Isolation and characterization of insertion sequence elements from ...

Vol. 173, No. 4

JOURNAL OF BACTERIOLOGY, Feb. 1991, p. 1502-1508

0021-9193/91/041502-07$02.00/0

Isolation and Characterization of Insertion Sequence Elements from Gram-Negative Bacteria by Using New Broad-Host-Range, Positive Selection Vectors REINHARD SIMON,* BARBARA HOTTE, BARBEL KLAUKE, AND BOB KOSIER Lehrstuhl far Genetik, Fakultat far Biologie, Universitat Bielefeld, Postbox 8640, D4800 Bielefeld 1, Federal Republic of Germany Received 20 July 1990/Accepted 10 December 1990

On the basis of an RSF1010-derived broad-host-range vector, three different systems which enable positive detection and isolation of insertion sequence (IS) elements from gram-negative bacteria were constructed. Vectors pSUP104-pheS, pSUP104-rpsL, and pSUP104-sac were used successfully in a number of Rhizobium strains and in Xanthomonas campestris. More than 20 different IS elements were isolated and characterized. The 16 IS elements from Rhizobium meliloti were further used to characterize various R. meliloti strains by hybridization. The resulting hybridization patterns were different for every strain and gave a dear and definite IS fingerprint of each strain. These IS fingerprints can be used to identify and characterize R. meliot strains rapidly and unequivocally, as they proved to be relatively stable. Some of the IS elements were found to be identical when the IS fingerprints from a given strain were compared. This method of IS fingerprinting can also establish whether IS elements are the same, related, or different.

Insertion sequence (IS) elements are defined as relatively small mobile genetic entities which, unlike drug resistance transposons, do not contain selectable genes (5). This definition makes it clear that direct selection for the presence of an IS element is usually not possible. IS elements in procaryotes were first identified as causative agents of spontaneous mutations in Escherichia coli (21, 36). Since then, various classes of transposable elements have been found to be natural constituents of many bacterial chromosomes, plasmids, and bacteriophages. As far as gram-negative bacteria are concerned, most IS elements have been isolated from enteric bacteria (mainly E. coli; for reviews and lists of isolated elements, see references 14 and 20). However, there have been a substantial number of reports on IS elements identified in other gram-negative bacterial species, such as Agrobacterium tumefaciens (3, 15, 24, 42), Xanthomonas campestris (22, 23), and various Rhizobium (9, 28, 32) and Pseudomonas (6, 13, 35, 41, 45) strains. In general, these elements have been found more or less by chance because of the capacity to inactivate or activate particular genes. However, there have been successful attempts to screen for transposable elements by means of direct selection procedures (8, 15, 30, 35). In this report, we present the construction and use of three broad-host-range vectors designed to enable systematic searches for the presence of transposable elements in gramnegative bacteria. Advantage was taken of two direct selection systems initially constructed to simplify cloning experiments with E. coli (7, 17), as well as a system described by Gay and coworkers (15). The common principle of these systems is based upon insertional inactivation of a vectorborne dominant sensitivity marker. With pSUP104-pheS and pSUP104-rpsL, this leads to drug resistance. Inactivation of pSUP104-sac allows its host to grow on medium containing 5% sucrose. Both of these events are positively selectable phenotypes in the hosts. *

(A preliminary report of parts of this work has been presented previously [38].)

