Park et al. 10.1073/pnas.0600943103. |
Fig. 5. TMD1 of S2168 facilitates release of P1 Lyz to the periplasm. Periplasmic and spheroplast fractions were prepared and analyzed by SDS/PAGE and immunoblotting as described in Materials and Methods. Above each panel, the primary antibody used is indicated. Lane 1 in all panels contains molecular mass standards. periplasmic fraction; s, spheroplasts (cytosol and membranes). (A) Lanes 2 and 3, S2168TMD1FLyzDSAR. (B) Lanes 2 and 3, PhoA. (C) Lanes 2 and 3, Rl. PhoA and Rl were used as internal controls for periplasmic and spheroplast fractions, respectively.
Fig. 6. S2168 and its derivatives are localized to the membrane. Total membrane and soluble fractions were prepared and analyzed by SDS-PAGE and immunoblotting as described in Materials and Methods. Above each panel, the primary antibody used is indicated. Lane 1 in all panels contains molecular mass standards. m, membrane fraction; s, soluble fraction. (A) Lanes 2 and 6, S2168; lanes 3 and 7, S2168DTMD1; lanes 4 and 8, SARlyzFS2168DTMD1; lanes 5 and 9, ssPhoAFS2168. The two arrows to the right indicate the positions of (upper arrow) S2168, SARlyzFS2168DTMD1, and ssPhoAFS2168 and (lower arrow) S2168DTMD1. (B) Lanes 2 and 3, Rl. (C) Lanes 2 and 3, FtsIcmyc. Rl and FtsIcmyc were used as internal controls for soluble and membrane fractions, respectively.
Fig. 7. Kinetics of formation of disulfide-linked dimers with ssphoA-S2168S16C, S2168S16C, and S2171S16C alleles. The Western blots shown in Fig. 2 E and F were quantified by using ImageJ software suite (http://rsb.info.nih.gov/ij/index.html). Each lane was analyzed for the monomer and dimer bands and the result expressed as percentage of dimer [100× dimer/(monomer +dimer)].
Fig. 8. RYIRS-tagged S2168 can block S2168 holin triggering in trans. Cultures of MG1655 lysogens carrying lD(stf tfa)::cat cI857 S2168 R21Rz21Rz121 and transactivation plasmids carrying pTP4 (S2168R21E35Q), pTP10 (RYIRS-S2168R21E35Q) or the empty vector, pRE, were grown to A550 ~0.2 at 30°C, thermally induced for 15 min by aerating at 42°C, and then aerated at 37°C, with monitoring for culture mass as A550.
Table 1. Plasmids used in this work
Plasmid | Features | Source or reference |
pS105 | pBR322 origin, pR′ promoter and S105RRzRz1 from | (1) |
pJF118EH | ColE1 origin, lacIQ, and multiple cloning sites (EcoRI, SmaI, BamHI, SalI, PstI, HindIII) under Ptac promoter | (2) |
pRE | A derivative of pJF118EH replacing lacIQ and Ptac with pR′ promoter region from pS105 | M. Xu and R.Y., unpublished data |
pQ | pSC101 origin with modification, Plac/ara-1 promoter, Q from | (1) |
pJFLyz | pJF118EH with P1 lyz | (4) |
pJFFtsI | pJF118EH encoding c-myc tag fused to FtsI | (4) |
pJFPhoA | pJF118EH encoding PhoA | (4) |
pR | pJF118EH encoding R | (4) |
pBP71 | pBR322 origin, pR′ promoter and QS71RRzRz1 from phage 21, expressing only S2171 from S21 gene | (3) |
pBP68 | pBR322 origin, pR′ promoter and QS68RRzRz1 from phage 21, expressing only S2168 from S21 gene | (3) |
pTP1 | S105RRzRz1 of pS105 replaced with S71RRzRz1 of pBP71 | This study |
pTP2 | S105RRzRz1 of pS105 replaced with S68RRzRz1 of pBP68 | This study |
pTP3 | pTP2 with S2168amber | This study |
pTP4 | pTP2 with R21E35Q | This study |
pTP5 | pTP2 with codons of S2168TMD1 replaced with ones of SAR domain of P1 Lyz | This study |
pTP6 | pTP2 with codons of signal sequence of alkaline phosphatase inserted between codons 1 and 2 of S2168 | This study |
pTP7 | pTP2 encoding RYIRS fusion to N terminus of S2168 | This study |
pTP8 | pJFLyz encoding S2168TMD1 fusion to SAR domainless P1 Lyz | This study |
pTP9 | pTP5 encoding RYIRS fusion to N terminus of SARlyz?S2168TMD1 | This study |
pTP10 | pTP4 encoding RYIRS fusion to N terminus of S2168 | This study |
pETS2168his | pET11a carrying S2168 gene of pBP68 at NdeI-HindIII restriction enzyme site with introduction of codons of GGH6GG between codons 66 and 67. | J.F.D. and M. Barenboim, unpublished data |
1. Gründling A, Manson MD, Young R (2001) Proc Natl Acad Sci USA 98:9348-9352.
2. Fürste JP, Pansegrau W, Frank R, Blöcker H, Scholz P, Bagdasarian M, Lanka E (1986) Gene 48:119-131.
3. Barenboim M, Chang C-Y, dib Hajj F, Young R (1999) Mol Microbiol 32:715-727.
4. Xu M, Struck DK, Deaton J, Wang IN, Young R (2004) Proc Natl Acad Sci USA 101:6415-6420.
Supporting Materials and Methods
Bacterial Strains and Culture Conditions.
