PLASMID
27, 141-154 (1992)
Heterologous
Gene Expression
in Bacteroides
fagi/&
C. JEFFREY SMITH, MARC B. ROGERS, AND MARIAN L. MCKEE’ Department of Microbiology and Immunology, East Carolina University, Greenville, North Carolina 27858 Received August 26, 199 1; revised September 20, 199 1
Eacteroides jkagilis and other gastrointestinal tract Bacteroides are unusual gram-negative eubacteria in that genes from other gram-negative eubacteria are not expressed when introduced into these organisms. To analyze gene expression in Bucteroides, expression vector and promoter probe (detection) vector systems were developed. The essential feature of the expression vector was the incorporation of a Bacteroides insertion sequence element, IS4351, which possessespromoter activity directed outward from its ends. Genes inserted into the multiple cloning site downstream from an IS4351 DNA fragment were readily expressed in B. fragilis. The chloramphenicol acetyltransferase (cut) structural gene from Tn9 was tested and conferred chloramphenicol resistance on B. frugiks. Both chloramphenicol resistance and CAT activity were shown to be dependent on the IS4351 promoters. Similar results were obtained with the Escherichiu coli &glucuronidase gene (uidA) but activity was just 30% ofthe levels seen with cat. Two tetracycline resistance determinants, tetM from Streptococcusagalactiae and tetC from E. co/i, also were examined. tetC did not result in detectable tetracycline resistance but the grampositive tetMgene conferred high-level resistance to tetracycline and minocycline in Bacteroides hosts. Based on the cut results, promoter probe vectors containing the promoterless cut gene were constructed. These vectors were used to clone random B. fragilis promoters from partial genomic libraries and the recombinants displayed a range of CAT activities and chloramphenico1MICs in B. fragilis hosts. In addition, known E. coli promoters (Ptet, Ptac, Ptrc, Psyn, and Pl P2rrnB) were tested for activity in B. fragilis. No chloramphenicol resistance or CAT activity was observed in B. frugilis with these promoters. 0 1992 Academic Press. Inc
The anaerobic intestinal tract Bacteroides species, including Bacteroides fragilis, are a phylogenetically unique group which diverged very early from the main eubacterial line (Paster et al., 1985; Weisburg et al., 1985). An expected consequence of this early divergence might be the evolution of unique genetic mechanisms for the control of gene expression either at the level of transcription or translation. In support of this are the numerous examples in which heterologous genesare not expressedin Bacteroides. In an extensive survey of tetracycline resistance genes, including tetA, B, C, M, and P, only the streptococcal tetM gene resulted in resistant Bacteroides but only to a level of 0.5 pg/ ml (Guiney et al., 1988). Similarly, Bacteroides shuttle vectors bearing a variety ofEscherichia coli markers such as P-lactam ’ Present address: Department of Microbiology, Uniformed Services University of Health Science, Bethesda, MD 208 14.
resistance, aminoglycoside resistance, and trimethoprim resistance do not appear to express these drug resistances in Bacteroides hosts (Guiney et al., 1984a; Shoemaker et al., 1986b, 1989; Smith, 1985b; Smith and Spiegel, 1987). Although the bulk of this work has dealt with the expression of foreign antibiotic resistance determinants, the phenomenon is not restricted to this classof genes.For example, endonuclease, EcoRZ, under control of the X pR promoter is not expressedwhen present on the shuttle vector pJST6 1 (Thompson and Malamy, 1990). Moreover, the lac promoter fails to activate ermFS transcription in Bacteroides (Smith, 1987) and R751 integrated into the Bacteroides chromosome does not appear to expressany of the conjugation functions or antibiotic resistances(Shoemaker et al., 1986a). There also seemsto be some restriction on the ability of E. coli to express genes of Bacteroides origin. Again much of the evidence for this comes from, but is not limited to,
141
0147-619X/92
$3.00
Copynght 0 1992 by Academic Press, Inc. All rights of reproduction in any form reeen ied.
142
SMITH,
ROGERS.
