Vol. 173, No. 23

JOURNAL OF BACTERIOLOGY, Dec. 1991, p. 7599-7606

0021-9193/91/237599-08$02.00/0 Copyright © 1991, American Society for Microbiology

Construction of an IS946-Based Composite Transposon in Lactococcus lactis subsp. lactist DENNIS A. ROMERO'1t AND TODD R. KLAENHAMMER2* Departments of Microbiology1 and Food Science,2 Southeast Dairy Foods Research Center, North Carolina State University, Raleigh, North Carolina 27695-7624 Received 17 June 1991/Accepted 23 September 1991 An artificial composite transposon was constructed based on the lactococcal insertion sequence IS946. A 3.0-kb element composed of the pC194 cat gene (Cmr) flanked by inversely repeated copies of IS946 was assembled on pBluescript KS+. When subcloned into the shuttle vector pSA3 (Emr), two putative transposons were created on the recombinant plasmid pTRK128: the 3.0-kb Cmr element (Tn-CmA) and an inverse 11.5-kb Emr element (Tn-EmA). pTRK128 was electroporated into the recombination-deficient strain Lactococcus lactis MMS362, which contains the self-transmissible plasmid pRS01. An MMS362 Cmr Emr transformant was used to assay for transposition events via conjugal mobilization of pTRK128-encoded Cmr or Emr to L. 1actis LM2345. Transfer of either marker alone occurred at frequencies of ca. 2 x 10-4 per input donor. Approximately 19% of the Emr transconjugants were Cms, indicating loss of the cat gene marker. No Cmr Ems transconjugants were recovered (n = 550). Plasmid analysis showed that the Cm' Emr isolates contained a single large plasmid that was determined to be a cointegrate between pRS01 and the Tn-EmA element. A 32P-labeled pSA3 probe hybridized specifically to pTRK128 sequences and revealed different junction fragments within each of the cointegrate plasmids. DNA sequence analysis of the Tn-EmA::pRS01 junctions from a representative cointegrate verified transposition by Tn-EmA. This represents the first example of a functional composite transposon in the genus Lactococcus and serves as an experimental tool and model for the genetic analyses of transposons in these organisms.

these phenotypes are associated with conjugally transmissible elements and, at least for the lactose genes, appear to be widely disseminated within lactococci, there have been speculations of an ISSJ-mediated composite transposon. However, no evidence has been reported that supports this hypothesis or indicates that ISSI is capable of forming a composite transposon. Regarding the use of transposable elements for genetic analyses, the heterologous gram-positive transposons Tn9J7, Tn9J6, and Tnl545 and their derivatives have been used with partial success in lactococci (13, 16, 40). Constraints that limit the utility of these elements include their large size, the low frequency at which insertion mutants are recovered, strain-dependent insertion specificity, and their nonlactococcal origin. Also, the use of transposable elements not indigenous to lactococci could pose a regulatory question if such elements were used to construct modified starter strains. The gram-negative transposon TnS has also been used to characterize phage resistance genes from plasmid pTR2030 (19). However, because Tn5 is inactive in lactococci, the target sequences were first cloned onto a shuttle vector and insertion mutants were then recovered in Escherichia coli. Considering the limitations of heterologous transposable elements, it would be advantageous to construct a transposon based on indigenous genetic material that could be used directly in lactococci. The lactococcal insertion sequence IS946, an iso-ISSJ element, has demonstrated random insertion into a target plasmid (34) or into the chromosome (36, 37). This study was undertaken to develop a composite transposon based on IS946 for use as an experimental tool in the genetic analysis of lactococci. We describe the construction of a model composite transposon composed of an antibiotic resistance marker flanked by inversely repeated modules of IS946. The element demonstrated random inser-

