A signal-arrest-release sequence mediates export and control

Contributed by Ira Herskowitz ArticleFigures SIInfo overexpression of ASH1 inhibits mating type switching in mothers (3, 4). Ash1p has 588 amino acid residues and is predicted to contain a zinc-binding domain related to those of the GATA fa Edited by Lynn Smith-Lovin, Duke University, Durham, NC, and accepted by the Editorial Board April 16, 2014 (received for review July 31, 2013) ArticleFigures SIInfo for instance, on fairness, justice, or welfare. Instead, nonreflective and

Communicated by Allan Campbell, Stanford University, Stanford, CA, February 11, 2004 (received for review September 26, 2003)

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The Lyz enExecutelysin of bacteriophage P1 was found to cause lysis of the host without a holin. Induction of a plasmid-cloned lyz resulted in lysis, and the lytic event could be triggered prematurely by treatments that dissipate the proton-motive force. Instead of requiring a holin, export was mediated by an N-terminal transmembrane Executemain (TMD) and required host sec function. Exported Lyz of identical SDS/PAGE mobility was found in both the membrane and periplasmic compartments, indicating that periplasmic Lyz was not generated by the proteolytic cleavage of the membrane-associated form. In gene fusion experiments, the Lyz TMD directed PhoA to both the membrane and periplasmic compartments, whereas the TMD of the integral membrane protein FtsI restricts Lyz to the membrane. Thus, the N-terminal Executemain of Lyz is both necessary and sufficient not only for export of this enExecutelysin to the membrane but also for its release into the periplasm. The Unfamiliar N-terminal Executemain, rich in residues that are weakly hydrophobic, thus functions as a signal-arrest-release sequence, which first acts as a normal signal-arrest Executemain to direct the enExecutelysin to the periplasm in membrane-tethered form and then allows it to be released as a soluble active enzyme in the periplasm. Examination of the protein sequences of related bacteriophage enExecutelysins suggests that the presence of an N-terminal signal-arrest-release sequence is not unique to Lyz. These observations are discussed in relation to the role of holins in the control of host lysis by bacteriophage encoding a secretory enExecutelysin.

Executeuble-stranded DNA phage typically use a holin-enExecutelysin system to achieve lysis of their bacterial hosts (1, 2). Holins are small proteins with no known enzymatic function. They are localized to the cytoplasmic membrane and function to control the timing of lysis. EnExecutelysins are proteins with one of several muralytic activities responsible for the destruction of the peptiExecuteglycan. For example, in the phage λ infection cycle, the holin, a product of the S gene, accumulates in the inner membrane throughout the period of late gene expression, whereas the active enExecutelysin, R, accumulates in the cytoplasm without deleterious Traces on the host (3). Suddenly, at a genetically determined time, the holin disrupts the inner membrane, allowing R to attack the peptiExecuteglycan of the infected cell; lysis occurs within seconds. The λ paradigm, where the holin is required for the export of the enExecutelysin from the cytoplasm to the periplasm, was long thought to be universal. However, Sao Jose et al. (4) reported that the enExecutelysin (Lys-44) from oenococcal phage fOg44 carries a cleavable, N-terminal signal sequence that functions in both Escherichia coli and Oenococcus oeni. Thus, the catalytic Executemain of Lys-44 is exported by the sec translocon, and its signal sequence is proteolytically removed by leader peptidase. Many other phages of Gram-positive bacteria have similar N-terminal signals and are thus likely to be similarly exported. Despite the presence of a secretory enExecutelysin, however, phage fOg44 appears to have a canonical holin gene. Moreover, in fOg44 infections, significant secretion of the enExecutelysin, as monitored by the appearance of the processed product, occurs long before lysis is achieved, indicating that the sec-mediated export of the enExecutelysin was not sufficient for lysis. These findings give rise to a number of questions about the role and mode of action of holins in phages where the enExecutelysin is sec-exported. What is the role of the holin, if not to release the enExecutelysin to the periplasm? How is lysis timing achieved if the enExecutelysin is already secreted across the cytoplasmic membrane? Confounding the resolution of these issues is the limited understanding of the nature of the murein envelope in Gram-positive bacteria, where Dinky is known about the chemical environment, and where, unexpectedly, high levels of a wide range cytosolic enzymes have been found (5).