antibiotic resistance determinants. The most common example is the lack of erm expression in E. cob, where cloned copies of the gene, even under control of the lac promoter, do not confer clindamycin or erythromycin resistance in E. coli (e.g., Guiney et al., 1984a; Smith, 1985b, 1987). This is complicated further by the fact that adjacent to ermF in Tn4351 is a tetracycline resistance gene which is expressed in E. coli but not in Bacteroides (Guiney et al., 1984a,b). More recently, cloned tetracycline and cefoxitin resistanceshave failed to be expressedin E. coli (Guiney et al., 1989; Shoemaker et al., 1989; Thompson and Malamy, 1990). Other types of cloned genes such as chondroitin lyase II (Guthrie et al., 1985) and neuraminidase (Russo et al., 1990) are expressedvery poorly or not at all from their indigenous promoters but the cloned glutamine synthetasewas both expressed and nitrogen-regulated in E. coli (Southern et al., 1986). Clearly the explanation for differential expression of genes in these two organisms will not be simple and may in fact involve several components. However, it has not been possible to carefully examine gene expression in Bacteroidesdue to the lack of genetic tools suitable for the analysis of promoter function and activity. Often in nature, antibiotic resistance determinants are disseminated horizontally across genus lines. Bacteroides is no exception to this and both erm (Rasmussen et al., 1986) and the P-lactamase, cfiA (Thompson and Malamy, 1990), appear to be of gram-positive origin. The successfulutilization of these genes must have required a genetic event which enhanced their expression in the Bacteroides host and in the case of erm, this event was the integration of a transcriptionally active 184352 element adjacent to the gene (Rasmussen et al., 1986, 1987; Smith, 1987). IS4351 is a component of the erm transposon Tn4351 (Rasmussen et al., 1987) and is identical to DRS455 1-R in Tn4552 (Smith, 1987). This element has promoters directed outward from both ends and is capable of activating transcription of neighboring genesin both E. coli and Bacteroides.In this
AND
MCKEE
report we have taken advantage of these IS4351 features for the construction of expression vectors designed to analyze heterologous gene expression in Bacteroides. Application of this system showed clearly that a variety of foreign genescan be expressedin B. fragilis when given the appropriate transcription initiation signals. This allowed us to create vectors with multiple selective markers for use in the analysis of Bacteroides promoter activity and to examine the activity of E. coli consensus promoters in B. fragilis. MATERIALS
AND METHODS
Bacterial strains and media. The relevant properties of the bacterial strains and plasmids used in this study are presented in Table 1. For experiments using Bacteroidesspecies, cultures were grown in an anaerobic chamber (Coy Laboratory Products, Inc., Ann Arbor, MI) in brain-heart infusion broth supplemented with hemin, menadione, cysteine, NaHCO,, and agar when required (Smith, 1985a). E. coli strains were grown aerobically in L-broth (agar) as described previously (Smith, 1985b). The following antibiotic concentrations (pg/ml) were used except where indicated in the text: clindamycin (Cc), 5.0; tetracycline (Tc), 5.0; chloramphenicol (Cm), 10; rifampicin (Rf), 20; gentamycin (Gn), 25; spectinomycin (Sp), 40; and ampicillin (Ap), 50. Cc was a gift from the Upjohn Co. (Kalamazoo, MI) and ah other antibiotics were from Sigma Chemical Co. (St. Louis, MO). The 5-Bromo-4-chloro3-indolyl @-glucuronide (X-glut) was added to a final concentration of 40 pg/ml. Antibiotic MICs were determined by agar dilution after 48 h of growth on supplemented brain-heart infusion plates (B. fragilis) or 24 h of growth on L-agar (E. coli). Transformation and conjugation conditions. Plasmid transformation of B. fragilis 638 was performed by the polyethylene glycol-facilitated method (Smith, 1985~) and E. coli transformations were done according to the protocol of Hanahan (1983). Conjugal transfer of plasmids from E. coli to Bacte-
B. jzgilis
143
GENE EXPRESSION TABLE 1
BACTERIALSTRAINSANDPLASMIDS Strain or plasmid
Relevant propertiesn
B. jiiagilis strains 638 v503 v531 IB143
Rf Cc’, Tc’, Ap’ Tc’, Ap’ Tc’, Rf; (progeny from 638 x IB 138 mating)
Privitera et al. (1979) Mays et al. ( 1982) Welch et al. (1979) Smith (1985b)
E. coli strains DH5ol Sp200
recA, hsdRl7, Iacu&l
GIBCO/BRL Bardonnet et al. ( 1988)
Cc’, Ap’, 6.7 kb Ap’, source of uidA Tc’, Km’, contains 5 kb HincII insert from pJI2 cloned into pACYCl77, source of tetM Sp’, Cc’, /UC+,on-f, a pUC19/pBI143 chimera, 8.8 kb Ap’, Cc’, 184351 promoter Ap’, Cc’, oriT, IS4351 promoter Sp’, Cc’, oriT, cut reporter gene Sp’, Cc’, oriT, rrnB terminators, cut reporter gene pFD290 with cut pFD290 with tetC pFD290 with tetM pFD340 with uidA
Smith, 198513 Bardonnet et al. ( 1988) Burdett et al. (1982)
Plasmids pFD167 pNB4 pJI3 pFD288 pFD290 pFD340 pFD325 pFD325TT pFD308 pFD309 pFD310 pFD344
Source or reference
Smith (1989) This This This This
report report report report
This This This This
report report report report
a Antibiotic resistance markers not described in text are Km, kanamycin; and Tp, trimethoprim.