Prokaryotic transposable elements are genetic entities that are capable of movement from one genomic location to another via insertion as a discrete integral unit (22, 23). They are found in bacterial chromosomes, plasmids, and bacteriophages. Their ability to mediate DNA rearrangements that affect genetic organization and gene expression and regulation has invited numerous theories regarding the role of transposable elements in developmental and evolutionary processes (7). Bacterial transposable elements have also become indispensable tools in molecular biology. Their properties have been exploited for genetic analyses, where they are used for insertional mutagenesis and delivery of genes and regulatory sequences (4, 5). Bacterial insertion sequences (ISs) range in size from 800 to 2,500 bp and generally encode only those genes necessary for transposition (14). Composite or compound transposons normally contain an intervening segment of nontransposable DNA flanked by IS elements that supply the transposition functions. Within the genus Lactococcus, application of transposable elements in genetic analyses has recently begun to receive attention. Four indigenous elements, all ISs, have been characterized. The first and most thoroughly investigated is ISSJ (32). Additional iso-ISSJ elements and three new ISs, IS904, IS981, and IS1076, have since been characterized (17, 21, 31, 34). The iso-ISSJ elements have been found in at least two copies on plasmids encoding lactosefermenting ability, proteolytic enzymes, and the determinants for abortive infection and restriction-modification activities (17, 21, 32, 34). In the latter example, the ISs were shown to flank the phage resistance determinants. Since * Corresponding author. t Paper no. FS91-18 of the Journal Series of the Department of Food Science, North Carolina State University, Raleigh. t Present address; Promega, Madison, WI 53711-5399.

7599

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ROMERO AND KLAENHAMMER

J. BACTERIOL.

TABLE 1. Bacterial strains and plasmidsa Strain or

Relevant

plasmid

characteristics

Comments

L. lactis

MMS362 LM2345 NCK408 NCK413 NCK415 E. coli DH5 Plasmids pRS01

p$A3 pTRK25 pTRK27 pTRK81 pTRK126 pTRK128 pTRK154 pTRK155

str-14, Rec- (pRS01) spc4 rif-S, plasmid free str-14, Rec- (pRS01, pTRK128) str-14, Rec- (pRS01, pTRK155) str-14, Rec- (pRS01, pTRK156) F- endAI recAl hsdRl7 (rK- mK-) deoR supE44 thi-J gyrA96 relA

Rec- conjugation host (2) Conjugation host (2) MMS362/pTRK128 MMS362/pTRK155

Tra+, 48.2 kb Emr Cmr Tcr, 10.2 kb Cmr Emr, 5.2 kb Cmr Emr, 5.2 kb Cmr Apr, 4.6 kb Cmr Apr, 6.3 kb Emr Cmr, 13.0 kb Cmr Apr, 4.7 kb Emr Cmr, 12.3 kb

Resident plasmid of MMS362 (2) Shuttle vector (9) pGK12::IS946M (34) pGK12::IS946V (34)

MMS362/pTRK156 Bethesda Research Laboratories, Gaithersburg, Md.

pBluescript KS+::IS946 pBluescript KS+::IS946-cat-IS946 (Tn-CmA) pSA3::Tn-CmA (Tn-EmA) pBluescript KS+::AIS946-cat-IS946 (Tn-CmB) 0.7-kb NruI-EcoRV deletion in pTRK128 creating

Tn-EmB Emr Cmr, 12.0 kb pSA3::Tn-CmB a Cmr, Emr, spr, Smr, and Apr, resistance to chloramphenicol, erythromycin, spectinomycin, streptomycin, and ampicillin respectively; Tra, conjugation proficiency; Rec, host recombination. pTRK156

tion independent of the host's general recombination system.

(The preliminary results of this study were reported at the Third International American Society for Microbiology Conference on Streptococcal Genetics [35]). MATERIALS AND METHODS Bacterial strains and plasmids. The strains and plasmids used in this study are listed in Table 1. Lactococcus lactis cultures were propagated at 30°C in M17 broth (43) supplemented with 0.5% (wt/vol) glucose. Antibiotics were added at the following concentrations: chloramphenicol, 5 ,ug/ml; erythromycin, 5 ,ug/ml; rifampin, 50 ,ug/ml; spectinomycin, 300 ,ug/ml; and streptomycin, 1,000 p.g/ml. E. coli was propagated in LB mediumn (28) with 10 to 20 ,ug of chloramphenicol, 10 jig of tetracycline, and 50 to 100 ,ug of ampicillin per ml. Plasmid isolation and characterization. Isolation of plasmid DNA from L. lactis was performed as described by Anderson and McKay (1). Total genomic DNA from L. lactis was isolated as described by Hill et al. (18), with modifications. To 3 ml of mid-log-phase cells harvested into an Eppendorf tube, 400 RI of sucrose solution (6.7% sucrose, 50 mM Tris, 1 mM EDTA [pH 8.0]) and 25 p.l of lysozyme solution (10 mg of lysozyme per ml in 25 mM Tris [pH 8.0]) were added and mixed thoroughly. After 20 min at 37°C, 50 ,ul of sodium dodecyl sulfate solution (10% [wt/vol] in 50 mM Tris-20 mM EDTA [pH 8.0]) and 20 ,ul of proteinase K (20 mg/ml in water) were added and mixed by inversion, and the cells were incubated at 65°C for 20 min. The lysate was extracted with 500 ,ul of phenol saturated with 3% NaCl. The upper phase was removed and extracted with 500 ,ul of chloroformisoamyl alcohol (24:1). DNA was precipitated twice with 1/10 volume of 3 M sodium acetate and 2 volumes of ice-cold ethanol. The final pellet was washed with 70% ethanol and suspended in 100 ,u1 of 10 mM Tris-1 mM EDTA (pH 7.5); the samples were not allowed to dry completely during this

