Broad-Host-Range Shuttle Vectors for Screening of Regulated Promoter Activity in Viridans Group Streptococci: Isolation of a pH-Regulated Promoter

Bacterial strains, plasmids, and growth conditions.Bacterial strains and plasmids used in this study are listed in Tables1 and 2, respectively. Escherichia coli strains BHB2600 and DH5α were cultured in Luria-Bertani broth and on Luria-Bertani agar at 37°C. For maintenance of plasmids, ampicillin (50 μg/ml) or erythromycin (150 μg/ml for strain BHB2600 and 300 μg/ml for strain DH5α) was added to the growth medium. VS were cultured in Todd-Hewitt (TH) broth and on TH agar (Difco Laboratories, Detroit, Mich.) at 37°C in a 5% CO₂ atmosphere or anaerobically. When required, TH broth and TH agar were supplemented with erythromycin (5 μg/ml).

MIC and MBC determination.The MICs and minimal bactericidal concentrations (MBCs) of spectinomycin for VS were determined by broth microdilution assays (32). Dilution series of spectinomycin or kanamycin, ranging from 25 to 1,000 μg/ml, in TH broth supplemented with 5% horse blood were prepared in microtiter plates. To each well 100 μl of TH broth containing 10⁶ bacteria was added, and the plates were incubated overnight at 37°C in a 5% CO₂ atmosphere. The MIC was defined as the lowest antibiotic concentration at which no growth was visible. To determine the MBC, 1 μl from each well without visible bacterial growth was cultured on blood agar plates. The MBC was defined as the lowest antibiotic concentration that reduced the bacterial inoculum at least 1,000-fold.

DNA isolation.Plasmid DNA was isolated from E. coli using Qiagen GmbH (Hilden, Germany) plasmid DNA isolation kits and was isolated from VS as described previously (52). Chromosomal DNA from VS was isolated using the Puregene chromosomal DNA isolation kit for gram-positive bacteria and yeast (Gentra Systems Inc., Minneapolis, Minn.).

Molecular cloning and DNA sequence determination.DNA manipulations were done according to standard techniques (39). Plasmid DNA was introduced into E. coli by electroporation using the method of Dower et al. (11). DNA sequencing was performed with the PCR-mediated Taq Dye Deoxy Terminator Cycle sequencing kit (Perkin-Elmer, Foster City, Calif.) using an Applied Biosystems model 373 DNA sequencer.

Construction of selection vectors pMM223 and pMM225.Plasmids used for the construction of the selection vectors pMM223 and pMM225 are listed in Table 2. The 3.3-kbBamHI-HindIII fragment of pEC2A (5), containing the promoterless trp′-′lacZfusion gene (9), was ligated to theEcoRI-BamHI part of the pIC20R multiple cloning site (MCS) (29) and the product was introduced into theEcoRI- and HindIII-digested shuttle vector pMG36e (49), resulting in pMM201 (data not shown). To achieve higher cloning efficiencies, the MCS-trp′-′lacZfragment was excised from pMM201 as an EcoRI-KpnI fragment and ligated to EcoRI- and KpnI-digestedE. coli plasmid pLITMUS28 (14), creating pMM210 (Fig. 1). Self-annealed primer AV7 (Table3) was cloned into the KpnI site of pMM210, replacing this site by an SpeI site (pMM211). The gene aad(9), originating fromEnterococcus faecalis and conferring resistance to spectinomycin (27), was amplified as a promoterless gene from plasmid pORI19S (H. E. Smith, personal communication) using primers AV3 and AV4 (Table 3). The 795-bp PCR fragment was digested with BamHI and cloned into the BamHI site of pMM211, resulting in pMM214. A bidirectional transcriptional terminator of Streptococcus equisimilis H46A present on a 922-bpPstI-HindIII fragment of plasmid pSU31 (45) was amplified using primers AV8 and AV9 (Table 2). The terminator fragment was digested with SpeI andEcoRI and ligated in the reversed orientation in front of the selection cassette of pMM214 to generate plasmid pMM218 (Fig. 1). For the construction of the second selection cassette, theaphIII gene originating from E. faecalis plasmid pJH1 and conferring resistance to kanamycin (48) was selected. It was amplified from plasmid pMG36 (49) as a promoterless gene by using primers AV1 and AV2 (Table 3). The 855-bp product was digested with BamHI and used to replace theBamHI fragment of pMM218 comprising the promoterlessaad(9) gene. The resulting plasmid was designated pMM219. The selection cassettes were excised as SpeI fragments from pMM218 and pMM219 and ligated to XbaI-digested pMM206, a derivative of the lactococcal shuttle vector pGKV210 (50), to yield the vectors pMM223 [aad(9) Sp] and pMM225 (aphIII Km), respectively (Fig. 1).