roides was accomplished by triparental matings using standard filter mating protocols (Shoemaker et al., 1986a; Smith et al., 1982). Generally for these matings, plasmid-containing E. coli donors were sedimented on filters together with helper strains containing either RR231 or R751 and the recipient B. fragilis 638 in a ratio of 0.5/0.5/1.0. Mating plates were incubated aerobically for 18 h, and cells were washed from filters and then plated on selective media. For shotgun cloning experiments with pFD325, total genomic DNA from the appropriate B. jiragilis strains was completely digested with Sau3A, ligated to the BamHIdigested vector, and then transformed into E. coli DHSa with selection on spectinomycin. Recombinant E. coli clones were plated at a density of 100-300 colonies per plate and
after 18-24 h incubation, plates were scraped with a sterile spatula and suspended in 10 ml of L-broth. This was shaken at 37°C for l-2 h and aliquots were used for matings with RK231 and B. fragilis 638 as described above. Transconjugants from these matings were selected on plates containing Cm, Cc, Rf, and Gn. Preparation, analysis, and manipulation of DNA. Plasmid DNAs were purified from E. coli by density gradient centrifugation (Radloff et al., 1967) of crude lysates prepared by the alkaline lysis method (Maniatis et al., 1982). Routine screening of E. coli transformants was done according to Birnboim and Doly (1979) and B. fragilis strains were screened either by alkaline lysis (Birnboim and Doly, 1979) or by the high-salt-sodium dodecyl sulfate method (Welch and Macrina,
144
SMITH,
ROGERS,
1981). Total B. frugilis genomic DNA was purified from 50-ml cultures by CsCl density centrifugation asdescribed previously (Smith and Spiegel, 1987). DNA samples were analyzed by agarose gel electrophoresis using Tris-borate or Tris-acetate buffers (Maniatis et al., 1982). Analysis of small restriction fragments (~500 bp) was performed on 3-4s NuSieve GTG agarose (FMC BioProducts) slab gels. Restriction endonuclease digestions and T4 ligation reactions were performed according to the supplier’s (GIBCO/BRL, Gaithersburg, MD) specifications. Individual restriction fragments to be used for plasmid constructions were purified from agarosegels by absorption onto glassbeads (Gene Clean, Bio 101, La Jolla, CA). When required, DNA fragments were blunt-ended using the Klenow fragment of DNA polymerase I (Maniatis et al., 1982). Cloning of known promoter fragments in pFD325TT. E. coli promoters known to be highly active were cloned into pFD325TT as follows. The Tc’ promoter from pBR322 (Ptet) was isolated on a 188-bp EcoRI/ EcoRV fragment (bp 436 1-185; Sutcliffe, 1979), blunt-ended, and ligated into the SmaI site of pFD325TT. This placed the - 10 region about 180 bp from the cat start codon. The tat promoter (Ptac) was obtained by digestion of pKK223-3 (Pharmacia/LKB, Inc.) with BumHI (Brosius and Holy, 1984; deBoer et al., 1983). This 280-bp fragment was blunt-ended and cloned into the SmaI site. This placed the -10 sequence about 75 bp upstream from the cat start codon. The trc promoter (Ptrc) was obtained by digesting pKK233-2 (Amann and Brosius, 1985) with sph1 and &I. The resulting 5 17-bp fragment was ligated into pFD325TT digested with the same two enzymes. This construct placed the - 10 site about 80 bp from the cat start site. The synthetic promoter (Psyn; Rossi et al., 1983) was obtained by digestion of pYEJOO1 (Pharmacia/LKB, Inc.) with SspI and PvuII and the resulting 460-bp fragment was cloned into the pFD325TT SmaI site. The Psyn - 10 sequence was about 180 bp from the cat start codon. The Pl and P2 promoters
AND
MCKEE
from rrnB were ligated into pFD325TT on an 1800-bp PstI/SmuI fragment obtained from pKIS3535 (Brosius et al., 1981; Table 1). In this construct the - 10 regions for Pl and P2, respectively, were about 940 and 820 bp upstream from the cut start codon. Finally, the Staphylococcus uureus protein A promoter (Uhlen et al., 1984) was cloned into the pFD325TT SmuI site. The source of this promoter was a 450-bp SspI/RsuI fragment obtained from pRIT5 (Pharmacia/LKB, Inc.). The putative protein A promoter (Ppta) - 10 region(s) was located about 130- 135 bp upstream from the cut start codon. Individual clones bearing each of the promoters were obtained by transformation of E. coli with selection on media containing Sp and 200 pg/ml Cm. The orientation of all cloned promoter fragments was verified by restriction mapping. Preparation of cell extracts and enzyme assays. Cell-free extracts for chloramphenicol acetyltransferase (CAT) assayswere prepared from 100-200 ml midlogarithmic phase cultures. Cultures were sedimented and then washed in an equal volume of TD buffer (50 mM Tris, 0.03 mM dithiothreitol, pH 7.8). Cell pellets were then suspended in 3 ml of TD buffer and lysed by passage through a French pressure cell (12,000 lb/in.*). Cellular debris were removed by centrifugation for 15 min at 12,OOOg,4°C. The 5,5’-dithiobis(2-nitrobenzoic acid) assay of Shaw (1975) was used for measurement of CAT activity. Assays were performed at 37°C by recording (DU65, Beckman Instruments, Inc.) the increase in AdI of reaction mixtures with and without Cm added. Results were calculated using the 5-thio-2-nitrobenzoic acid molar extinction coefficient of 13,600 at 4 12 nm. One unit of activity is defined as the amount of enzyme catalyzing the formation of 1 nmol product per minute in the standard assay. Cell-free extracts for P-glucuronidase assayswere prepared from 1O-20 ml midlogarithmic phase cultures. Cultures were sedimented, washed in 50 mM sodium phosphate buffer (pH 7.0) and then suspended in 3 ml