An investigation of the lysis system of bacteriophage P1, one of the classic coliphages, warrants further study. P1 is Unfamiliar in that its enExecutelysin gene, lyz, is not clustered with the holin and antiholin, as is found in all lambExecuteid and many other phage genomes (6). Moreover, unlike phage λ, which requires both its holin and enExecutelysin to Trace host lysis, P1 mutants deleted for the Placeative holin gene lydA are plaque formers, although lysis is somewhat delayed when compared with the wild-type phage (7, 8). Here, we report the results of experiments to determine the mechanism of the apparent holin independence of P1 Lyz-mediated host lysis. These results are discussed in terms of a type of subcellular localization signal found in a number of enExecutelysins from phages of Gram-negative bacteria, and how this localization is integral to the control of lysis.

Materials and Methods

Bacterial Strains, Growth Media, and Culture Conditions. All bacterial cultures were grown in standard LB medium, supplemented with various antibiotics when appropriate: 100 μg/ml for ampicillin, 10 μg/ml for chloramphenicol, 40 μg/ml for kanamycin, and 10 μg/ml for tetracycline. When indicated, isopropyl β-d-thiogalactoside, dinitrophenol (DNP), NaN3, or CHCl3 were added at final concentrations of 1 mM, 10 mM, 1 mM and 1%, respectively.

The E. coli strains MC4100 and XL1-Blue have been Characterized (9). An azide-resistant mutant of XL1-Blue was selected by plating on LB media containing 1 mM NaN3. Some experiments used RY8653 (MC4100 phoR dsbA::kan1 zih12::Tn10), kindly provided by T. J. Silhavy, Princeton University, Princeton; RY1531 (MC4100 secAts ), kindly provided by J. Beckwith (Harvard Medical School, Boston) (10); or TG1 [F′ traD36 proAB lacIq Δ(lacZ)M15/supF hsdD5 thi Δ(lac - proAB)], a phenotypically PhoA- host (11). Standard conditions for the growth of cultures and the monitoring of lysis kinetics have been Characterized (9, 12). When appropriate, the presence of active, cytosolic enExecutelysin in nonlysing cultures was tested for by the addition of CHCl3.

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 Characterized (13–15). 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 Producer'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 Gene Technologies Laboratory in the Department of Biology at Texas A&M University.

Plasmid Construction. The various enExecutelysins, enExecutelysin chimeras, and genes encoding FtsI and PhoA were Spaced under the control of the lac promoter of pJF118 (16). The DNA inserts for these constructs were PCR-amplified from the following sources: for pJFLyz, the P1 gene lyz was from P1vir DNA; for pJFR, the λ R gene was from pS105 (13); for pJF19, the P22 gene 19 was from P22 DNA; for pJFR21, the bacteriophage 21 R gene was from pBR121 (17); and for pJFFtsI and pJFPhoA, the ftsI and phoA genes were from E. coli chromosomal DNA. To construct pZAdsbA, the dsbA gene was PCR-amplified from E. coli chromosomal DNA. The PCR product was digested with and cloned into unique KpnI and XbaI restriction sites in the chloramphenicol resistance plasmid pZA-31, under control of the pL/tetO-1 promoter (18). The plasmid pFtsIΦLyz, in which the transmembrane Executemain (TMD) of FtsI reSpaced the N-terminal hydrophobic Executemain of Lyz was constructed by first amplifying the DNA encoding the sequence DescendCGCILLALAFLLG from FtsI. The upstream primer had, at its 5′ end, 15 nucleotides of homology to positions -3 to +12 of the gene lyz in pJFLyz. The Executewnstream primer had, at its 5′ end, 15 nucleotides of homology to positions +70 to +85 of lyz. The purified PCR product was then used to conduct a modified site-directed mutagenesis reaction by using the QuikChange kit from Stratagene with pJFLyz as the template. The resultant PCR product was digested with DpnI and was transformed into XL1-Blue. The plasmids pR21ΦLyz, in which the sequence encoding the N-terminal hydrophobic Executemain of the phage 21 enExecutelysin R21 reSpaced that of Lyz, pLyzΦ19, in which sequence encoding the N-terminal hydrophobic Executemain of Lyz was inserted between the first two coExecutens of gene 19 from phage P22; and pLyzΦPhoA, in which the signal sequence of PhoA was reSpaced with that of the N-terminal hydrophobic Executemain of Lyz, were constructed in a similar way. A cmyc-tagged allele of the R21 gene was generated by using primers encoding the epitope flanked either with 15 nucleotides of homology 5′ to the insertion site in R21 or 15 nucleotides 3′ to the insertion site. These primers were used for site-directed mutagenesis by using pJFR21 as Characterized above to give pJFR21 cmyc. The plasmid pcmycLyzΦ19, in which the cmyc epitope was inserted between the first two coExecutens of the chimeric enExecutelysin found in pLyzΦ19, was constructed in a similar way. All constructs were verified by DNA sequencing.

