Expression of the sulfonamide resistance gene from plasmid R46

PLASMID

23, 35-4 1 ( 1990)

Expression of the Sulfonamide

Resistance Gene from Plasmid R46

FRANCOIS GUERINEAU,’ LOUISE BROOKS, AND PHILIP MULLINEAUX John Innes Institute, AFRC Instime of Plant ScienceResearch, Colney Lane, Norwich NR4 7UH. United Kingdom Received November 27, 1989; revised January

9, 1990

The expression of the sul I gene from plasmid R46, a wide host range plasmid of the IncN incompatibility group, was studied in Escherichia coli. Using a promoter test vector, a promoter was detected upstream of the sul Igene. From a nuclease protection experiment, the transcription was determined to start 360 bp upstream of the coding sequence. Two putative promoter -35 and - 10 sequences were found upstream from the predicted transcription start. The presence of this promoter sequence in other R factors was discussed in relation with previous data showing that the sul I genes were transcribed from other promoters. The translation product of the sul2 gene was detected in minicells. Its size indicates that the translation starts at the first ATG codon found in the open reading frame. o 1990 Academic PRSS, hc.

were reported in R factors, based on hybridization profiles (Swedberg and Skold, 1983). The nucleotide sequences of the sul I genes from plasmids R388 (Sundstom et al., 1988) and R46 (Guerineau and Mullineaux, 1989) are highly homologous. The sul Z genes from R6-5 and from R388 were reported to be transcribed from the promoters of the streptomycin/spectinomycin and the trimethoprim resistance genes, respectively (Swedberg, 1987; Sundstriim et al., 1988). We show here that the sul1 gene from R46 additionally possesses its own promoter, the activity of which is sufficient to confer sulfonamide resistance.

Sulfonamides are antibacterial compounds which interfere with the metabolism of folic acid (Brown, 1962). Their structure is close to that of paminobenzoic acid, a substrate for dihydropteroate synthase, so they can act as competitive substrates for this enzyme (Roland et al., 1979). Resistance to sulfonamides (SulR) is carried by many R plasmids of the Enterobacteriaceae and is found in transposons such as Tn2Z (De La Cruz and Grinsted, 1982) or Tn2603 (Yamamoto et al., 1981). It is frequently associated with the streptomycin/ spectinomycin resistance (Barth and Grinter, 1974). The sulfonamide resistance genes code for a modified dihydropteroate synthase which is insensitive to sulfonamides (Wise and AbouDonia, 1975). Plasmid R46, a member of the IncN group isolated from Salmonella typhimurium, is 5 1.7 kb long, is self transmissible, and carries resistances to ampicillin, tetracycline, spectinomycin/streptomycin, and sulfonamides (Datta and Kontomichalou, 1965) and to arsenite, arsenate, and antimony compounds (Silver et al., 1981); it also confers protection against uv-induced damage (Drabble and Stocker, 1968). Two types of sul genes

MATERIALS

AND

METHODS

Materials Restriction enzymes were purchased from GIBCO-BRL or New England Biolabs. Alkaline phosphatase and mung bean nuclease were purchased from Boehringer-Mannheim. Radioisotopes were purchased from New England Nuclear.

Bacteria, Plasmids, and Bacteriophages Escherichia coli strain JM 10 1 (genotype: supE, thi, A(lac-proAB), [F, traD36, proAB,

’ To whom correspondence should be addressed at Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, Scotland, United Kingdom.

lacIqAM15] combinant 35

was used as the host for the replasmids. Plasmid pED961 is 0147-619X/90

$3.00

Copyright Q 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

36

GUERINEAU. BROOKS. AND MULLINEAUX

composedof pBR322 (Sutcliffe, 1978)carrying a 2.8kb EcoRI-SalI fragment from plasmid R46(Brown, 1981).M13mp18 and M13mp19 (Yanisch-Perron et al., 1985) were used for cloning. E. coli strain DS410 (genotype: m&A, minB, rpsL, sup+) was used to produce minicells (Dougan and Sherratt, 1977).

that 0.25 &i of [14C]chloramphenicol (New England Nuclear) was used in each reaction. Nuclease Protection Assay