B. frugilis GENE EXPRESSION of the same buffer. Cells were disrupted by sonication and cellular debris were removed by centrifugation for 15 min at 12,OOOg, 4°C. Activity was measured using an endpoint assay with the substrate pnitrophenyl #I-D&curonide (Novel and Novel, 1976). One-milliliter reaction mixtures containing 50 mrvfsodium phosphate buffer (pH 7.0), 4 mM 2-mercaptoethanol, 2.5 mM p-nitophenyl /3D-glucuronide, and enzyme were incubated at 37°C and the reaction was terminated by the addition of 0.5 ml of 1 M Na$ZO,. The AdI0 was recorded and activity determined from the nitrophenol molar extinction coefficient (E = 4680). One unit of activity is the amount of enzyme required for the formation of 1 nmol nitrophenol per minute. For whole cell assays,cultures were washed and suspendedin buffer as above, l/20 vol of toluene was added, and the mixture was vortexed for 15-30 s. The ,&glucuronidase reaction conditions were as described above except that following the addition of Na,CO,, cells were removed by centrifugation. Protein concentrations of cell-free extracts were determined by the method of Bradford (1976). The method of Lowry et al. (195 1) was used to measure the protein concentration of whole cells solubilized in 0.2 N NaOH. RESULTS
Construction of expression vectorspFD290
and pFD340. We have shown previously that IS4351 contains at least two promoters active in Bacteroides (Smith, 1987). This has allowed us to design a vector incorporating these promoter sequencesand to test the expression of heterologous genes in Bacteroides. The starting point for construction of the expression vector was pFDl67 (Smith, 1985b), a 6.3-kb shuttle plasmid composed of the E. coli replicon pUC 19 and the cryptic Bacteroides plasmid pBIl43. For selection in Bacteroides, the ermFS structural gene (Ccr) was inserted into a unique ClaI site within the pBI143 portion of the molecule. One end of this gene fragment starts just 25 bp upstream from the ermFS ATG start codon,
145
and presumably ermFS is transcribed by an endogenous promoter located within the pBIl43 sequences(Smith, 1987). To provide promoter activity for foreign genes, a l-6-kb PstI/EcoRI fragment from pFDl36 (Smith, 1985a) was inserted by blunt-end ligation into the Hind1 site located in the MCS of pFDl67. This DNA fragment contains an entire copy of IS4351 less27 bp from the end of the element. The exact endpoint of the blunt-ended EcoRI site in this fragment (3’ end) corresponds to base number 1159 (see Fig. 2 in Smith, 1987) and the endpoint for the PstI site (5’ end) is not known precisely but lies about -600 bp from bp 1 in our previously published sequence(Smith, 1987). This construct, pFD290 (Fig. IA), has the IS4351 promoters conveniently placed upstream of unique cloning sites for BamHI, SmaI, KpnI, and SstI. Further, the blunt-end ligation junction produced at the EcoRI end of the IS fragment resulted in the formation of a translation stop signal, TGA, in the putative transposase reading frame. Translation stop signals in the other two reading frames are located 8 and 101 bp upstream from the 3’ end of the IS element. Although pFD290 can be mobilized from E. coli to Bacteroides using RR23 1 or R75 1 (Smith, unpublished observations; Shoemaker et al., 1986b), we sought to improve the mobilization frequency by insertion of an E. coli transfer origin into pFD290. This was accomplished by the isolation of a 0.75kb HaeII fragment from pDP1 (Guiney et al., 1984a) bearing the RK2 oriTand insertion of this into a HaeII site on pFD290. The resulting plasmid, pFD340 (Fig. IA), was otherwise identical to pFD290. Expression of cat in B. jiiagilis. Naturally occurring Cm’ Bacteroides possessa low level CAT activity (Britz and Wilkinson, 1978b; Martinez-Suarez et al., 1985); thus, it seemed reasonable that a foreign cat gene should confer a detectable phenotype in Bacteroides if it could be expressed.Therefore the expression vector was first tested by insertion of the Tn9 cat structural gene into the unique pFD290 SmaI site. This was accomplished
146
ROGERS, AND MCKEE
A
8600
bp
‘rnFS
B
0 kbl
1
2
3
FIG. 1. Restriction cleavage site maps of the expression vectors pFD290, pFD340, and their derivatives. (A) Circular maps of the plasmids are shown with the approximate locations of several relevant restriction sites. The restriction site locations were deduced by standard mapping techniques (Maniatis et ab, 1982) and from published sequence data hut for the sake of clarity not all restriction sites are shown. The Smal (MCS) region contains the following unique cloning sites: BarnHI, Seal, Kpnl, and Sstl. The Pstl (MCS) region contains unique sites for Pstl and Sphl. pB1143 sequences are indicated by the stippled boxes; pUC 19sequencesare indicated by the thin line; ermFS and Apr (AMP) sequencesare indicated by the open boxes with arrows showing the direction of transcription; IS4351 sequencesare indicated by the solid boxes with an arrow showing the approximate location and direction of transcription of the putative transposase; oriT sequences are indicated by the hashed-marked box; and the pUC19 replication origin, oriV, is indicated by an arrow. (B) Partial restriction maps of expression vector derivatives showing the insertion sites of the cloned indicator genes. Vector sequences are shown by the striped boxes and the IS4351 segments are labeled IS; the indicator genesare shown by the open boxes (structural genes) and thin lines (flanking sequences).The MCS designation indicates the location of the Smal junction used for construction of these derivatives and corresponds to bp 4060 in the maps shown in (A). The scale in kb is given at the bottom of the figure.