process. Routinely, 5- to 10-pd samples were used for restriction digests. Rapid plasmid isolation from E. coli was performed by a modified alkaline lysis method (41), and the protocol of Rodriguez and Tait (33) was used for large-scale plasmid purification. DNA from agarose gels was transferred onto MSI Magnagraph nylon membranes (Micron Separations, Inc., Westboro, Mass.) as described by Southern (42). [32P]dCTPlabeled probes were prepared by using a Multiprime DNA labeling kit (Amersham, Arlington Heights, Ill.). Radiolabeled probes were eluted from unincorporated nucleotides by using a Sephadex G50 spin column (Boehringer Mannheim Biochemicals, Indianapolis, Ind.). Hybridization reactions were performed with an Omni-blot apparatus (American Bionetics, Inc., Emeryville, Calif.) as described by Luchansky et al. (27). Conjugation and transformation. Solid-surface conjugal matings on M17 agar were performed as described by McKay et al. (29). The recombination-deficient (Rec-) strain L. lactis MMS362 containing the self-transmissible plasmid pRS01 was used as the conjugal donor for mobilization of various plasmids to the plasmid-free recipient strain L. lactis LM2345. Conjugal transfer frequencies were calculated as the numbers of transconjugants recovered per input donor from at least two independent trials. Transposition was assayed via conjugal mobilization as described previously (34). Transconjugants were selected for either Cmr or Emr and then scored for acquisition of the unselected antibiotic marker. Transfer of one phenotype but not the other was indicative of transposition by a composite transposon. Transformation was accomplished by electroporation with a Gene Pulser (Bio-Rad Laboratories, Richmond, Calif.) as previously described for L. lactis (34) or as outlined by Dower et al. (12) for E. coli. Molecular cloning and DNA sequencing techniques. The general procedures for DNA manipulations were essentially those described by Maniatis et al. (28). Nucleic acid se-

VOL. 173, 1991

pTRK81

I

Bam

BM A a |

II

BM . .

