Rec- Tcr Emr Laff- Rec- Tcr Fusr Rif Tcr LaffFusr LaffLaff- Sm'
MG1363
Laff+
MG1363Sm MMS373
Laff+ Smr Emr Laff- Rec-
MMS373Fus
Emr Fusr Laff- Rec-
BL812 BL824 BL1211 BL1212 BL1911 BL1914 BL1921 BL1924 EFL201 FMCB1 LL511 LL3422 LL3511 LL3522 LL4011 LL4012 LL6421
Conjugative transposon(s)b
Streptococcus pyogenes JRS75 Kmr Smr
MEN205 x FB2002
26 3
25
a Abbreviations for antibiotic resistance and sensitivity phenotypes: Em, erythromycin; Fus, fusidic acid; Km, kanamycin; Rif, rifampin; Tc, tetracycline. Rec-, UV sensitivity. b The number of copies of the conjugative transposons is indicated within parentheses if there is more than one copy present. c Rearrangements of the transposon have been observed in this strain by Southern blot analysis with the donor strain.
pAM641 contains a transfer-deficient mutant of Tn916, Tn916 tra-641::TnS (32). Plasmid pFI431 harbors a 2-kb PstI fragment encoding Clu from the chromosome of L. lactis MG1363 (17). Bacterial strains are listed in Table 1. L. lactis MG1363, LM0230, and MMS373 are derivatives of strains 712, C2, and ML3, respectively, which are closely related to each other (8). L. lactis recombination deficiency was assessed by UV sensitivity as described by Anderson and McKay (1). The introduction of Tn916 tra-641 and Tn916AE (27) into B. subtilis BB1421 was done in two steps. First, pAM641 was transformed into competent B. subtilis W168 cells (43). The Tcr transformant FB2002, which had only one copy of Tn916 tra-641 as determined by Southern blot analysis (34) (Fig. 1, lane 1), was used as a recipient into which Tn916AE was introduced by conjugation. This was possible because a strain containing a conjugative transposon presents no barrier to the introduction of a second, differently marked
conjugative transposon (26). FB2002 (Tcr gene on Tn916 tra-641) was crossed with MEN205 (Emr gene on Tn9J6AE), and Tcr Emr transconjugants were obtained. BB1421 is one of these transconjugants and harbored only one copy of each transposon (data not shown). Polymerase chain reaction. The presence and location of the TnS in the Tn916 tra-641 was checked by using the polymerase chain reaction. The primer A303075 (5'GACGCTACTTIGTGTA-3') is homologous to the right end of TnS and reads out of TnS. The primer T301726 (5'lTlTGGTGGGTGTTTl-3') is homologous to the left end of Tn5 and also reads to the outside of TnS. The primer OTRC1 (5'-TATCAAGGTCAAAACCACTC-3') is homologous to the right end of Tn916 and reads toward the inside of Tn916. After denaturation at 94°C for 3 min, a three-step cycle was repeated 30 times. The cycle consisted of denaturation at 94°C for 1 min, annealing at 37°C for 1 min, and extension at 72°C for 6 min. When primers A303075 and OTRC1 were used to locate TnS in Tn916 tra-641, a fragment of 5.9 kb was
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J. BACTE RIOL.
BRINGEL ET AL. I
3
TABLE 2. Transfer of Tn916 tra-641 in conjugation between strains derived from L. lactis MG1363a
II
-.
5
4
6
7
8
kb)
9 10 11 12 13 14 15 16
16
*
Donorb
Frequency of recombinantsc
..
....~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~. 79 ...* ......I 7.9
BL1911 BL1914 BL1921 BL1924
1.0 2 2 1.1
x x x x
10-4 8 x 10-5 per recipient (4) 10-8 + 1 x 10-8 per donor (2) 10-9 + 1 x 10-9 per recipient (4) 10- ± 1 x 10-6 per donor (2)
The recipient was FMCB1, a Fusr Rif' derivative of strain MG1363. Tn916 tra-641 is present in one copy at a different site in each donor. c The frequency of recombinants was calculated by dividing the number of Tcr transconjugants by the number of parental cells present in limiting amounts (see Materials and Methods). Numbers within parentheses indicate the number of repetitions of each mating. a
b
FIG. 1. Southern blot analysis (34) of L. lactis transconjugants harboring the mutant transposon Tn916 tra-641. Chromosomal DNA was digested with either HaeIII (lanes 1 through 8) or PvuII (lanes 9 through 16). After electrophoresis on a 0.4% agarose gel, DNA fragments were hybridized with a radioactive probe specific to Tn916 (plasmid pAM120). The sizes of the DNA segments indicated in the center of the figure were deduced from the super marker Dynazyme. Lanes: 1, DNA from B. subtilis FB2002; 2 and 10, DNA from the donor BL1914; 5 and 13, DNA from the donor BL1924; 9, DNA from the recipient FMCB1; 3, 4, 11, and 12, DNA from transconjugants from cross I (BL1914 x FMCB1); 6, 7, 8, 14, 15, and 16, DNA from transconjugants from cross II (BL1924 x FMCB1).
amplified. No fragment was detected when primer T301726 (5'-lTTGGTGGGTGTTTT-3') replaced A303075. Therefore, the left end of Tn5 is facing the left end of Tn916. Plate matings. Donor and recipient cells were mixed on GM17 medium and incubated at 30°C overnight. When the recipient strain was S. pyogenes, the mating was performed on Todd-Hewitt yeast extract medium at 37°C (3). The frequency of transconjugant formation was determined by dividing the number of transconjugants per milliliter by the number of the parental cells (donor or recipient) found to be present in limiting amounts after the mating. DNA isolation. Total DNA was purified from B. subtilis as described by Saito and Miura (28). L. lactis chromosomal DNA was obtained by the procedure of Hayes et al. (21). PFGE and Southern blotting. For PFGE, DNA was prepared in situ in agarose blocks (37) and then digested with SmaI. Electrophoresis was monitored with a Bio-Rad CHEF DRII electrophoresis cell apparatus set up at 200 V with a pulse time increasing linearly from 40 to 80 s for 22 h at 15°C. Saccharomyces cerevisiae chromosomes were used as molecular weight markers. For Southern hybridizations (34), DNA was transferred to a positively charged Hybond-N membrane (Amersham) and hybridized with a randomprimed labeled DNA probe (DIG DNA labeling and detection kit) as recommended by the manufacturer (Boehringer Mannheim). RESULTS Introduction of Tn916 tra-641 into L. lactis by complementation with wild-type Tn916. The Tra-641 function, defined by an insertion of TnS into the conjugative transposon Tn916 (32), is necessary for conjugation from one E. faecalis strain to another. We find that it is also required for conjugative transposition between B. subtilis and E. faecalis, since when B. subtilis FB2002 (which carries Tn916 tra-641) was crossed with E. faecalis JH2-2,