by blunt-end ligation of a 77 5-bp TaqI restriction fragment obtained from pACYC 184 (Chang and Cohen, 1978). This gene fragment starts 29 bp upstream from the start of cat and includes the ribosome binding site but no obvious promoter signals (Alton and Vapnek, 1979). Becausepromoter sequences
located within the IS element also function in E. coli, cloning was simplified by the capability of direct selection of E. cob transformants on Cm. The resulting plasmid, pFD308 (Fig. lB), was then introduced into B. jiiagilis via transformation with selection on a Cc-containing medium. The plasmid content of sev-
147
B. frugilis GENE EXPRESSION TABLE 2 CHLQRAMPHENICOL MICs AND CHLORAMPHENICOL ACETYLTRANS~RA~EACTIVITIESFORB. fragilis 638 CONTAINING pFD29O:cut FUSIONS Plasmid
MIC”
None pFD290 pFD308 pFD308AIS
4 4 128 4
CAT (SPa#
27, 141-154 (1992)
Heterologous
Gene Expression
in Bacteroides
fagi/&
C. JEFFREY SMITH, MARC B. ROGERS, AND MARIAN L. MCKEE’ Department of Microbiology and Immunology, East Carolina University, Greenville, North Carolina 27858 Received August 26, 199 1; revised September 20, 199 1
Eacteroides jkagilis and other gastrointestinal tract Bacteroides are unusual gram-negative eubacteria in that genes from other gram-negative eubacteria are not expressed when introduced into these organisms. To analyze gene expression in Bucteroides, expression vector and promoter probe (detection) vector systems were developed. The essential feature of the expression vector was the incorporation of a Bacteroides insertion sequence element, IS4351, which possessespromoter activity directed outward from its ends. Genes inserted into the multiple cloning site downstream from an IS4351 DNA fragment were readily expressed in B. fragilis. The chloramphenicol acetyltransferase (cut) structural gene from Tn9 was tested and conferred chloramphenicol resistance on B. frugiks. Both chloramphenicol resistance and CAT activity were shown to be dependent on the IS4351 promoters. Similar results were obtained with the Escherichiu coli &glucuronidase gene (uidA) but activity was just 30% ofthe levels seen with cat. Two tetracycline resistance determinants, tetM from Streptococcusagalactiae and tetC from E. co/i, also were examined. tetC did not result in detectable tetracycline resistance but the grampositive tetMgene conferred high-level resistance to tetracycline and minocycline in Bacteroides hosts. Based on the cut results, promoter probe vectors containing the promoterless cut gene were constructed. These vectors were used to clone random B. fragilis promoters from partial genomic libraries and the recombinants displayed a range of CAT activities and chloramphenico1MICs in B. fragilis hosts. In addition, known E. coli promoters (Ptet, Ptac, Ptrc, Psyn, and Pl P2rrnB) were tested for activity in B. fragilis. No chloramphenicol resistance or CAT activity was observed in B. frugilis with these promoters. 0 1992 Academic Press. Inc
The anaerobic intestinal tract Bacteroides species, including Bacteroides fragilis, are a phylogenetically unique group which diverged very early from the main eubacterial line (Paster et al., 1985; Weisburg et al., 1985). An expected consequence of this early divergence might be the evolution of unique genetic mechanisms for the control of gene expression either at the level of transcription or translation. In support of this are the numerous examples in which heterologous genesare not expressedin Bacteroides. In an extensive survey of tetracycline resistance genes, including tetA, B, C, M, and P, only the streptococcal tetM gene resulted in resistant Bacteroides but only to a level of 0.5 pg/ ml (Guiney et al., 1988). Similarly, Bacteroides shuttle vectors bearing a variety ofEscherichia coli markers such as P-lactam ’ Present address: Department of Microbiology, Uniformed Services University of Health Science, Bethesda, MD 208 14.
resistance, aminoglycoside resistance, and trimethoprim resistance do not appear to express these drug resistances in Bacteroides hosts (Guiney et al., 1984a; Shoemaker et al., 1986b, 1989; Smith, 1985b; Smith and Spiegel, 1987). Although the bulk of this work has dealt with the expression of foreign antibiotic resistance determinants, the phenomenon is not restricted to this classof genes.For example, endonuclease, EcoRZ, under control of the X pR promoter is not expressedwhen present on the shuttle vector pJST6 1 (Thompson and Malamy, 1990). Moreover, the lac promoter fails to activate ermFS transcription in Bacteroides (Smith, 1987) and R751 integrated into the Bacteroides chromosome does not appear to expressany of the conjugation functions or antibiotic resistances(Shoemaker et al., 1986a). There also seemsto be some restriction on the ability of E. coli to express genes of Bacteroides origin. Again much of the evidence for this comes from, but is not limited to,
141
0147-619X/92
$3.00
Copynght 0 1992 by Academic Press, Inc. All rights of reproduction in any form reeen ied.
142
SMITH,
ROGERS.