pTRK126 Tn-CmA

12--Ul-

Bam H I I a__

t

_,

, bo

LACTOCOCCAL COMPOSITE TRANSPOSON

Ca

...........

~~~~~~~~~~-.... ^^-.

1-12

-

..

- Pstl

I

CI

Ir

E I

-

----------

:.--.--..--..-..--..-.;-.:::

Int t -_ I -Iqff-- %.a -

.! .,. ....... ::;,*.: .9,0;................ .-Illl : : : : .-.-.--..-.-.-.-.-.: .. : --..Am -: I DI EPTI-1-1-1-1-

I

-Sall I II

EHC

pTRK154 Tn-CmB

7601

E

BamHI -

-Sall

a-

I II

2

n

Ili 111112 ]UNIII

B-BarnHl CC-la

E-EcoRV H-HuiSf M-MspI FIG. 1. Plasmid maps of pBluescript KS+-derived vectors pTRK81, pTRK126, and pTRK154. The shaded boxes represent IS946M (left-flanking MspI site) and IS946V (internal EcoRV site), and the triangles depict the 18-bp terminal inverted repeats. The arrows denote the direction of transcription of the IS946 putative transposase and that of the pC194 cat gene. The broken arrow shown in pTRK154 indicates a nonfunctional transposase in the truncated IS946V left module. The pBluescript KS+ map was adapted from that supplied by Stratagene (La Jolla, Calif.). cat, chloramphenicol; amp, ampicillin.

quencing was performed from double-stranded plasmid DNA templates with a Sequenase kit (U.S. Biochemical Corp., Cleveland, Ohio) as outlined by McMaster et al. (29a). Junction sequences were determined by using two IS946-specific 17-mer oligonucleotide primers, p60out and p707out (34). RESULTS Construction of composite transposons Tn-CmA and TnEmA. The putative composite transposon Tn-CmA encoding Cmr was assembled on pBluescript KS+. The elements were derived from pTRK25 and pTRK27 (pGK12::IS946). The functional iso-IS946 elements, hereafter designated IS946M (from pTRK25) and IS946V (from pTRK27 [GenBank accession no. M33868]), are distinguished by an EcoRV site in IS946V (34). From pTRK25, a 1.7-kb HaeIII fragment containing IS946M was cloned into the SmaI site of pBluescript KS +, creating pTRK81 (Fig. 1). The Tn-CmA element was completed by insertion of a 1.8-kb HpaII-ClaI fragment from pTRK27, containing the cat gene (Cmr) and IS946V, into the ClaI site of pTRK81 (pTRK126, Fig. 1). Tn-CmA is a 3.0-kb element composed of the cat gene (originally from pC194) flanked by inversely repeated copies of IS946. To use conjugal mobilization to assay for transposition, the Tn-CmA element was subcloned onto shuttle vector pSA3, which is able to replicate in lactococci. Tn-CmA was liberated from pTRK126 as an XbaI-SalI fragment and

cloned into similarly digested pSA3. The resulting plasmid, pTRK128 (13.8 kb), contains Tn-CmA and an 11.5-kb inverse transposon that encodes Emr (Fig. 2). This putative inverse transposon, designated Tn-EmA, comprises most of pSA3 and the gram-positive ori and erm genes from pGB305 and the gram-negative ori and cat genes from pACYC184 (9). Conjugal mobilization of pTRK128-encoded Cmr and Emr. pTRK128 was electroporated into the Rec- strain L. lactis MMS362, and a transformant (NCK408) was used as a conjugal donor for the transfer of Cmr or Emr to L. lactis LM2345 (Table 2). Prior experiments have shown that pSA3::IS946 derivatives transfer via rec-independent cointegrate formation with pRS01 and that pSA3 itself is unable to mediate the recombination event (34). Transfer of either Cmr or Emr from NCK408 occurred at ca. 2 x 10-4 transformant per input donor; these results are comparable to those with pSA3 derivatives containing a single copy of IS946 (34). From three independent matings, 550 transconjugants selected only for Cmr were Emr, indicating transfer of the entire pTRK128 plasmid and no transposition by Tn-CmA alone. In contrast, 93 (19%) of 494 transconjugants selected only for Emr were Cms. These Cms Emr transconjugants indicate that Tn-EmA inserted into pRS01 and formed a Tn-EmA::pRS01 cointegrate that was conjugally transferred. Examination of randomly selected Cmr Emr and Cms Emr transconjugants showed that they contained a single plasmid of ca. 60 kb that was not found in the parental strains. Physical and phenotypic analyses of selected plas-

7602

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ROMERO AND KLAENHAMMER

Hind III Hind III

Xba I Hi ~~~~~~Barn

Tn-EmA

Tn-EmB

~~~~~~~Tn-CmB

Tn-CffiA

Eco RV

Hind III

NruI Sal I

pTRK155

pTRK128

pTRK156

12.3 kb 13.0 kb 12.0 kb The lines and vectors of depict the pSA3 portion of open pTRK156. pTRK155, pTRK128, FIG. 2. Circular plasmid maps pSA3-derived the plasmid, and the closed line shows the Sall-Xbal fragment from pTRK126 or pTRK154. The arrows represent IS946 modules (IS946M [open arrow] and IS946V [solid arrow]). showing the direction of transcription of the putative transposase. Two complete IS elements are present in pTRK128, and truncated modules are shown in pTRK155 (5' deletion) and pTRK156 (3' deletion). The broken lines in pTRK155 and pTRK156 indicate the remaining portion of pSA3 that is identical to pTRK128. Note that the cat gene on pSA3 is not functional in gram-positive bacteria.