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Fig. 1.

Schematic representation of the construction of selection vectors pMM223 and pMM225. Details of the construction are given in the text. TT, streptococcal transcription terminator; Ap^R, ampicillin resistance gene; Em^R, erythromycin resistance gene; Cm, promoterless chloramphenicol acetyltransferase gene; B, BamHI; Bg, BglII; C,ClaI; E, EcoRI; Ec, EcoRV; H,HindIII; K, KpnI; N, NruI; P,PstI; Sc, SacI; Sa, SalI; Sm,SmaI; S, SphI; Sp, SpeI; X,XbaI; Xh, XhoI. Endonucleases printed in bold represent unique sites in the MCS.

Electrotransformation of VS.Electrocompetent VS cells were prepared according to the method of Smith et al. (44), with minor modifications. Briefly, bacteria harvested from mid-log cultures in TH broth were washed three times in ice-cold, sterile distilled water and three times in ice-cold 0.3 M sucrose–10% glycerol. Bacteria were resuspended in 1/500 volume of the latter solution, and 50 μl of this suspension was used for electroporation with a Gene-Pulser and Pulse Controller (Bio-Rad Laboratories B.V., Veenendaal, The Netherlands). Immediately after electroporation, 0.95 ml of TH broth supplemented with 0.3 M sucrose was added, and the bacteria were incubated for 2 h at 37°C and subsequently plated on the appropriate selective agar medium. Colonies of VS transformants were visible after 24 to 48 h of incubation at 37°C in a 5% CO₂ atmosphere. To obtain maximal transformation frequencies, cuvettes with different electrode gap sizes (0.1 or 0.2 cm) were tested, and resistance setting (100 or 200 Ω), field strength (10 to 25 kV/cm), and type of electrical pulse (decayed pulse or squared pulse) were varied. In addition, the influence of procedures affecting VS cell wall integrity on electroporation efficiencies was tested. dl-Threonine (40 mM), 1% (vol/vol) glycine, or a sub-MIC concentration of penicillin G was added to the growth medium used for the preparation of competent VS cells, or competent VS were enzymatically treated with lysozyme (0.5 mg/ml; Sigma Chemical Co., St. Louis, Mo.) and mutanolysin (2.5 U/ml; Sigma) for 1 h.

Natural transformation of S. gordonii CH1. S. gordonii CH1 cells were made competent for natural transformation according to the method of Jenkinson (24). Briefly, an overnight culture in brain heart yeast (brain heart infusion medium [Difco Laboratories] with 5 g of yeast extract [Difco] per liter) was diluted 100-fold in fresh brain heart yeast supplemented with 5% horse serum and 1% glucose and then incubated for 3 h at 37°C. This culture was diluted 100-fold in fresh medium and incubated for 1 h at 37°C. From this culture 1-ml aliquots were taken, 1.5 μg of plasmid DNA was added, and incubation was continued for another 3 h at 37°C. Aliquots of 10 μl were plated onto selective TH agar and incubated for 24 to 48 h at 37°C in a 5% CO₂ atmosphere to select for plasmid-containing transformants.