All bacterial cultures were grown in standard LB medium, supplemented with various antibiotics when appropriate: ampicillin, 100 mg/ml; chloramphenicol, 10 mg/ml; kanamycin, 40 mg/ml; and tetracycline, 10 mg/ml. When indicated, isopropyl b-D-thiogalactoside (IPTG), dinitrophenol (DNP) or CHCl3 were added at final concentration of 1 mM, 2 mM, or 1%, respectively.Standard conditions for the growth of cultures and the monitoring of lysis kinetics have been described (1, 2). All experiments, except for the work described in Fig. 8, were done with a lacIq1 tonA::Tn10 derivative of MG1655, the sequenced wild-type strain of Escherichia coli K-12 (3). For the experiment reported in Fig. 8, the bacterial host used was E. coli K-12 MDS12, which is isogenic with MG1655 except for 12 deletions of nonessential DNA (4).
Standard DNA Manipulation, PCR, and DNA Sequencing.
Procedures for the isolation of plasmid DNA, DNA amplification by PCR, PCR product purification, DNA transformation, and DNA sequencing have been described (5-7). Oligonucleotides were obtained from Integrated DNA Technologies (Coralville, IA) and were used without further purification. Ligation reactions were performed by using the Rapid DNA ligation kit from Roche Molecular Biochemicals according to the manufacturer's instructions. All other enzymes were purchased from Promega, with the exception of Pfu polymerase, which was from Stratagene. Automated fluorescent sequencing was performed at the Laboratory for Plant Genomic Technologies in the Crop Biotechnology Center at Texas A&M University.Plasmids and Induction.
The key features of the plasmids used in this work are listed in Table 1 (supporting information). All of the plasmids listed carry the selectable AmpR determinant except for pQ, which carries KanR. For most experiments, the bacterial strains carried two plasmids, the low copy plasmid pQ, carrying the gene for the l late gene activator, Q, under Plac/ara-1 control, and a medium copy plasmid with a pBR322 origin and the lysis gene cassette, SRRzRz1, under the control of the late promoter, pR' (8). The lysis gene cassettes were either from l or phage 21, as indicated. Induction of the lysis genes was accomplished by adding 1 mM IPTG to the culture. In experiments where the wt or modified alleles of P1 lyz were induced, the only plasmid present was a derivative of pJF118, a medium copy plasmid carrying the lyz allele under control of the tac promoter (9). In the experiment shown in Supporting Fig. 8, the host was MDS12 carrying the hybrid prophage lD(stf tfa)::cat cI857 S2168 R21am Rz21Rz121. In this prophage, the phage 21 lysis cassette, with the S2168 holin allele and a null allele of R21, replaces the l lysis cassette and is under the control of the l late promoter, pR'. This lysogen was transformed with transactivation plasmids pTP4 (S2168 R21E35Q), pTP10 (RYIRSFS2168 R21E35Q), described below, or an empty vector, pRE (Table 1). In these transformants, thermal induction of the lysogen results in transactivation of the plasmid borne lysis cassette when the induced prophage enters late gene expression.The various S21 genes used in this study were expressed in constructs based on the plasmid pS105 (10), which is a pBR322 derivative carrying the l late promoter, pR', and the entire l lysis cassette, SRRzRz1, on an EcoRI-ClaI fragment. The promoter is activated when the pR' activator Q is supplied from the IPTG induction of pQ. The plasmid pQ is a low copy vector with Q under a lac-ara promoter (11). The source of phage 21 lysis genes was the plasmid pBP71 (12), which has an EcoRI-ClaI fragment carrying the genes S2171R21Rz21Rz121. S2171 is an allele of the holin gene S21 in which the Met-4 codon has been changed to Leu (CUG) so that only the S2171 antiholin gene product is expressed (Fig. 1C). To construct pTP1, the EcoRI-ClaI fragment in pS105 was replaced by the corresponding EcoRI-ClaI fragment from pBP71. The expression level of the S2171 gene was increased by changing the Shine-Dalgarno sequence from cggaggc to aggaggt (positions -13 to -7 upstream of the S21 start codon), using site-directed mutagenesis. The plasmid pTP2, carrying the genes S2168R21Rz21Rz121, has an identical structure except that the first three codons of the S21 gene are deleted. The sole S21 gene product from this allele is S2168 (Fig. 1C). The spacing between the modified Shine-Dalgarno sequence and the start codon of the S21 reading frame is identical for both plasmids. Missense and nonsense changes in the S21 and R21 genes of pTP1, pTP2, and others were introduced by conventional site-directed mutagenesis using the QuikChange kit from Stratagene. To make the plasmid pTP3, codon 46 of S2168 in pTP2 was changed to the amber codon UAG. The R21E35Q allele in pTP4 was created by conventional site-directed mutagenesis using pTP2 as the template. The plasmid pTP10 is identical to pTP4 except the S2168 gene has been modified to encode the epitope-tagged RYIRS-S2168.