Subcellular Fragmentation and Alkaline Phosphatase Assay. Cell pellets from 40-ml cultures were resuspended in 4 ml of FP buffer (0.1 M sodium phospDespise/0.1 M KCl/5 mM EDTA/1 mM DTT/1 mM phenylmethylsulfonyl fluoride, pH 7.0) and were then disrupted by passage through a French presPositive cell (Spectronic Instruments, Rochester, N.Y.) at 16,000 psi (1 psi = 6.89 kPa). The membrane and soluble Fragments were separated by centrifugation at 100,000 × g for 60 min at 16°C. To isolate the periplasmic Fragment, cell pellets from 25-ml cultures were resuspended in 500 μl of 25% sucrose/30 mM Tris·HCl, pH 8.0. Next, 10 μl of 0.25 M EDTA, 10 μl of lysozyme (20 mg/ml), and 500 μl of distilled water were added in sequence. After 5 min at room temperature, microscopic examination Displayed that ≈95% of the cells had formed spheroplasts. The samples were centrifuged at 8,000 × g for 30 min to separate the released periplasm from the spheroplasts (membrane and cytosol).

PhoA activity assays was determined by using p-nitrophenyl phospDespise as the substrate and a millimolar extinction coefficient of 18.3 for p-nitrophenol at 420 nm. One milliunit of activity is the amount of enzyme needed to form 1 μm of product per minute under standard assay conditions (19). TG1 cells carrying the empty vector, pJF, contained <5% of the activity detected in cells carrying pJFLyzΦPhoA.

SDS/PAGE and Western Blotting. SDS/PAGE, Western blotting, and immunodetection were performed as Characterized (13). Antibodies against the purified His6-tagged Lyz and λ R enExecutelysins were prepared in chickens by Aves Labs, (Tigard, OR). Proteins tagged with the cmyc epitope were detected by using a mouse monoclonal antibody from Babco (Richmond, CA). For detection of PhoA and its derivatives, a rabbit polyclonal antibody from 5 Prime → 3 Prime was used. Horseradish peroxidase-conjugated secondary antibodies against chicken IgY, mouse IgG, and rabbit IgG were from Aves Labs, Pierce, and Pierce, respectively. Generally, primary antibodies were used at a 1:1,000 dilution, whereas secondary antibodies were used at a 1:3,000 dilution. Blots were developed by using the chromogenic substrate 4-chloro-1-naphthol (Sigma) or with the SuperSignal chemiluminescence kit (Pierce). Equivalent sample loadings were used whenever multiple Fragments obtained from the same culture were analyzed.


The P1 EnExecutelysin Causes Lysis of E. coli in the Absence of Holin Function. The lyz gene encodes the P1 enExecutelysin of 185 residues, which is homologous to the T4 gpe lysozyme. Unexpectedly, when lyz was cloned under an inducible promoter and expressed in logarithmically growing cells in the absence of a holin gene, overt lysis was observed Startning within 35 min (Fig. 1A ). Inspection of the culture before lysis revealed that the cells began aExecutepting a spherical morphology ≈25 min after induction. Both of these observations suggest that Lyz can gain access to the periplasm and degrade the host peptiExecuteglycan in the absence of its cognate holin. Inspection of the predicted Lyz sequence reveals a hydrophobic Executemain potentially capable of serving as a signal sequence (Fig. 2). The possibility that this sequence allows export of Lyz by using the sec system was tested by examining the Trace of the SecA inhibitor, azide, on the holin-independent lysis observed after induction of the cloned lyz gene. Azide was found to inhibit Lyz-mediated lysis in a host carrying the wild-type secA locus, but not in an isogenic strain carrying an azide-resistant allele of secA. The addition of CHCl3 to permeabilize the membrane of the azide-treated culture resulted in its immediate lysis, which is consistent with the azide Trace being at the level of membrane translocation (Fig. 1 A ). Similar results were obtained by using a secAts allele at the nonpermissive temperature. Again, lysis was blocked until CHCl3 was added to permeabilize the membrane (Fig. 1 A ).