antibiotic resistance determinants. The most common example is the lack of erm expression in E. cob, where cloned copies of the gene, even under control of the lac promoter, do not confer clindamycin or erythromycin resistance in E. coli (e.g., Guiney et al., 1984a; Smith, 1985b, 1987). This is complicated further by the fact that adjacent to ermF in Tn4351 is a tetracycline resistance gene which is expressed in E. coli but not in Bacteroides (Guiney et al., 1984a,b). More recently, cloned tetracycline and cefoxitin resistanceshave failed to be expressedin E. coli (Guiney et al., 1989; Shoemaker et al., 1989; Thompson and Malamy, 1990). Other types of cloned genes such as chondroitin lyase II (Guthrie et al., 1985) and neuraminidase (Russo et al., 1990) are expressedvery poorly or not at all from their indigenous promoters but the cloned glutamine synthetasewas both expressed and nitrogen-regulated in E. coli (Southern et al., 1986). Clearly the explanation for differential expression of genes in these two organisms will not be simple and may in fact involve several components. However, it has not been possible to carefully examine gene expression in Bacteroidesdue to the lack of genetic tools suitable for the analysis of promoter function and activity. Often in nature, antibiotic resistance determinants are disseminated horizontally across genus lines. Bacteroides is no exception to this and both erm (Rasmussen et al., 1986) and the P-lactamase, cfiA (Thompson and Malamy, 1990), appear to be of gram-positive origin. The successfulutilization of these genes must have required a genetic event which enhanced their expression in the Bacteroides host and in the case of erm, this event was the integration of a transcriptionally active 184352 element adjacent to the gene (Rasmussen et al., 1986, 1987; Smith, 1987). IS4351 is a component of the erm transposon Tn4351 (Rasmussen et al., 1987) and is identical to DRS455 1-R in Tn4552 (Smith, 1987). This element has promoters directed outward from both ends and is capable of activating transcription of neighboring genesin both E. coli and Bacteroides.In this
AND
MCKEE
report we have taken advantage of these IS4351 features for the construction of expression vectors designed to analyze heterologous gene expression in Bacteroides. Application of this system showed clearly that a variety of foreign genescan be expressedin B. fragilis when given the appropriate transcription initiation signals. This allowed us to create vectors with multiple selective markers for use in the analysis of Bacteroides promoter activity and to examine the activity of E. coli consensus promoters in B. fragilis. MATERIALS
AND METHODS
Bacterial strains and media. The relevant properties of the bacterial strains and plasmids used in this study are presented in Table 1. For experiments using Bacteroidesspecies, cultures were grown in an anaerobic chamber (Coy Laboratory Products, Inc., Ann Arbor, MI) in brain-heart infusion broth supplemented with hemin, menadione, cysteine, NaHCO,, and agar when required (Smith, 1985a). E. coli strains were grown aerobically in L-broth (agar) as described previously (Smith, 1985b). The following antibiotic concentrations (pg/ml) were used except where indicated in the text: clindamycin (Cc), 5.0; tetracycline (Tc), 5.0; chloramphenicol (Cm), 10; rifampicin (Rf), 20; gentamycin (Gn), 25; spectinomycin (Sp), 40; and ampicillin (Ap), 50. Cc was a gift from the Upjohn Co. (Kalamazoo, MI) and ah other antibiotics were from Sigma Chemical Co. (St. Louis, MO). The 5-Bromo-4-chloro3-indolyl @-glucuronide (X-glut) was added to a final concentration of 40 pg/ml. Antibiotic MICs were determined by agar dilution after 48 h of growth on supplemented brain-heart infusion plates (B. fragilis) or 24 h of growth on L-agar (E. coli). Transformation and conjugation conditions. Plasmid transformation of B. fragilis 638 was performed by the polyethylene glycol-facilitated method (Smith, 1985~) and E. coli transformations were done according to the protocol of Hanahan (1983). Conjugal transfer of plasmids from E. coli to Bacte-
B. jzgilis
143
GENE EXPRESSION TABLE 1
BACTERIALSTRAINSANDPLASMIDS Strain or plasmid
Relevant propertiesn
B. jiiagilis strains 638 v503 v531 IB143
Rf Cc’, Tc’, Ap’ Tc’, Ap’ Tc’, Rf; (progeny from 638 x IB 138 mating)
Privitera et al. (1979) Mays et al. ( 1982) Welch et al. (1979) Smith (1985b)
E. coli strains DH5ol Sp200
recA, hsdRl7, Iacu&l
GIBCO/BRL Bardonnet et al. ( 1988)
Cc’, Ap’, 6.7 kb Ap’, source of uidA Tc’, Km’, contains 5 kb HincII insert from pJI2 cloned into pACYCl77, source of tetM Sp’, Cc’, /UC+,on-f, a pUC19/pBI143 chimera, 8.8 kb Ap’, Cc’, 184351 promoter Ap’, Cc’, oriT, IS4351 promoter Sp’, Cc’, oriT, cut reporter gene Sp’, Cc’, oriT, rrnB terminators, cut reporter gene pFD290 with cut pFD290 with tetC pFD290 with tetM pFD340 with uidA
Smith, 198513 Bardonnet et al. ( 1988) Burdett et al. (1982)
Plasmids pFD167 pNB4 pJI3 pFD288 pFD290 pFD340 pFD325 pFD325TT pFD308 pFD309 pFD310 pFD344
Source or reference
Smith (1989) This This This This
report report report report
This This This This
report report report report
a Antibiotic resistance markers not described in text are Km, kanamycin; and Tp, trimethoprim.