MATERIALS AND METHODS Bacterial strains. The strains and plasmids used in this study are listed in Table 1. Culture, mating, and selection conditions. E. coli strains were grown on Penassay medium (Difco) at 37°C. R. meliloti and Agrobacterium strains were grown on either TY (2) or rhizobial minimal medium (4) at 30°C. Matings were performed on solid media as outlined by Simon (37). Sucrose was added to media to give a final concentration of 5% (a 50% sucrose stock solution was filter sterilized separately). p-Fluorophenylalanine (PFP) was purchased from Sigma and used at 350 ,ug/ml. Antibiotics were used at the following concentrations (micrograms per milliliter): chloramphenicol, 100; kanamycin, 50; neomycin, 100; rifampicin, 100; streptomycin, 800; and tetracycline, 5. DNA methods. Restriction enzymes and ligase were obtained from Pharmacia or BRL and used as stipulated by the suppliers or by Maniatis et al. (25). For quick analysis of plasmid length, an in-gel lysis technique was used. Cells (4 x 107) of a fresh overnight culture were pelleted and suspended in 20 ,ul of SR solution (25% sucrose, 10% Ficoll, 1 mg of RNase per ml in Tris-borate [25]) to which lysozyme (2 mg/ml) had been freshly added. The suspension was immediately pipetted into the wells of a horizontal gel (0.7% agarose, 2% sodium dodecyl sulfate in Tris-borate). The cells were allowed to lyse for 15 min (or until the wells cleared) and then electrophoresed at 15 V for 25 min, followed by a further 2.5 h at 100 V. Before ethidium bromide staining and photography, the gels were thoroughly

rinsed in water.

Plasmid DNA was isolated by using a rapid boiling lysis technique described by Arnold and Piihler (1). Total DNA was isolated by lysozyme-sarcosyl-proteinase

Corresponding author. 1502

VOL. 173,

1991

IS ELEMENTS FROM GRAM-NEGATIVE BACTERIA

TABLE 1. Bacterial strains and plasmids Strain or plasmid R. meliloti 2011 MVII

220-12 220-13

Relevant characteristic(s)

Source or reference

Sm' derivative of the wild type (SU47)

J. Denarie

Field isolate Field isolate Field isolate

Erlangen, FRGa Bielefeld, FRG Bielefeld, FRG S. Long

USDA1024 USDA1029

S. Long

L5-30 AK631

S. Long A. Kondorosi

Long

NGR185

S.

102F34

D. Helinski

R. leguminosarum 3853 VF39 F7

X. campestris B100

Field isolate Field isolate

England Bielefeld, FRG; 27 W. Selbitschka

Smr derivative of wild-type DSM1526

18

Mobilizing strain

40

E. coli S17-1

with

RR28

chromosomally integrated RP4 derivative recA pheS PFPr

17

Plasmids

pSUP104

pSUP104-pheS pSUP104-rpsL pSUP104-sac pUM24 RP4-7

pNO1523 pHE8

pUC8 pUC4K a

RSF1010 derivative; Cmr Tcr Tcr pheS Tcr rpsL Cmr Nmr sac cassette of pUM24 npt-l sacB sacR

This work This work This work

cassette RP4 Tc::Tn7 Apr Nmr Tpr

39

Apr rpsL Cmr pheS Apr lacZ Apr lacZ Kmr

7 17 43 43

29

31

FRG, Federal Republic of Germany.

K lysis, followed by phenol-chloroform extraction and isopropanol precipitation (modified from reference 26). EcoRI-generated fragments of total DNA were separated by horizontal gel electrophoresis (0.8% agarose in Trisacetate [25] buffer) for 17 h at 20 V. After ethidium bromide staining, the gel was vacuum blotted onto a nylon filter (Hybond-N [Amersham] or Biodyne A [Pall]) by using a vacuum blotting system (Pharmacia) according to the man-

ufacturer's instructions. The probes (vector and IS element) were partially digested with Sau3A and then labeled with digoxigenin-11-dUTP (Boehringer). Hybridization was done with 5 ml of hybridizing solution in a cylinder which rotated at a slow and constant speed in an oven (Bachofer) for 12 to 24 h at 68°C. Washing and immunological and color reactions were also performed in the hybridization oven at the temperatures

specified by Boehringer.