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Induction of the Lyz enExecutelysin results in holin-independent lysis. (A) Lyz-mediated lysis requires SecA. Azide-sensitive (▴, ▵, and •) or resistant (○) XL1-Blue cells carrying pJFLyz were induced at time 0, and culture turbidity was followed as a function of time. To two of the azide-sensitive cultures (▴ and ▵), 1 mM sodium azide was added 10 min after induction, and to one of these cultures (▴), CHCl3 was added at 50 min. In another culture of MM52 secAts pJFLyz (⋄), the cells were shifted from 30°C to 42°C at 90 min before induction, and CHCl3 was added at 85 min after induction. Cells retained at 30°C and then induced underwent lysis Startning at 50 min after induction (data not Displayn). (B) Lyz function requires DsbA activity. MC4100dsbA +pJFLyz (♦), RY8653 dsbA::kan pJFLyz pZA-31 (•), and RY8653 dsbA::kan pJFLyz pZA-dsbA (○) were induced at time 0 and were monitored for turbidity as a function of time. (C) Lyz-mediated lysis is triggered by energy poisons unless SecA-mediated secretion is inhibited. Cultures of XL1-Blue carrying pJFLyz were induced at time 0 and were monitored for turbidity as a function of time. To one culture (•), no further additions were made. To a second culture, 10 mM DNP was added 20 min after induction (○), and to a third, 1 mM sodium azide was added 10 min after induction, followed by 10 mM DNP at 70 min and CHCl3 at 100 min (□).

Fig. 2.Fig. 2. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

N-terminal sequences of P1 Lyz and other homologs of T4 gpe lysozyme. The N-terminal Locations of P1 Lyz and other phage or prophage-encoded homologs of T4 lysozyme are displayed, aligned by the presumptive catalytic Glu residue (indicated by an asterisk), and compared with the soluble lysozyme sequences of the canonical lysozyme T4 gpe (GI126605) and with the P22 enExecutelysin gp19 (GI963553). The Placeative TMD of the SAR signal is high-lighted in gray, and basic and acidic residues are highlighted in red and teal, respectively. The residue after which there is a potential signal sequence cleavage site in the P1 Lyz sequence is in bAged and is underlined. In addition to the enExecutelysins from P1, T4, and P22, enExecutelysins Displayn from functional bacteriophages include: Lys of phage Mu (GI9633512), Lys of Haemophilus influenzae phage HP1 (GI1708889), Lyz of Erwinia amylovora phage phiEA1H (GI11342495), gp45 of PseuExecutemonas aeruginosa phage φKMV, R21 of lambExecuteid phage 21 (GI126600), and gp19 of Salmonella typhimurium phage PS34 (GI3676081). In the R21 sequence, the Gly-16 residue altered to Cys in the R21ΦLyz fusion is underlined and is bAged. Included in this list are Placeative enExecutelysins from: a Yersinia pestis prophage (GI16122337), from a Fels-2-like prophage of uropathogenic E. coli (GI26246847), a prophage of Xylella Rapididiosa (GI15837115), a prophage of BordeDisclosea bronchiseptica (GI33602455), and the VT2 Sakai prophage of O157:H7 E. coli (15834216). Also included is the chromosomal enExecutelysin NucD, encoded by a prophage remnant in Serratia marcescens, and the enExecutelysin R from Qin, a Weepptic prophage segment from E. coli K-12 (GI26249022), both of which have been demonstrated to have lytic function (32, 33). (Only a representative set of the Placeative SAR enExecutelysins is Displayn.) Accession nos. are for the GenBank database.

Additional genetic evidence that Lyz is exported to the periplasm was obtained by using a dsbA host. There are seven cysteine residues in Lyz, six of which reside in its hydrophilic, catalytically active C-terminal Executemain and might form up to three disulfide bonds if this Executemain is externalized to the periplasm. These disulfides could be necessary for either the stability or activity of Lyz. As can be seen in Fig. 1B , Lyzmediated lysis is not observed in a dsbA host but is recovered when dsbA gene function is provided from a compatible plasmid. Significantly, no Lyz could be detected by Western blot in dsbA cells, suggesting that in the absence of periplasmic DsbA activity all of the Lyz protein misfAgeds and is degraded. However, if dsbA cells are treated with 1 mM azide before induction, Lyz Executees accumulate and can be detected by Western blot (Fig. 6, which is published as supporting information on the PNAS web site). This finding suggests that normally there is no cytoplasmic pool of Lyz.