Bacterial RNA was prepared as described by Ebina and Nakazawa (1983). A nuclease protection assaywas done using a [35S]dATPlabeled probe made from a Ml3 clone used Bacterial Cultures as a template and subsequently cut by restricTo monitor their resistanceto sulfonamides, tion enzymes, as described in Aldea et al. bacteria were cultured in liquid minimal salts (1988). A sequence ladder generated from a containing 1%glucose and asulam (methyl(C Ml3 clone was run in parallel with the proaminobenzenesulfonyl) carbamate, Grey- tected fragment. Mung bean nucleasewas used hound Chromatography and Allied Chemi- at 500 units/ml. cals, West Birkenhead, UK) at 500 pg/ml. The bacterial growth was monitored by measuring In Vivo Translation Assay the optical density at 600 nm. Expression of plasmid-encoded genes was carried out in E. coli minicells. Plasmids were transformed into E. coli strain DS4 10 (Dougan Small-scale plasmid preparations were per- and Sherratt, 1977). Minicells were prepared formed using a method described by Ish-Ho- using a protocol derived from Adler et al. rowicz and Burke (198 1) except that an ex- (1967). Proteins were labeled with [3’S]traction with phenol/chloroform (1 vol/ 1 vol) methionine and separated by SDS-PAGE’ was performed before precipitation with using standard procedure. Gels were fluoroethanol. Restriction enzymes were used ac- graphed and exposed to X-ray film. cording to the manufacturer’s instructions. RESULTS AND DISCUSSION Blunt ends were created from single-stranded extremities by treatment with T4 DNA poly- Localization of a Promoter Upstream of the merase.Vectors were dephosphorylated by alsul I Coding Sequence kaline phosphataseduring their digestion with Deletions were made in pED96 1, upstream restriction enzymes. In some cases, DNA of the sul Z gene (Fig. 1). Plasmid pBRsulA1 fragments were purified by electroelution from was obtained after a Ba131 deletion of 550 bp agarose gels as described in Maniatis et al. initiated at the EcoRI site of pED96 1. The de(1982). T4 DNA ligase was used to ligate the leted fragment was cloned into pBR322 (Fig. fragments before transformation of E. coli by 1). The EcoRI-ClaI fragment from pBR322, the CaC12method (Mandel and Higa, 1970). containing the -35 sequence of the TcR promoter (Sutcliffe, 1978), was deleted in Assayfor CAT Activity pBRsulA 1 to give pBRsulA2 (Fig. 1). Bacteria After incubation overnight, bacteria were harboring pBRsulA2 had a growth rate in the washed once in reaction buffer (Tris 0.25 M, presence of asulam at 500 pg/ml similar to pH 7.7) and lysed by sonication. The lysate those harboring pED96 1. These data indicate was centrifuged at 10,OOOg for 5 min. Protein that the deletion introduced into pED96 1 did in the supernatant was assayedby the method not affect the resistance to asulam. Conseof Bradford (1976). Aliquots containing 0.5 or 5 pg of protein were processed for the assay ’ Abbreviations used:SDS-PAGE, sodium dodecyl sulof chloramphenicol acetyltransferase activity fate-polyacrylamide gel electrophoresis;ORF, open readas described in Gorman et al. ( 1982), except ing frame.

Plasmid Preparation and Cloning

37

SULFONAMIDE RESISTANCE GENE

pBRsulA5 pBRsulA6

’

:I SUll 500bp

FIG. 1. Deletions in the sul2 upstream region. Thick lines represent R46 sequence. In pBRsulA4,5,6, thin lines show deleted regions. The vector, not represented in pBRsulA2,4,5,6, is the same as that in pBRsulA1 from the SalI site to the EcoRI site. The triangle in pBRsulA2 showsthe transcription start point, as determined in Fig. 3. T4 Pal, T4 DNA polymerase. lig., T4 DNA ligase.

quently, the sequenceremaining upstream of the sull gene in pBRsuZA2contains all the information required for the expression of the gene. Deletions were made in pBRsulA2 upstream from the putative sul I coding sequence (Fig. 1). Bacteria harboring the different constructs were incubated overnight in liquid minimal medium containing asulam at 500 pg/ml and their growth was monitored. It appeared that growth of bacteria harboring pBRsulA4 or pBRsulA5 was totally inhibited whereas bacteria carrying pBRsulA6 grew normally under the same conditions, as revealed by the optical density of the suspension. No decreasein yield was apparent from the preparation of the various deleted plasmids, suggestingthat the deletions did not affect significantly the plasmid copy number. These data could indicate that an element located in the 400-bp EcoRI-Hind111 fragment present