roides was accomplished by triparental matings using standard filter mating protocols (Shoemaker et al., 1986a; Smith et al., 1982). Generally for these matings, plasmid-containing E. coli donors were sedimented on filters together with helper strains containing either RR231 or R751 and the recipient B. fragilis 638 in a ratio of 0.5/0.5/1.0. Mating plates were incubated aerobically for 18 h, and cells were washed from filters and then plated on selective media. For shotgun cloning experiments with pFD325, total genomic DNA from the appropriate B. jiragilis strains was completely digested with Sau3A, ligated to the BamHIdigested vector, and then transformed into E. coli DHSa with selection on spectinomycin. Recombinant E. coli clones were plated at a density of 100-300 colonies per plate and
after 18-24 h incubation, plates were scraped with a sterile spatula and suspended in 10 ml of L-broth. This was shaken at 37°C for l-2 h and aliquots were used for matings with RK231 and B. fragilis 638 as described above. Transconjugants from these matings were selected on plates containing Cm, Cc, Rf, and Gn. Preparation, analysis, and manipulation of DNA. Plasmid DNAs were purified from E. coli by density gradient centrifugation (Radloff et al., 1967) of crude lysates prepared by the alkaline lysis method (Maniatis et al., 1982). Routine screening of E. coli transformants was done according to Birnboim and Doly (1979) and B. fragilis strains were screened either by alkaline lysis (Birnboim and Doly, 1979) or by the high-salt-sodium dodecyl sulfate method (Welch and Macrina,
144
SMITH,
ROGERS,
1981). Total B. frugilis genomic DNA was purified from 50-ml cultures by CsCl density centrifugation asdescribed previously (Smith and Spiegel, 1987). DNA samples were analyzed by agarose gel electrophoresis using Tris-borate or Tris-acetate buffers (Maniatis et al., 1982). Analysis of small restriction fragments (~500 bp) was performed on 3-4s NuSieve GTG agarose (FMC BioProducts) slab gels. Restriction endonuclease digestions and T4 ligation reactions were performed according to the supplier’s (GIBCO/BRL, Gaithersburg, MD) specifications. Individual restriction fragments to be used for plasmid constructions were purified from agarosegels by absorption onto glassbeads (Gene Clean, Bio 101, La Jolla, CA). When required, DNA fragments were blunt-ended using the Klenow fragment of DNA polymerase I (Maniatis et al., 1982). Cloning of known promoter fragments in pFD325TT. E. coli promoters known to be highly active were cloned into pFD325TT as follows. The Tc’ promoter from pBR322 (Ptet) was isolated on a 188-bp EcoRI/ EcoRV fragment (bp 436 1-185; Sutcliffe, 1979), blunt-ended, and ligated into the SmaI site of pFD325TT. This placed the - 10 region about 180 bp from the cat start codon. The tat promoter (Ptac) was obtained by digestion of pKK223-3 (Pharmacia/LKB, Inc.) with BumHI (Brosius and Holy, 1984; deBoer et al., 1983). This 280-bp fragment was blunt-ended and cloned into the SmaI site. This placed the -10 sequence about 75 bp upstream from the cat start codon. The trc promoter (Ptrc) was obtained by digesting pKK233-2 (Amann and Brosius, 1985) with sph1 and &I. The resulting 5 17-bp fragment was ligated into pFD325TT digested with the same two enzymes. This construct placed the - 10 site about 80 bp from the cat start site. The synthetic promoter (Psyn; Rossi et al., 1983) was obtained by digestion of pYEJOO1 (Pharmacia/LKB, Inc.) with SspI and PvuII and the resulting 460-bp fragment was cloned into the pFD325TT SmaI site. The Psyn - 10 sequence was about 180 bp from the cat start codon. The Pl and P2 promoters
AND
MCKEE
from rrnB were ligated into pFD325TT on an 1800-bp PstI/SmuI fragment obtained from pKIS3535 (Brosius et al., 1981; Table 1). In this construct the - 10 regions for Pl and P2, respectively, were about 940 and 820 bp upstream from the cut start codon. Finally, the Staphylococcus uureus protein A promoter (Uhlen et al., 1984) was cloned into the pFD325TT SmuI site. The source of this promoter was a 450-bp SspI/RsuI fragment obtained from pRIT5 (Pharmacia/LKB, Inc.). The putative protein A promoter (Ppta) - 10 region(s) was located about 130- 135 bp upstream from the cut start codon. Individual clones bearing each of the promoters were obtained by transformation of E. coli with selection on media containing Sp and 200 pg/ml Cm. The orientation of all cloned promoter fragments was verified by restriction mapping. Preparation of cell extracts and enzyme assays. Cell-free extracts for chloramphenicol acetyltransferase (CAT) assayswere prepared from 100-200 ml midlogarithmic phase cultures. Cultures were sedimented and then washed in an equal volume of TD buffer (50 mM Tris, 0.03 mM dithiothreitol, pH 7.8). Cell pellets were then suspended in 3 ml of TD buffer and lysed by passage through a French pressure cell (12,000 lb/in.*). Cellular debris were removed by centrifugation for 15 min at 12,OOOg,4°C. The 5,5’-dithiobis(2-nitrobenzoic acid) assay of Shaw (1975) was used for measurement of CAT activity. Assays were performed at 37°C by recording (DU65, Beckman Instruments, Inc.) the increase in AdI of reaction mixtures with and without Cm added. Results were calculated using the 5-thio-2-nitrobenzoic acid molar extinction coefficient of 13,600 at 4 12 nm. One unit of activity is defined as the amount of enzyme catalyzing the formation of 1 nmol product per minute in the standard assay. Cell-free extracts for P-glucuronidase assayswere prepared from 1O-20 ml midlogarithmic phase cultures. Cultures were sedimented, washed in 50 mM sodium phosphate buffer (pH 7.0) and then suspended in 3 ml
