Generalización de la Base de Murphy

All the constructions described below were confirmed by dye-terminator sequencing at Secugen (Centro de Investigaciones Biológicas, Madrid).

4.6.1. Construction of plasmids pET24b-maEfa and pET24b-maEfa-His

To overproduce and purify the MAEfa and MAEfa-His proteins, plasmids pET24b-maEfa and pET24b-MaEfa-His were constructed (see Section 7). In both cases, the pET24b expression vector (Novagen) was used. To obtain pET24b-maEfa

55 plasmid, a 1,502-bp region of the V583 chromosme containing the maEfa gene was amplified by PCR using the UpmaEfa and DwmaEfa primers. These primers contained a single restriction site for NdeI and XhoI, respectively. The amplified product was digested with both enzymes, and the 1,470-bp digestion product was inserted into the pET24b expression vector. To overproduce the MAEfa-His protein, the maEfa gene was engineered to encode a MAEfa protein fused to a C-terminal His6-tag. Specifically, a 1,481-bp region of the V583 genome was amplified by PCR using the UpmaEfa and DwmaEfaHis primers. DwmaEfaHis oligonucleotide was designed to change the stop codon of maEfa gene by a restriction site for XhoI. Subsequently, the PCR product was digested with NdeI and XhoI and the 1,448-bp digestion product was cloned into the pET24b expression vector.

4.6.2 Construction of the pAS terminator-probe vector and the pAST promoter- probe vector

To construct the terminator-probe vector, pAS, an 833-bp DNA region of the pGreenTIR plasmid (Miller and Lindow, 1997), which contains the gfp reporter cassette, was amplified by PCR using the oligonucleotides F-gfp and R-gfp. Both oligonucleotides include a HindIII restriction site. The PCR-synthesized DNA fragment was digested with HindIII and the 802-bp digestion product was inserted into HindIII- linearized plasmid pLS1 (Lacks et al., 1986). The promoter-probe vector, pAST, was constructed introducing the transcriptional termination sites T1T2 of the E.coli rrnB ribososomal RNA operon (Brosius, 1981) into the multicloning site of pAS plasmid. For PCR amplification of a DNA region containing such terminators we used the pPR54 plasmid as template (Serrano-Heras et al., 2005) and the oligonucleotides F-T1T2rrnB and R-T1T2rrnB as primers. The 286-bp DNA fragment was further digested with SalI, and the 246-bp digestion product was cloned into the SalI site of plasmid pAS in both orientations, obtaining plasmid pAST (orientation T1T2rrnB; promoter-probe vector) and plasmid pAS-T2T1rrnB (opposite orientation).

4.6.3. Construction of pAS and pAST derivatives

To assess whether pAS functions as a terminator-probe vector, several derivatives were constructed, including pAST, whose construction was described in the previous section. To generate pAS-TrsiV, a 305-bp region of the enterococcal genomic DNA that contains the putative terminator of the sigV–rsiV operon (Benachour et al., 2005) was amplified with the F-TrsiV and R-TrsiV primers. Then, the PCR-amplified DNA was digested with SalI, and the 265-bp restricted fragment was inserted into the

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SalI site of the pAS vector. For the construction of plasmid pAS-TpolA, a 278-bp region

of the pneumococcal genome containing the terminator of the polA gene (López et al., 1989) was amplified with the F-TpolA and R-TpolA primers. After SalI digestion, the resulting 238-bp fragment was cloned into the SalI site of the pAS vector.