mids indicated they were pTRK128::pRSO1 or Tn-EmA:: pRSO1 cointegrates (data not shown). An IS946 outer end is necessary for transposition of TnEmA. Transposition by Tn-EmA was examined by constructing analogs deleted for an outer or inner IS end. These analog transposons are shown in Fig. 2, where they reside on their respective pSA3-based plasmids, pTRK155 and pTRK156. IS946 modules are depicted as arrows that indicate the direction of transcription of the putative transposase. Relative to the Emr marker, the outer and inner IS ends are denoted by the head and tail of the arrows, respectively. The first analog transposon, designated TnEmB, was constructed by digestion of pTRK128 with NruI and EcoRV and self-ligation to create plasmid pTRK155. In Tn-EmB, a portion of the IS946V module has been truncated, removing one inner IS end. The Emr marker is flanked

TABLE 2. Conjugal transfer of pTRK128-, pTRK155-, and pTRK156-encoded Cmr or Emr by pRS01 from L. lactis MMS362

donors and segregation of antibiotic resistance markers Donor (plasmid)

NCK408(pTRK128) NCK413(pTRK155)

NCK415(pTRK156)

Selection

Chloramphenicol Erythromycin Chloramphenicol Erythromycin Chloramphenicol Erythromycin

No. of transTransfer conjugants/total no. frequency Cmr Ems Cm' Em'

2 2 5 5

x 10-4

x 10-4 x 10-4 x 10-4 9 X 10 1 x 104

0/550 93/494

0/100 23/92 0/234

()/200

by one inactivated IS and one functional IS; the remaining portion of pTRK155 is identical to pTRK128. To delete an outer IS end relative to the Emr marker, a second Cmr transposon, designated Tn-CmB, was first assembled on pBluescript KS+ (pTRK154, Fig. 1). The truncated IS946V module and upstream sequences were originally derived from pTRK27 (34). As depicted in Fig. 1, the left and right ends of the Tn-CmB are identical. Tn-CmB was cloned as a BamHI-SalI fragment into pSA3 to create pTRK156 (Fig. 2). The net result is that the putative inverse Em' transposon formed on pTRK156 has been deleted for an outer IS end. pTRK155 and pTRK156 were electroporated into the Rec- strain L. lactis MMS362. Transformants containing pTRK155 (NCK413) or pTRK156 (NCK415) were used as conjugal donors in mobilization experiments analogous to those described with pTRK128. Transfer of Cmr or Emr by either plasmid occurred at frequencies comparable to those in pTRK128 (Table 2), and the transconjugants harbored putative cointegrates (data not shown). No Cmr Ems isolates were recovered from pTRK155 matings. However, 25% of the selected Emr transconjugants examined were Cm'. This indicated that the transposase encoded by the functional IS copy recognized the distal IS946 terminus, enabling Tn-EmB transposition into pRS01. No Cms Emr transconjugants were recovered with pTRK156, indicating that deletion of an outer IS946 end eliminates the ability of the Emr element on pTRK156 to transpose. Despite the orientation of the IS946 termini favoring transposition by Tn-CmB from pTRK156, no Cmr Ems transconjugants were recovered. Examination of Tn-EmA::pRS01 junctions. The Tn-EmA:: pRS01 junctions from several cointegrates were examined to

VOL. 173, 1991

LACTOCOCCAL COMPOSITE TRANSPOSON

A BC D E F G H I

9.2

using a combination of restriction sites within Tn-EmA and pRS01. To isolate the 6.0-kb HindIII junction, pTRK152 was digested with HindIII, self-ligated, and transformed into E. coli DH5. The 6.0-kb junction contains the pACYC184 ori and gram-negative cat gene that enables its isolation in E. coli as a free plasmid. E. coli DH5 Cmr clones harbored a 6.0-kb plasmid with a single HindIIl site. A representative clone containing a 6.0-kb plasmid designated pTRK157 was chosen for sequencing reactions. To isolate the 2.8-kb HindIII junction fragment, pTRK152 was double digested with BamHI and BgIII, self-ligated, and transformed into E. coli DH5. pRS01 is known to contain a single BamHI site (2). BglII, which generates a compatible end for BamHI ligation, was included to generate a potentially smaller plasmid clone, assuming that a BglII site is also present in pRS01. Tn-EmA does not contain a BglII site. A Cmr clone harboring a ca. 20-kb plasmid designated pTRK158 was isolated. pTRK158 possessed an internal 2.8-kb HindIll fragment that corresponded to the 2.8-kb IS junction. The IS946-specific primer p707out (34) was used to sequence the Tn-EmA: :pRS01 junctions directly from pTRK157 and pTRK158. The outer IS946 junctions of pTRK157 and pTRK158, representative of the outer junction in the pTRK152 cointegrate, were compared with the TnEmA IS946 junctions in pTRK128 (Fig. 5). The Tn-EmA IS junction sequences from pTRK128 were taken from the resolution plasmids pTRK25 and pTRK27 that were determined previously (34). In pTRK152, the outer IS946 junctions are flanked by 8-bp direct repeats characteristic of IS946-ISSI insertions (32, 34). The 8-bp direct repeats differed from the outer junctions in pTRK128, therefore confirming Tn-EmA transposition into pRS01 as an integral element.