1503

RESULTS Construction of vectors and selection procedures. The structures of the positive-selection vectors used in this study

are shown in Fig. 1. Construction of pSUP104-pheS and pSUP104-rpsL involved several subcloning steps to adapt

the restriction sites at the ends of the marker gene-containing fragments to the target sites on the vector molecules and is briefly summarized in the legend to Fig. 1. One vector, pSUP104-pheS (Fig. iA), contains the E. coli pheS gene which encodes the a subunit of phenylalanyl tRNA synthetase. This enzyme is sensitive to the amino acid analog p-fluorophenylalanine (PFP). E. coli mutant RR28 (17), carrying a mutation in the chromosomal pheS gene, is PFP resistant (PFPr). In the merodiploid situation, the vector-borne, dominant pheS gene needs to be inactivated to allow growth of RR28 cells on medium containing PFP. To allow insertions to occur, pSUP104-pheS was mobilized from E. coli donor S17-1 (40) into various Smr derivatives of wild-type R. meliloti strains. Transconjugants containing pSUP104-pheS were selected and purified on TY medium containing streptomycin and tetracycline. Tcs R4 derivative RP4-7 (39) was introduced into individual R. meliloti(pSUP104-pheS) clones by conjugation from E. coli. The resulting Tcr Nmr transconjugants were grown to saturation in liquid medium containing tetracycline and neomycin to select for the presence of both plasmids. As no PFPr R. meliloti mutants were available, pSUP104pheS was mobilized by RP4-7 back to E. coli RR28 (PFP') to

select for spontaneous inactivation of the pheS gene. These transconjugants were selected on LB medium containing tetracycline to verify the presence of pSUP104-pheS, as well as PFP to select for inactivation of pheS. Selected clones

further characterized as described below. The second vector, pSUP104-rpsL (Fig. 1B), carries the E. coli rpsL (strA) gene which encodes the ribosomal protein RpsL, which is responsible for Sms. The rpsL gene is transdominant, so that Smr mutants of E. coli carrying pSUP104-rpsL become Smis. Surprisingly, the rpsL marker of pSUP104-rpsL was expressed not only in E. coli but also in non-E. coli bacteria, such as R. meliloti, R. leguminosarum, and X. campestris. Thus, in spontaneous Smr mutants of these strains, inactivation of the vector-borne rpsL gene was positively selected for and used as a method to entrap IS elements. For this purpose, pSUP104-rpsL was mobilized from E. coli S17-1 into Smr derivatives of various R. meliloti and R. leguminosarum strains and into X. campestris. Transconjugants were selected and purified at 30°C on minimal medium were

containing tetracycline. Individual clones were grown in liquid minimal medium, and 5 x 10' cells were plated on rhizobial minimal mediurn containing tetracycline and streptomycin. Smr clones were

selected and further characterized. The series of pSUP104-based entrapment vectors was complemented by construction of pSUP104-sac (Fig. 1C), which carries an npt-l sacB sacR cassette (31). The sacB gene from Bacillus subtilis codes for the exoenzyme levansucrase, which confers sucrose susceptibility to cells containing the intact gene (15). Matings were performed as described above but with various R. leguminosarum wildtype and laboratory strains. Transconjugants were selected and purified on rhizobial minimal medium plates containing neomycin. After several rounds of purification, single colonies were grown in liquid TY medium with neomycin and 1.5 x 109 to 5 x 109 cells were plated on TY plates which

J. BACTERIOL.

SIMON ET AL.

1504

Eco RI (a

A

I(01o°ioc Cla

( 830StPSE (8948st EI I

I

H.in

d III(iso)

Sam

Sal

TABLE 2. Average percentages of inactivation of the rpsL gene due to deletions (shorter than orginal vector size), point mutations (same vector size), and insertions (arger vector size)a Avg % inactivation of rpsL due to:

StrainPon mutations Deletions

HI(1850 I

Insertions

(2150)

R. meliloti 2011 R. meliloti MVII R. meliloti 102F34 R. meliloti USDA1024 R. meliloti 220-12 R. meliloti 220-13R. leguminosarum 3853 R. leguminosarum VF39 X. campestris B100

B

a

1 82 0 4 3 0

50 100 32

Plasmid sizes were determined by in-gel-lysis as

4 8

13 19 4 3 50 0

18

95 10 87 81 93 97 0 0 50

described in Materials

and Methods. (aso) Pst I Bst E I I _

(sioo)

c (11350 8st EII Hin d III (saoo) Pst I (100)

Eco RI (stool