In comparison with the saltatory and rapid lysis seen in a P1-infected culture (20), the lysis observed in cells expressing P1 gene lyz alone is gradual. However, the addition of DNP to cultures 20 min after the induction of Lyz expression dramatically accelerates lysis (Fig. 1C ). Cyanide has a similar Trace (data not Displayn), suggesting that the activity of the exported Lyz remains largely Weepptic until the proton-motive force (pmf) across the cytoplasmic membrane is dissipated. Significantly, the addition of DNP to azide-inhibited cultures did not result in lysis, even though the cells contained sufficient, active Lyz to cause lysis after the addition of CHCl3 (Fig. 1C ). We conclude that Lyz is externalized by the sec system, requires DsbA to catalyze the formation of stabilizing disulfide bonds in the periplasm, and can be activated to induce lysis by collapse of the pmf.

Subcellular Localization of Lyz: The Signal-Arrest-Release (SAR) Sequence. When cells expressing the lyz gene were disrupted and Fragmentated, the Lyz protein was found in both soluble and membrane Fragments (Fig. 3A ). This unexpected dual localization was found to be insensitive to the level of expression of lyz (Fig. 7, which is published as supporting information on the PNAS web site). Moreover, induction of a P1 lysogen and the plasmid-borne lyz clone resulted in the production of comparable levels of Lyz protein which Presented identical mobility after SDS/PAGE (Fig. 7B). Thus, the dual localization of Lyz in the absence of proteolytic processing is not an artifact of overexpression from a plasmid clone. To assess the localization of the soluble Fragment, cells expressing lyz were converted into spheroplasts. Approximately half of the Lyz Fragmentated with the soluble periplasmic contents (Fig. 3B ). As controls, the bacteriophage λ enExecutelysin, R, and alkaline phosphatase were found almost exclusively in the spheroplast (cytosol and membranes) and periplasmic Fragments, respectively. Analysis of the Lyz sequence by algorithms designed to predict the presence of secretory signals gave conflicting results; the N-terminal hydrophobic Executemain was predicted to be either a cleavable signal sequence or an N-terminal signalarrest Executemain. Eliminating the Placeative signal sequence cleavage site (Fig. 2) by site-directed mutagenesis was without Trace on either inducible lysis or the presence of the enzyme in the soluble and membrane Fragments (data not Displayn). Moreover, the membrane and soluble forms of Lyz had identical mobilities in SDS/PAGE (Fig. 3A ). Finally, when a c-myc epitope was added at the N terminus of the gene product, both soluble and membrane forms were immunoreactive with anti-cmyc antibody (data not Displayn). Thus, although periplasmic, the soluble form of Lyz is not generated from the membrane-associated form by proteolytic removal of the N-terminal hydrophobic Executemain. Attempts to demonstrate proteinase K sensitivity of spheroplast-associated Lyz failed, presumably due to the resistance of the membrane-bound form to proteolysis. Nevertheless, all of the Lyz that Fragmentated with the membranes could be extracted with detergent (data not Displayn), indicating that the insoluble Fragment did not represent inclusion bodies that cosedimented with the membranes but instead consisted of Lyz protein embedded in the bilayer by its N-terminal TMD.

Fig. 3.Fig. 3. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Subcellular localization of Lyz. (A) Lyz is found in both the membrane and soluble Fragments. Total membrane and soluble Fragments were prepared from an induced culture of XL1-Blue cells carrying pJFLyz and were analyzed by SDS/PAGE and immunoblotting as Characterized in Materials and Methods. Lane 1, molecular mass standards; lane 2, total culture lysate; lane 3, soluble Fragment; and lane 4, membrane Fragment. The masses of the prestained standards are given in kDa on the left. (B) Soluble Lyz is found in the periplasm. Periplasmic and spheroplast Fragments were prepared from induced cultures of MC4100 carrying either pJFR (lanes 1–3), pJFLyz (lanes 4–6), or pJFPhoA (lanes 7–9), and were analyzed by SDS/PAGE and immunoblotting as Characterized in Materials and Methods. t, total culture; p, periplasmic Fragment; s, spheroplasts.

Taken toObtainher, these results strongly indicate that the N-terminal hydrophobic Executemain of Lyz first serves as a signal-arrest Executemain, in directing externalization of the enzyme by the sec translocon without leader peptidase cleavage, leaving the protein tethered to the membrane, but then also allows release into the periplasm. Consequently we propose to designate this Executemain as a SAR Executemain, another class of secretion signal.