in pBRsulA6 but missing in pBRsulA4 and pBRsulA5 was required for the expression of the sul I gene. To determine whether or not a promoter is present upstream from the sul Z coding sequence, the whole 660-bp upstream sequence was inserted into the promoter-test plasmid pKK232-8 (Brosius, 1984) in the same orientation with respect to the cat gene as it is with respect to the suf I gene in plasmid R46 (Fig. 2a). Bacteria having this construct (pKK.sulEw showed CAT activity (Fig. 2b), suggesting that a transcriptional promoter is present in the sul I upstream sequence.A 180bp D&I subfragment from the upstream sequence was cloned into pKK232-8 in the proper orientation with respectto the cat gene, giving pKK&D (Fig. 2a). The CAT activity present in bacteria harboring this construct was close to the one recorded when the whole sul Z upstream sequence was cloned into the promoter-test plasmid (Fig. 2b). This means that the promoter activity upstream from the sul Z gene is located in the 180-bp DdeI fragment. On the basis of the amount of protein used in the CAT assaysand the relative intensity of the spots given by acetyl-chloramphenicol (Fig. 2b) determined by densitometry (data not shown), bacteria having pKK&EN or pKKmlD showed a specific CAT activity at least 20 times lower than that of bacteria having pBR328, suggestingthat the sul I promoter is much weaker than the cut promoter. Mapping of the Transcription

Start

For making the probe, a second strand complementary to the mRNA strand was synthesized using klenow polymerase and a mixture of nucleotides containing [35S] dATP, from the sequencing universal primer annealed on the single strand of a M 13mp 18 clone containing the EcoRI-NcoI fragment from pBRsulA2 (Fig. 1). The mixture was digestedwith EcoRI and NcoI and the DNA was denatured, generating a labeled single-stranded fragment corresponding to sequences1 to 666 (Guerineau and Mullineaux, 1989). This probe was used in a nuclease protection assay

38

GUERINEAU, BROOKS, AND MULLINEAUX

a pKKsulEN pKKsulD

b 1,3-AC-Cm

3-At-Cm l-AC-Cm

Cm origin $I4326

pKKZ3S8 pKKdEN

pKkulO

FIG. 2. Promoter activity in sul I upstream sequences.(a) Organization of pKK.ruZENand pKKsulD. The filled-in EcoRI-NcoI fragment or the filled-in 180-bp DdeI fragment from pBRsulA2 (Fig. I) was purified and inserted into the SmaI site of pKK232-8, creating, respectively,pBR.sulENand pBR.sulD.The orientation of the inserts was checked with Hind111 or by nucleotide sequencing. The numbers refer to the nucleotide sequence (Guerineau and Mullineaux, 1989). mcs, multiple cloning sites. (b) Autoradiograph after assays for CAT activity in bacteria harboring the recombinant plasmids. Extracts were prepared and assayswere performed as indicated under Materials and Methods. 0.5 .ugof protein was used for the assay in the case of pBR328, 5 rg in the other cases.AC-Cm, acetyl-chloramphenicol.

with RNA prepared from bacteria harboring pED96 1 or not. A protected fragment of approximately 363 baseswas visible only in the case of bacteria harboring pED961 (Fig. 3), giving a transcription start presumably at the adenine residue in position 304, 360 bp upstream from the sul I coding sequence. Examination of the sequenceupstream from the predicted transcription start reveals the presence of weak consensuswith E. coli -35 and - 10 promoter regions (Harley and Reynolds, 1987; Oliphant and Struhl, 1988). Two candidates are suggestedas putative -35 and - 10 sequences(Fig. 4). This experiment confirms the presenceof a promoter upstream from the ml I coding sequence. The low homology of the sul I sequences with the consensus sequence might explain the low CAT activity detected in the promoter test experiment.