The first step in constructing plasmid pTP5, encoding SARlyzFS2168DTMD1, was amplifying the sequence encoding MKGKTAAGGGAICAIAVMITIVM from lyz using a upstream primer that had, at its 5' end, homology to positions -21 to -1 and a downstream primer that had, at its 5' end, homology to positions +93 to +73 of the S2168 gene of pTP2. The purified PCR product was used to prime a site-directed mutagenesis reaction using pTP2 as its template. In a similar fashion, the following plasmids were constructed using pTP2, pTP4, pTP5 or pJFLyz as the template for the final PCR reaction, as appropriate: pTP6, encoding ssPhoAFS2168, with the signal sequence (residues 1-26) of PhoA replacing the N-terminal Met of S2168; pTP7, encoding RYIRSFS2168, with the amino acid sequence RYIRS inserted after the N-terminal Met residue of S2168; pTP8, encoding S2168TMD1FLyzDSAR, with the SAR domain of Lyz replaced by TMD1 of S2168; pTP9, encoding RYIRS-SARlyzFS2168DTMD1, with the amino acid sequence RYIRS inserted after N-terminal Met residue of SARlyz. Construction of pJFLyz, pJFFtsI, pJFPhoA, and pRl, in which lyz, ftsI, phoA, and Rl are under tac promoter control, respectively, was previously described (9). Nucleotide sequences encoding c-myc tag were introduced to ftsI gene by site-directed mutagenesis to construct pJFFtsIcmyc.
The construction of the plasmid pETS2168his was carried out as described (2) with minor modifications. Briefly, the coding sequence of S2168 was amplified by PCR with NdeI and HindIII sites at its 5' and 3' end, respectively. Both purified PCR products and the pET11a vector (Novagen, San Diego, CA) were cut with NdeI and HindIII and ligated by using T4 DNA ligase (New England Biolabs, Beverly, MA). To make pETS2168his, a nucleotide sequence encoding the residues GGH6GG was inserted between codons 66 and 67 of the S21 gene by site-directed mutagenesis.
All constructs were verified by DNA sequencing.
Subcellular Fractionation.
Cell pellets from 25-ml cultures were resuspended in 2 ml of 0.1 M sodium phosphate/0.1 M KCl/5 mM EDTA/1 mM DTT/1 mM phenylmethylsulfonyl fluoride, pH 7.0 and then disrupted by passage through a French pressure cell (Spectronic Instruments, Rochester, NY) at 16,000 psi (1 psi = 6.89 kPa). The membrane and soluble fractions were separated by centrifugation at 100,000 ´g for 60 min at 16°C. The membrane proteins were extracted in 2 ml of 1% Empigen BB (Fluka)/20 mM BES/0.5 M NaCl/10% glycerol/35 mM MgCl2, pH 8.0 with gentle stirring overnight at 4°C. Periplasmic fractions were isolated from spheroplasted cells, as described (9). As controls, Rl, the endolysin encoded by bacteriophage l, was used for the soluble and spheroplast fraction, FtsIcmyc was used for the membrane fraction, and PhoA was used for the periplasmic fraction (Figs. 5 and 6). In general, control samples were prepared in parallel to the experimental samples.1. Chang C-Y, Nam K, Young R (1995) J Bacteriol 177:3283-3294.
2. Smith DL, Struck DK, Scholtz JM, Young R (1998) J Bacteriol 180:2531-2540.
3. Guyer MS, Reed RR, Steitz JA, Low KB (1981) Cold Spring Harb Symp Quant Biol 135-140.
4. Kolisnychenko V, Plunkett G, III, Herring CD, Feher T, Posfai J, Blattner FR, Posfai G (2002) Genome Res 12:640-647.
5. Smith DL, Chang C-Y, Young R (1998) Gene Expr 7, 39-52.
6. Smith DL, Young R (1998) J Bacteriol 180:4199-4211.
7. Gründling A, Bläsi U, Young R (2000) J Biol Chem 275:769-776.
8. Gründling A, Smith DL, Bläsi U, Young R (2000) J Bacteriol 182:6075-6081.
9. Xu M, Struck DK, Deaton J, Wang IN, Young R (2004) Proc Nat Acad Sci USA 101:6415-6420.
10. Gründling A, Bläsi U, Young R (2000) J Bacteriol 182:6082-6090.
11. Gründling A, Manson MD, Young R (2001) Proc Natl Acad Sci USA 98:9348-9352.
12. Barenboim M, Chang C-Y, dib Hajj F, Young R (1999) Mol Microbiol 32:715-727.