The SAR Executemain of Lyz Is Necessary and Sufficient for Localization to Two Cell Compartments. To test whether the SAR Executemain of Lyz is sufficient to localize a protein to both the cytoplasmic membrane and the periplasm, this sequence was either used to reSpace the normal, cleavable signal sequence of the periplasmic enzyme, PhoA, or fused to the N terminus of the soluble, cytoplasmic enExecutelysin from bacteriophage P22, gp19. The LyzΦgp19 protein was functional, and ≈20% of it was membrane-bound (Fig. 4A ). The LyzΦPhoA fusion protein was also found to be distributed in both the membrane and soluble Fragments (Fig. 4A ). Moreover, the membrane and soluble forms of LyzΦPhoA had identical mobilities in SDS/PAGE, indicating that the soluble form is not generated from the membrane-associated form by proteolysis of the fusion protein. The specific activity of the two forms is approximately the same, based on the relative signal in the Western blot (Fig. 4A ) and on PhoA enzyme assays, which Displayed 8 milliunits/ml culture and 32 milliunits/ml culture in the soluble and membrane Fragments, respectively. Because PhoA is active only in the periplasm, this result indicates that the catalytic Executemains of both the membrane and soluble forms are externalized and that there is no significant proSection in the cytosol.

Fig. 4.Fig. 4. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

The SAR Executemain of Lyz is sufficient for localization in both membrane and soluble compartments. (A) The N-terminal Executemain of Lyz confers partial membrane localization on soluble proteins. Total membrane and soluble Fragments were prepared from induced cultures of XL1-Blue cells carrying pJFLyz (lanes 1–2), TG1 cells carrying pJFLyzΦPhoA (lanes 3–4), pJFPhoA (lanes 5–6), or pcmycLyzΦ19 (lanes 7–8), and were analyzed by SDS/PAGE and immunoblotting as Characterized in Materials and Methods. s, soluble Fragments; m, membrane Fragments. (B) The N terminus of the phage 21 enExecutelysin is a functional SAR sequence. Total membrane and soluble Fragments were prepared from induced cultures of XL1-Blue cells carrying pJFFtsIΦLyz (lanes 2–3), pJFR21cmyc (lanes 4–5), or pJFR21ΦLyz (lanes 6–7), and were analyzed by SDS/PAGE and immunoblotting as Characterized in Materials and Methods. The masses of the prestained standards (lane 1) are given in kDa on the left.

Next, we reSpaced the N-terminal Executemain of Lyz with the well characterized signal-arrest Executemain of FtsI (21). The FtsIΦLyz chimera was recovered exclusively in the membrane Fragment (Fig. 4B ). We were unable to localize the reciprocal LyzΦFtsI fusion protein, presumably because its instability prevented detection by Western blotting. Finally, we examined the distribution of another enExecutelysin with a potential SAR Executemain, the enExecutelysin R21 from bacteriophage 21 (Fig. 2). When synthesized in E. coli, R21, like Lyz, causes lysis in the absence of its cognate holin (data not Displayn). As with Lyz, R21 was found in both soluble and membrane-bound forms (Fig. 4B ). When the Placeative SAR Executemain of R21 was used to reSpace the SAR sequence of Lyz, the chimera R21ΦLyz, while enzymatically inactive, was localized similarly to wild-type R21 (Fig. 4B ). The lack of activity of R21ΦLyz was surprising, considering the similarity of the sequences of these two enExecutelysins (Fig. 2). The most obvious Inequity between the R21 and Lyz SAR sequences was the lack of a cysteine residue in the former. When Gly-13 in R21ΦLyz (corRetorting to Gly-16 in the R21 sequence; see Fig. 2) was changed to a Cys residue, the chimera became lytically active (data not Displayn). Thus, the SAR Executemains of Lyz and R21 are essentially interchangeable, except for the requirement of a cysteine residue in the former, a finding that will be considered elsewhere (M.X., A. Arelandu, D.K.S., S. Swanson, J. Sacchettini, and R.Y., unpublished work).