β-Galactosidase activity assay.β-Galactosidase activities of the VS clones were determined using the fluorescent substrate fluorescein di-β-galactopyranoside (FDG; Molecular Probes Europe BV, Leiden, The Netherlands). Bacteria cultured overnight in TH broth containing erythromycin (5 μg/ml) were harvested by centrifugation, washed, and resuspended in STES buffer (10 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, 20% sucrose) to an optical density at 620 nm (A₆₂₀) of 1.0. Of this suspension 1.5 ml was centrifuged, the pellet was resuspended in 1 ml of STES buffer supplemented with 50 mg of lysozyme per ml and 200 U of mutanolysin per ml, and this suspension was incubated at 37°C. After 2 h, 50 μl of 1% sodium dodecyl sulfate and 50 μl of chloroform were added and the samples were mixed for 10 s and then were left standing for 15 min at room temperature. Six replicate samples of 50 μl of this suspension were transferred to wells of microtiter plates, 150 μl of Z buffer (40 mM NaH₂PO₄, 60 mM Na₂HPO₄, 10 mM KCl, 1 mM MgSO₄, 40 mM β-mercaptoethanol [pH 7.0]) containing 33 μM FDG was added to each replicate, and microtiter plates were incubated at 37°C. The emission was measured at different times at 530 nm (band-pass wavelength = 30 nm) after excitation at 485 nm (band-pass wavelength = 20 nm) using a Cytofluor II fluorescence multiwell plate reader (PerSeptive Biosystems, Inc., Framingham, Mass.). The β-galactosidase activity was plotted as arbitrary fluorescence units over time, and results are the averages of six reactions.

Construction of an S. gordonii CH1 genomic library.A genomic library of S. gordonii CH1 was constructed using the selection vector pMM223. Vector DNA was digested with BglII and dephosphorylated using calf intestine alkaline phosphatase (Boehringer Mannheim GmbH, Mannheim, Germany). Genomic DNA isolated from S. gordonii CH1 was digested to completion with Sau3A. Vector and chromosomal fragments were ligated, and the ligation mixture was introduced intoE. coli BHB2600 by electroporation. Plasmid DNA was isolated from erythromycin-resistant transformants constituting the genomic library and introduced into the homologous streptococcal strain CH1 by electroporation.

Selection of pH-regulated promoters.The streptococcal genomic bank was plated onto TH agar of pH 7.3 supplemented with erythromycin (5 μg/ml) for plasmid maintenance and spectinomycin (500 μg/ml) for selection of active streptococcal promoters. After anaerobic incubation at 37°C for 36 h, colonies resistant to erythromycin as well as spectinomycin were replated onto TH agar of pH 6.2 supplemented with erythromycin and spectinomycin. As a control for the viability of the isolated S. gordonii clones, these were also restreaked onto TH agar plates of pH 6.2 supplemented with erythromycin only and onto plates of pH 7.3 supplemented with erythromycin and spectinomycin. Plasmids were isolated from clones that failed to grow on the pH 6.2 agar but that did grow on the agar of pH 7.3 in the presence of spectinomycin. The fragments cloned in these plasmids were amplified by PCR using primers AV4 and AV9 (Table 3), and the PCR products were sequenced using primer AV19 (Table 3). The obtained sequences were analyzed using the BLAST program (2).

Measurement of in vitro growth rate.To determine the relative activities of promoters at different pH values, the growth rates of selected clones were determined in TH broth of pH 6.2 and pH 7.3, in the presence and absence of spectinomycin. A single colony of each clone was cultured at 37°C in TH broth supplemented with erythromycin for plasmid maintenance. Overnight cultures were diluted 100-fold in fresh TH broth containing both erythromycin (5 μg/ml) and spectinomycin (500 μg/ml) or containing erythromycin alone. Growth was monitored by measuring the A₆₂₀ over time, and the mid-log-phase doubling time (t_½) was determined. Relative promoter activity at pH 6.2 and 7.3 was expressed as the ratio of growth in the presence and absence of spectinomycin at each pH [t_½(+spec)/t_½(−spec)].

Statistical evaluation.The significance of the differences between the growth rate ratios at either pH was calculated with Student's t test.

Nucleotide sequence accession numbers.The sequences of the vectors pMM223 and pMM225 have been assigned GenBank accession numbers AF076212 and AF076213 , respectively. The sequence of the isolated pH-regulated promoter fragment from S. gordonii CH1 pMM243 has been assigned GenBank accession number AF127175 .