J K L

-

1.91.6 -

p

7603

a.3

FIG. 3. Identification of random Tn-EmA::pRS01 junction fragments: autoradiogram of Hindlll-digested DNA probed with 32p_ labeled pSA3. Lanes: A, lambda DNA; B, pTRK128; C, total genomic DNA from donor NCK408; D, total genomic DNA from recipient LM2345; E through L, total genomic DNA from eight Tn-EmA transconjugants. The 1.6-kb bands in lanes J, K, and L were visible upon extended exposure of the autoradiograms.

determine whether Tn-EmA insertions into pRS01 were random. Total genomic DNA from 12 Cms Emr transconjugants was isolated, digested with HindlIl, and probed with 32P-labeled pSA3. The results for eight isolates are shown in Fig. 3. Hybridization occurred specifically to the Tn-EmA component of the cointegrates (Fig. 3, lanes E through I) and not to the recipient L. lactis LM2345 genome (Fig. 3, lane D). Of the three HindlIl fragments specifically generated from pTRK128, the 1.6- and 1.9-kb fragments, corresponding to the region containing the gram-positive ori, were present in all plasmids. These data confirmed that this region of Tn-EmA was not interrupted in the formation of the respective cointegrates. The 9.2-kb HindIll fragment containing the Tn-EmA termini was missing in all cointegrates and was replaced by two new fragments that represent the junctions created upon insertion of Tn-EmA into pRS01. The 12 cointegrates exhibited varied junction fragments, indicating that Tn-EmA inserted randomly into pRS01. Sequence determination of the Tn-EmA::pRS01 outer junctions. A representative Tn-EmA::pRS01 cointegrate designated pTRK152 (Fig. 3, lane E) was further characterized to verify transposition by Tn-EmA. As outlined in Fig. 4, the 6.0- and 2.8-kb HindlIl fragments containing the IS946 junctions were separately isolated as individual plasmids by

DISCUSSION An in vitro-constructed composite transposon, Tn-EmA, comprising an Emr marker flanked by inversely repeated modules of the lactococcal element IS946, generated random insertions into a target plasmid in L. lactis. Transposition by a second element, Tn-EmB, demonstrated that a phenotypic marker need only be flanked by one functional IS946 module and a distal IS terminus for transposition. Tn-EmA and Tn-EmB represent the first examples of transposition by a composite transposon in lactococci. They will provide new experimental tools for genetic analyses of lactococci and pTRK157 &o k

I H

(B) ___

1_______

L-.

-______4 A* i --4IIIiI

H

e*I .

k

H

HH

B

H

yu1iSi~Li--

I

dLqp-gwpT IlI pTRK158 I1 20kb FIG. 4. Schematic diagram of the Tn-EmA::pRS01 junction region of pTRK152 showing the fragments generated to isolate pTRK157 and pTRK158. Solid lines depict Tn-EmA, and dashed lines depict pRS01 DNA. The open (IS946M) and solid (IS946V) arrows represent the inversely repeated flanking IS modules. The internal 2.8-kb HindlIl junction fragment in pTRK158 is indicated. erm, erythromycin. Chloramphenicol (cat) and the origin of replication (ori) are derived from pACYC184. Restriction site abbreviations: H, Hindlll; B, BamHI; (B), BamHI or BglII. I

7604

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ROMERO AND KLAENHAMMER

TAACATAAATAT ATTGTATTTATA

ATAAATATATAT --TATTTATATATA

_GTTTAAATTTCC

AAATTTCCTAAA

--CAAATTTAAAGG

TTTAAAGGATTT

CATGAT--CAG--J-

-

.A

-nq

,ATTTCAGTACG

------CTAAAGTCATGC

GTAACTAAAGTC

"