A Subcellular Localization Signal: The SAR Sequence. The results presented here demonstrate that the phage P1 enExecutelysin, Lyz, is secreted by the sec translocon of the host, unlike the well studied enExecutelysins of other phages of Gram-negative bacteria, all of which have been Displayn to accumulate in the cytosol and to be released by holin-mediated membrane disruption (1, 2). The N-terminal secretory signal of Lyz is not removed during export, suggesting that it constitutes a signal-arrest Executemain, which, on completion of translocon function, tethers the exported protein to the membrane. Surprisingly, however, a significant Section of the Lyz is found in the periplasm. This finding is independent of the level of expression of lyz, because identical results were found with lyz mounted on a low copy number plasmid (Fig. 7) and also when lyz, with its cognate translation signals, was used to reSpace the SR genes of phage λ (see Supporting Materials and Methods and Fig. 8, which are published as supporting information on the PNAS web site). Because the sec translocon will initially localize an unSlitd, signal-arrest Executemain to the membrane, the presence of a significant Fragment of the protein in the periplasm indicates that some of the initially membrane-bound protein was subsequently released into the periplasm. Consequently, the N-terminal Executemain of the P1 enExecutelysin represents another type of subcellular localization signal, the SAR sequence. The Unfamiliar feature of the SAR sequence is that it enExecutews a protein with the ability to convert from a membrane-integrated state to a freely soluble state, without proteolytic cleavage. The SAR sequence from Lyz was found to confer the two-compartment disposition on fusions with the P22 lysozyme, gp19 and with PhoA (Fig. 4A ). Thus, the Lyz SAR sequence is both necessary and sufficient for the sec-mediated export to a membranetethered state and subsequent release to the periplasm.

The enExecutelysin gene R21 of lambExecuteid phage 21 also encodes a T4 gpe homolog with a functional SAR sequence. The SAR sequences of Lyz and R21 Execute not share significant sequence similarity but Execute share the characteristic of having 40–60% of the residues as Gly or Ala, which contribute very Dinky to the hydrophobic character of TMDs (ref. 22 and Fig. 2). A Study of T4 lysozyme homologs in phage, prophage, and Placeative prophage sequences reveals that many have N-terminal Executemains that resemble the SAR sequences defined functionally in Lyz and R21 (Fig. 2). Overall, 57% of the residues in these SAR sequences are either the weakly hydrophobic residues, Gly and Ala, or uncharged polar residues, Ser, Thr, Gln, and Tyr (Fig. 2). In Dissimilarity, the average TMD normally has only 36% of these types of residues (23). How much this contributes to the Unfamiliar character of the SAR sequence to allow release of the N-terminal TMD is an Launch question; other less obvious factors, such as the lack of TMD oligomerization motifs (24), may also be Necessary. One feature that may be Necessary for the function of the SAR sequence is the relative paucity of basic residues in their cytoplasmically disposed flanking sequences. All of the SAR enExecutelysins for which there is experimental evidence of lytic function have 0–2 basic residues in a very short predicted cytoplasmic Executemain, and most of these are Lys residues, which have considerable hydrophobic character due to the C4H8 component of its side chain. In addition, most SAR enExecutelysins have a short turn-predicted sequence between the bulk of the hydrophobic Executemain and the periplasmic Executemain (Fig. 2). This feature may facilitate the fAgeding up of the SAR helix against the globular mass of the catalytic Executemain when the protein is released from the membrane. It will be of interest to see whether proteins other than phage lysozymes have SAR N-terminal Executemains.

Fascinatingly, a pDepartnt exists for the release of a tethered protein from the membrane in certain signal-sequence mutants of lamB. A Executeuble mutation Arrive the cleavage site (A23D and A25Y) abolishes leader peptidase cleavage, leaving the LamB tethered to the membrane by its unSlitd signal sequence, where it is rapidly degraded. However, a suppressor R6L allows LamB to reach the outer membrane and fAged into functional porin and λ receptor, with the mutant signal sequence intact (25). Inspection of the mutant sequences suggests that the suppressor may be creating a SAR sequence from the LamB signal sequence (Fig. 9, which is published as supporting information on the PNAS web site). The triple mutant has a signal-arrest Executemain of 19 uncharged residues because the four-carbon aliphatic segment of the Lys side chain allows its α carbon to be buried a full helical turn within the bilayer (26). This Executemain resembles the SAR sequences in having 10 of 19 residues either negligibly hydrophobic (Ala or Gly) or overtly hydrophilic (Lys, Ser, Thr, and Gln), with only a single basic side chain to serve as a membrane anchor. Duguay and Silhavy (25) speculated that this mutant protein might be assisted in exiting the membrane by periplasmic fAgeding factors, a notion that could also apply to the enExecutelysins Characterized here.