The sul Z promoter sequence,also present in plasmids R538-1 (Hollingshead and Vapnek, 1985), R1767 (Nucken et al., 1989), and pSa (Tait et al., 1985) and in the IncC plasmid pDGOlO0 (Cameron et al., 1986), is presumably conserved between all plasmids carrying Tn21-like transposons (Ouellette et al., 1987; Hall and Vockler, 1987), indicating that the sul I genespresent in these plasmids are probably transcribed from their own promoter. This finding is to be related to the data presented by Swedberg(1987) and Sundstrom et al. (1988) which show that the ml I genesfrom plasmid R388 and R6-5 are transcribed, respectively, from the promoters of the trimethoprim resistance (d/z$%) and streptomycin/ spectinomycin resistancegenes(aadA), located further than 1.5 kb upstream from the sul Z gene. The fact that the sequencein which we

39

SULFONAMIDE RESISTANCE GENE

a

b

TCGA

consensus

sequence: -35 -10 (6-9 TTGACA...(16-1*. . . . . . . bp) . . . . . . ..TATAAT........CAT

bpl

&

~ATTATTTTTMGCGTGCCTAAGCCCTACAC 306

265,

FIG. 4. Putative -35 and -10 sequencesof the sul I promoter. E. coli -35 and - 10 consensussequencesare from Harley and Reynolds (1987) and the transcription start consensussequence is from Hawley and McClure (1983). Candidates for -35 and -10 sequencesin the sul I sequenceare underlined. Arrows indicate transcription starts. Numbers refer to nucleotide sequence(Guerineau and Mullineaux, 1989).

cated downstream from the oxa (@-lactamase) and aadA genes,suggeststhat the transcription of the ml Z genes present in these plasmids can also occur from two promoters. Translation in a Minicell System

FIG. 3. Transcript mapping. RNA prepared from bacteria harboring no plasmid (lane a) or pED961 (lane b) was hybridized with a probe prepared asdescribedin Aldea et al. ( 1988) from a M 13 clone containing the I&RINcoI fragment from pBRsulA2 (Fig. 1) and treated with mung bean nucleate. The mixtures were run on a 6% (w/ v) polyacrylamide-urea gel, in parallel with sequencing reaction mixtures (lanes T, C, G, and A) of a M I3 clone. The sizes in nucleotides are indicated on the right of the ladder. The band in lane a and the upper band in lane b are made by the probe, and the lower band shown by an arrow in lane b is made by the protected fragment. The nucleotide sequencearound position 360 is indicated on the right of the coordinate.

In vivo translation experiments were done using minicells prepared from bacteria harboring pBR322, pBRsulA2, pBRsulA5, pBRsulA6, or no plasmid. The major band seenon Fig. 5 (lanes 2 to 5) is given by the plactamase encoded by pBR322. Its molecular weight is about 27 kDa (Sutcliffe, 1978). Another protein of approximately 30 kDa was synthesized in the minicells harboring 1

2

3

4

5

75502717-

found a promoter is also present in plasmid R388 (Sundstrem et al., 1988) suggeststhat the sul Z gene present in this plasmid may be transcribed from two distinct promoters. A similar organization in various R plasmids (Ouellette et al., 1987), including R46 (Brown and Willetts, 1981) where the sul Z gene is lo-

FIG. 5. Translation in minicells harboring no plasmid (lane I), pBR322 (lane 2), pBRsulA2 (lane 3), pBItrulA6 (lane 4), or pBRsulA5 (lane 5). Minicells were incubated with 10&i of [?$I methionine and labeled proteins were separated by PAGE. Sizes in kDa, indicated on the left side of the panel, were given by bovine serum albumin (75 kDa), ovalbumin (50 kDa), soybean trypsin inhibitor (27 kDa), and lysozyme (17 kDa).

40

GUERINEAU, BROOKS, AND MULLINEAUX

pBRsuZA2or pBRsuZA6but not in the caseof bacteria harboring pBRsuZA5in which the sequence containing the ml Zpromoter is absent. The size of 30 kDa is consistent with the predicted size of the protein when the translation is initiated at the first ATG codon of the longest ORF present in the sequence.Initiation at the secondATG would give a size of 26.5 kDa. Although a small ORF present upstream from the sul Z gene is transcribed and is in a favorable context for being translated, no corresponding band could be seen on the overexposed fluorogram. The size of the E. coli dihydropteroate synthase was found to be approximately 50 kDa (Richey and Brown, 1969). The size of 30 kDa reported here either could mean that the enzyme is a dimer or could indicate that the mutation creating the resistanceto sulfonamides has affectedthe size of the product. Alternatively, the resistant enzyme could be unrelated to the sensitive host enzyme. ACKNOWLEDGMENTS We thank N. Willetts for pED96 1, D. Sherratt for strain DS4 10, and G. Machray for critical reading of the manuscript. This work was funded by the DTI consortium “Plant Gene Tool Kit.” REFERENCES ADLER, H. I., FISHER,W. D., COHEN,A., AND HARDI-

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