B. frugilis GENE EXPRESSION of the same buffer. Cells were disrupted by sonication and cellular debris were removed by centrifugation for 15 min at 12,OOOg, 4°C. Activity was measured using an endpoint assay with the substrate pnitrophenyl #I-D&curonide (Novel and Novel, 1976). One-milliliter reaction mixtures containing 50 mrvfsodium phosphate buffer (pH 7.0), 4 mM 2-mercaptoethanol, 2.5 mM p-nitophenyl /3D-glucuronide, and enzyme were incubated at 37°C and the reaction was terminated by the addition of 0.5 ml of 1 M Na$ZO,. The AdI0 was recorded and activity determined from the nitrophenol molar extinction coefficient (E = 4680). One unit of activity is the amount of enzyme required for the formation of 1 nmol nitrophenol per minute. For whole cell assays,cultures were washed and suspendedin buffer as above, l/20 vol of toluene was added, and the mixture was vortexed for 15-30 s. The ,&glucuronidase reaction conditions were as described above except that following the addition of Na,CO,, cells were removed by centrifugation. Protein concentrations of cell-free extracts were determined by the method of Bradford (1976). The method of Lowry et al. (195 1) was used to measure the protein concentration of whole cells solubilized in 0.2 N NaOH. RESULTS
Construction of expression vectorspFD290
and pFD340. We have shown previously that IS4351 contains at least two promoters active in Bacteroides (Smith, 1987). This has allowed us to design a vector incorporating these promoter sequencesand to test the expression of heterologous genes in Bacteroides. The starting point for construction of the expression vector was pFDl67 (Smith, 1985b), a 6.3-kb shuttle plasmid composed of the E. coli replicon pUC 19 and the cryptic Bacteroides plasmid pBIl43. For selection in Bacteroides, the ermFS structural gene (Ccr) was inserted into a unique ClaI site within the pBI143 portion of the molecule. One end of this gene fragment starts just 25 bp upstream from the ermFS ATG start codon,
145
and presumably ermFS is transcribed by an endogenous promoter located within the pBIl43 sequences(Smith, 1987). To provide promoter activity for foreign genes, a l-6-kb PstI/EcoRI fragment from pFDl36 (Smith, 1985a) was inserted by blunt-end ligation into the Hind1 site located in the MCS of pFDl67. This DNA fragment contains an entire copy of IS4351 less27 bp from the end of the element. The exact endpoint of the blunt-ended EcoRI site in this fragment (3’ end) corresponds to base number 1159 (see Fig. 2 in Smith, 1987) and the endpoint for the PstI site (5’ end) is not known precisely but lies about -600 bp from bp 1 in our previously published sequence(Smith, 1987). This construct, pFD290 (Fig. IA), has the IS4351 promoters conveniently placed upstream of unique cloning sites for BamHI, SmaI, KpnI, and SstI. Further, the blunt-end ligation junction produced at the EcoRI end of the IS fragment resulted in the formation of a translation stop signal, TGA, in the putative transposase reading frame. Translation stop signals in the other two reading frames are located 8 and 101 bp upstream from the 3’ end of the IS element. Although pFD290 can be mobilized from E. coli to Bacteroides using RR23 1 or R75 1 (Smith, unpublished observations; Shoemaker et al., 1986b), we sought to improve the mobilization frequency by insertion of an E. coli transfer origin into pFD290. This was accomplished by the isolation of a 0.75kb HaeII fragment from pDP1 (Guiney et al., 1984a) bearing the RK2 oriTand insertion of this into a HaeII site on pFD290. The resulting plasmid, pFD340 (Fig. IA), was otherwise identical to pFD290. Expression of cat in B. jiiagilis. Naturally occurring Cm’ Bacteroides possessa low level CAT activity (Britz and Wilkinson, 1978b; Martinez-Suarez et al., 1985); thus, it seemed reasonable that a foreign cat gene should confer a detectable phenotype in Bacteroides if it could be expressed.Therefore the expression vector was first tested by insertion of the Tn9 cat structural gene into the unique pFD290 SmaI site. This was accomplished
146
ROGERS, AND MCKEE
A
8600
bp
‘rnFS
B
0 kbl
1
2
3
FIG. 1. Restriction cleavage site maps of the expression vectors pFD290, pFD340, and their derivatives. (A) Circular maps of the plasmids are shown with the approximate locations of several relevant restriction sites. The restriction site locations were deduced by standard mapping techniques (Maniatis et ab, 1982) and from published sequence data hut for the sake of clarity not all restriction sites are shown. The Smal (MCS) region contains the following unique cloning sites: BarnHI, Seal, Kpnl, and Sstl. The Pstl (MCS) region contains unique sites for Pstl and Sphl. pB1143 sequences are indicated by the stippled boxes; pUC 19sequencesare indicated by the thin line; ermFS and Apr (AMP) sequencesare indicated by the open boxes with arrows showing the direction of transcription; IS4351 sequencesare indicated by the solid boxes with an arrow showing the approximate location and direction of transcription of the putative transposase; oriT sequences are indicated by the hashed-marked box; and the pUC19 replication origin, oriV, is indicated by an arrow. (B) Partial restriction maps of expression vector derivatives showing the insertion sites of the cloned indicator genes. Vector sequences are shown by the striped boxes and the IS4351 segments are labeled IS; the indicator genesare shown by the open boxes (structural genes) and thin lines (flanking sequences).The MCS designation indicates the location of the Smal junction used for construction of these derivatives and corresponds to bp 4060 in the maps shown in (A). The scale in kb is given at the bottom of the figure.
by blunt-end ligation of a 77 5-bp TaqI restriction fragment obtained from pACYC 184 (Chang and Cohen, 1978). This gene fragment starts 29 bp upstream from the start of cat and includes the ribosome binding site but no obvious promoter signals (Alton and Vapnek, 1979). Becausepromoter sequences
located within the IS element also function in E. coli, cloning was simplified by the capability of direct selection of E. cob transformants on Cm. The resulting plasmid, pFD308 (Fig. lB), was then introduced into B. jiiagilis via transformation with selection on a Cc-containing medium. The plasmid content of sev-
147
B. frugilis GENE EXPRESSION TABLE 2 CHLQRAMPHENICOL MICs AND CHLORAMPHENICOL ACETYLTRANS~RA~EACTIVITIESFORB. fragilis 638 CONTAINING pFD29O:cut FUSIONS Plasmid
MIC”
None pFD290 pFD308 pFD308AIS
4 4 128 4
CAT (SPa#