*

pTRK128

---

pTRK158

A

l

pTRK152

pTRK157

h

*

FIG. 5. Comparison of the outer 1S946 junction sequences present in Tn-EmA (derived from pTRK128) and pTRK152 (reconstructed from pTRK157 and pTRK158). The arrows represent IS946V (solid) and IS946M (open). The upper junction sequence is from pTRK128. The lower sequence for pTRK152 was derived from sequence data from pTRK157 and pTRK158. The relevant 8-bp direct repeats at the junction sites are in boldface type.

form the basis for the construction of potential food-grade transposons. In contrast to our results with Tn-EmA and Tn-EmB, we were unable to demonstrate transposition by the corresponding Cmr transposons. Comparable transposons based on ISJ

and ISIO exhibit length-dependent transposition frequency (8, 30). The transposition frequency generally decreased with increasing length. However, this was apparently not the case with IS946. The Emr transposons are almost four times the size of the Cmr elements. Several possible explanations can be forwarded for the lack of transposition observed with the Cmr transposons. An insufficient number of (at gene copies may exist for the expression of Cmr in transconjugants. When used as a marker in homologous recombination, the pC194 (at gene required more than one copy per cell for expression in lactococci (25). Cointegrate plasmids composed of Tn-CmA or Tn-CmB and pRSO1 would rely upon the low-copy pRS01 ori for cell maintenance (less than one copy per cell [3]), which may be insufficient to confer Cmr. The copy number of pTRK128::pRS01 cointegrates, which also exhibit resistance to 5 Kg of chloramphenicol per ml, are likely under the control of the pSA3 ori gene and therefore exist at a higher copy number. Our inability to recover Tn-CmA Tn-CmB transposition events may also be due to differential activity of the IS946 ends. Dodson and Berg (11) have shown that the ends of IS50 in Tn5 exhibit different activities with respect to transposition. The 5' IS946 termini (5' with respect to the direction of transposase transcription) that serve as the outer ends of the Cmr transposons may show less activity than the 3' termini (outer ends for Emr transposons). To address these questions, we have constructed several IS946-based transposons that are currently under evaluation. We have configured the IS elements and IS termini in various orientations, and in some constructs we have incorporated the tetM gene from Tn919, which is expressed in single copy in lactococci (20). These transposons have also been assembled on various replicons, such as suicide vectors that do not replicate in L. lactis, to generate chromosomal insertions (38). There are numerous examples of naturally occurring and in vitro-constructed composite transposons. Among the most studied are Tn5, Tn9, and TnlO, which are flanked by insertion sequences IS50, IS, and IS1O. respectively. The efficient nature of transposition by Tn5 and Tn 10 in partic-

ular, has made these elements extremely useful in molecular biology. Studies have shown that minimum DNA sequences at the termini of IS50, IS1, and ISIO are sufficient for transposition activity (6, 15, 24). This has enabled the construction of various derivatives containing different combinations of phenotypic markers and regulatory sequences for increased utility as genetic tools (4, 5). The ability of IS946 to produce random insertions (34, 36, 37) made it a logical candidate to construct transposons for lactococci. The first application was the construction of IS946-based integration vectors that demonstrated random genomic insertions in lactococci (36, 37). These suicide vectors lack a gram-positive origin of replication and insert via IS946mediated transposition resulting in directly repeated copies of the IS. The results of Leenhouts et al. (26) with integration vectors based on homologous recombination indicate that the direct repeats generated upon insertion are stably maintained. Homologous recombination between directly repeated IS elements, however, has been shown to result in deletions (14) and represents a potential problem for longterm stability. In contrast, insertions generated by the composite transposons described in this study result in the flanking of intervening DNA with inversely repeated IS modules. Recombination between inversely repeated ISs results in inversions and no loss of genetic information and, therefore, should be inherently more stable (14). The ability of ISs to mediate the transposition of any segment of DNA provides increased plasticity to the genome. In addition to mediating various DNA rearrangements, ISs can promote the movement of nontransposable genes. Many IS elements are found only as parts of composite transposons and have not been observed independently in the chromosome (14). IS elements closely related to IS946 and ISSl flank several composite transposons. These include IS257 and IS431 in staphylococci and the IS6 family of elements (ISISdel, IS26, IS46, IS140, IS160) in gram-negative bacteria (14, 39). Transposition of an in vitro-constructed IS946-based element implies that any DNA sequence flanked by IS946 can form a composite transposon. In lactococci, ISSl and IS946 elements are associated with phenotypically important traits that are encoded on conjugally mobilizable plasmids. They include lactose metabolism (21, 32), proteinase activity (17), and bacteriophage resistance (34). The lactose plasmid pSK08 harbors two copies