To analyse whether pAST is suitable as a promoter-probe vector, various DNA regions containing defined or predicted promoters were inserted into the multicloning site of the plasmid. From the enterococcal genome, two regions of 192-bp and 190-bp containing the promoter of the uppS and EF2493 genes (Hancock et al., 2003), respectively, were amplified with the F-PuppS and R-PuppS primers or the F-P2493 and R-P2493 primers. After SacI digestion, the 164-bp (PuppS promoter) and 160-bp (P2493 promoter) restriction fragments were cloned into the SacI site of the pAST vector, generating plasmids pAST-PuppS and pAST-P2493, respectively. Moreover, to construct plasmid pAST-P2962, a 191-bp region of the enterococcal genome that contains the putative promoter of the EF2962 gene was amplified with the F-P2962 and R-P2962 primers. After BamHI digestion, the 158-bp restriction fragment (P2962 promoter) was inserted into the BamHI site of the pAST vector. Using pneumococcal genomic DNA as template, two regions of 199-bp and 195-bp containing the promoter of the sulA (Lacks et al., 1995; López et al., 1987) and ung (Méjean et al., 1990) genes, respectively, were amplified with the F-PsulA and R-PsulA primers or the F-Pung and R-Pung primers. The PCR-synthesized DNAs were further digested with BamHI. The 166-bp (PsulA promoter) and 159-bp (Pung promoter) digestion products were inserted into the BamHI site of the pAST vector, generating plasmids pAST-PsulA and pAST-

Pung, correspondingly. Regarding the inducible pneumococcal PfcsK promoter (Chan

et al., 2003), a 150-bp region was amplified using pneumococcal genomic DNA as template and the oligonucleotides F-PfcsK and R-PfcsK as primers. After XbaI digestion, the 117-bp restriction fragment (PfcsK promoter) was cloned into the XbaI site of the pAST vector in both orientations: plasmid pAST-PfcsK (gene gfp under the control of the PfcsK promoter) and plasmid pAST-oPfcsK (opposite orientation).

4.6.4. Construction of plasmids pAST-Pma, pAST-PmaΔ19 and pAS-PmaΔ19

The pAST-Pma recombinant plasmid was used to identify the transcription start site of the maEfa gene. To construct pAST-Pma, a 242-bp region of the V583 enterococcal genome was amplified by PCR using the UpPma and DwPma primers. Both oligonucleotides contained a restriction site for SacI. The amplified DNA was digested with SacI, and the 214-bp digestion product (coordinates 288864-2889078)

57 was inserted into the SacI site of pAST. In pAST-Pma, gfp expression is under the control of the Pma promoter. To construct pAST-PmaΔ19 and pAS-PmaΔ19, a 228-bp region of the V583 genome was amplified with the UpPma and DwPma2 primers. After

SacI digestion, the 197-bp restriction fragment (coordinates 2888864-2889059) was

cloned into the SacI site of both pAST (pAST-PmaΔ19) and pAS (pAS-PmaΔ19).

4.6.5. Construction of pDL287 derivatives plasmids

Several plasmids were constructed to provide the maEfa gene in trans. They are based on the pDL287 plasmid (LeBlanc et al., 1993), a derivative of pVA380-1, that

carries a Km resistance gene. First, two expression vectors for E. faecalis were developed, pDLF and pDLS. These plasmids contain an engineered unique restriction site for SphI downstream of the P2493 and PsulA promoters, respectively. For the construction of pDLF, a 194-bp region of the V583 enterococcal genome, which contains the P2493 promoter, was amplified using the oligonucleotides P2493Cla-F and P2493Cla-R. Both oligonucleotides carry a restriction site for ClaI. The P2493Cla- R carries also a SphI restriction site. The PCR product was digested with ClaI generating a 171-bp fragment which was inserted into ClaI-linearized pDL287. For the construction of pDLS, a 202-bp fragment that carries the PsulA promoter was amplified using pneumococcal chromosomic DNA and the primers PsulCla-F and PsulCla-R. Both primers contained a restriction site for ClaI. In addition, PsulCla-R includes a restriction site for SphI. After ClaI digestion, the 181-bp DNA fragment was cloned into the ClaI site of pDL287. The next step was to introduce a promoterless maEfa gene from V583 strain (maEfaV583) in the expression vectors. To this end, a 1,546-bp region of the V583 enterococcal genome was amplified using the oligonucleotides maSph-F and maSph-R. After SphI digestion, the 1,514-bp restriction fragment was inserted into the SphI site of pDLF and pDLS, obtaining pDLFmaEfaV583 and pDLSmaEfaV583, respectively. In addition, the maEfa gene of strain OG1RF (maEfaOG1RF), which was amplified by PCR with the same primers, was introduced into pDLF, obtaining pDLFmaEfaOG1RF.