VOL. 173, 1991

of ISSJ, which led Polzin and Shimizu-Kadota (32) to suggest the existence of a lactose transposon. The phage resistance plasmid pTR2030 also contains two copies of IS946 flanking the genes encoding abortive phage resistance and restriction-modification (34). Additionally, probes have identified several genomic copies of ISSJ and IS946 in various strains of lactococci (38). Furthermore, the lactococcal IS904 was located just upstream of the nisin structural gene (10), which is believed to be part of a larger transposable and/or self-transmissible element. All these traits are particularly advantageous for adaptation to a milk environment. Coupled with self-transmissible elements, ISs provide a mechanism for genetic recombination and subsequent mobility of important genes between various organisms. The accumulating evidence would argue that the IS elements are intimately involved with the evolution and dissemination of iihportant genetic traits among lactococci. Continued examination of the biology of IS946 and its relation to genetic exchange and rearrangements in lactococci will further our understanding of these organisms. ACKNOWLEDGMENTS This work was supported in part by U.S. Department of Agriculture Animal Molecular Biology Program agreement 87-CRCR-12547 and by Miles, Inc., Biotechnology Products Division, Elkhart, Ind. We thank J. J. Ferretti and L. L. McKay for providing strains and plasmids and C. Hill for helpful scientific discussion. REFERENCES 1. Anderson, D. G., and L. L. McKay. 1983. Simple and rapid method for isolating large plasmid DNA from lactic streptococci. Appl. Environ. Microbiol. 46:549-552. 2. Anderson, D. G., and L. L. McKay. 1984. Genetic and physical characterization of recombinant plasmids associated with cell aggregation and high-frequency conjugal transfer in Streptococcus lactis ML3. J. Bacteriol. 158:954-962. 3. Anderson, D. G., and L. L. McKay. 1984. In vivo cloning of lac genes in Streptococcus lactis ML3. Appl. Environ. Microbiol. 47:245-249. 4. Berg, C. M., and D. E. Berg. 1987. Uses of transposable elements and maps of khown insertions, p. 1071-1109. In F. C. Niedhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella typhimurium: cellular and molecular biology. American Society for Microbiology, Washington, D.C. 5. Berg, C. M., D. E. Berg, and E. A. Groisman. 1989. Transposable elements and the genetic engineering of bacteria, p. 879925. In D. E. Berg and M. M. Howe (ed.), Mobile DNA. American Society for Microbiology, Washington, D.C. 6. Berg, D. E. 1989. Transposon TnS, p. 185-210. In D. E. Berg and M. M. Howe (ed.), Mobile DNA. American Society for Microbiology, Washington, D.C. 7. Berg, D. E., and M. M. Howe (ed.). 1989. Mobile DNA. American Society for Microbiology, Washington, D.C. 8. Chandler, M., M. Clerget, and D. J; Galas. 1982. The transposition frequency of ISJ-flanked transposons is a function of their size. J. Mol. Biol. 154:229-243. 9. Dao, M. L., and J. J. Ferretti. 1985. Streptococcus-Escherichia coli shuttle vector pSA3 and its use in cloning of streptococcal genes. Appl. Environ. Microbiol. 49:115-119. 10. Dodd, H. M., N. Horn, and M. J. Gasson. 1990. Analysis of the genetic determinant for production of the peptide antibiotic nisin. J. Gen. Microbiol. 136:555-566. 11. Dodson, K. W., and D. E. Berg. 1989. Factors affecting transposition activity of IS50 and TnS ends. Gene 76:207-213. 12. Dower, W. J., J. F. Miller, and C. W. Ragsdale. 1988. High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res. 16:6127-6145. 13. Fitzgerald, G. F., and M. J. Gasson. 1988. In vivo gene transfer

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Construction of an IS946-based composite transposon in Lactococcus lactis subsp. lactis.

An artificial composite transposon was constructed based on the lactococcal insertion sequence IS946. A 3.0-kb element composed of the pC194 cat gene ...
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