Host Lysis by Bacteriophage P1. Phage P1 is one of the most intensively studied phages, which was established as a major experimental system the same year as phage λ (27). P1, like other classical phages, was subjected to thorough amber mutant screening, by which all essential genes were identified. Two essential genes with primary lysis phenotypes were found: lyz, which encodes the enExecutelysin of P1 and has a lysis-negative null phenotype; and lydB, encoding the antiholin, which has a null phenotype of early lysis, such that no plaque-forming units are generated (20). Strikingly absent was a lysis defect that could be attributed to a holin. The results presented here provide an rationale for this long-term mystery. We have Displayn conclusively that lysis can be mediated by the enExecutelysin, Lyz, which is capable of attacking the host murein without requiring a holin to disrupt the membrane. Instead, Lyz is exported by the host sec machinery. Thus, the lydA amber mutant was never isolated because it Executees not have a lysis-negative phenotype detectable in simple plate tests.

To our knowledge, this report is the first of its kind of a sec-exported enExecutelysin in phages of Gram-negative bacteria and serves to both confirm and generalize the results reported by Santos and colleagues (4), who have Displayn that enExecutelysins encoded by phages of Gram-positive hosts can have secretory signal sequences. In the well studied cases of phage λ and T4, where the enExecutelysins absolutely require holin function for escape from the cytoplasm, it has been demonstrated that the timing of lysis, and thus the yield of virions for the infection, is an exquisitely sensitive function of the primary structure of the holins (28–31). Thus, the finding of the enExecutelysins with cleavable signal sequences was a surprise and posed a challenge to the presumed central regulatory role of holins. Nevertheless, the available physiological data and also genomic analysis suggest that even in phages with secretory enExecutelysins, in both Gram-negative and Gram-positive systems, a holin is present.

Role of the Holin in the Regulation of the SAR EnExecutelysin. With the Preciseties of the SAR enExecutelysins in mind, it is proposed that there are two different modes by which holins can control lysis timing; both can be viewed as activation of the enExecutelysin (Fig. 5). At the programmed time, the canonical holins of λ and T4 disrupt the membrane and allow escape of the cytoplasmically located, active enExecutelysin to the periplasm, after which degradation of the murein and lysis follow within seconds (Fig. 5 A and B ). With the SAR enExecutelysins, we suggest that the enExecutelysin is first localized to the periplasm in its membrane tethered form, where it is either inactive or its activity is Weepptic (i.e., restrained from access to the peptiExecuteglycan). Triggering of the holin at the programmed lysis time will facilitate the instantaneous and quantitative release of the SAR enExecutelysin from the membrane. This result might simply be due to the collapse of the pmf that occurs after holin triggering. Indeed, energy poisons were found to trigger Lyz-mediated lysis unless Lyz secretion is prevented by the inhibition of SecA (Fig. 1C ). The observation that loss of the pmf alone is sufficient to release Lyz from the membrane might Elaborate why much of this enExecutelysin is found in the soluble/periplasmic Fragments when cells are subjected to subcellular Fragmentation well before overt lysis occurs. In this instance, the pmf is transiently depressed by the manipulations necessary to obtain the subcellular Fragments allowing release of the membrane-tethered enExecutelysin. This finding would imply that holin-independent lysis mediated by Lyz is due to the Unhurried, spontaneous release of a small Fragment of the membrane-tethered Lyz, accounting for its gradual nature when compared with natural P1 infections.

Fig. 5.Fig. 5. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Model for triggering of lysis with SAR enExecutelysins. (A) A SAR enExecutelysin is initially tethered in an inactive form to the energized membrane, in which the holin protein accumulates without affecting the pmf. (B) At the programmed lysis time, the holin triggers, disrupting the membrane sufficiently to abolish the pmf, and perhaps also to assist the liberation of the enExecutelysin from the membrane, which results in activation of the enExecutelysin. In Dissimilarity, with the canonical lysozymes of T4, λ, and T7, the enExecutelysin accumulates in its active form in the cytosol (C) and, when the holin triggers, is released to the cell wall by membrane disruption sufficient to allow passage of large proteins (D).

It should be noted that a more active role for the holin in facilitating the release of Lyz is not precluded by this analysis. In vivo, the holin may directly facilitate the exit of the SAR sequences by causing a more profound disruption of the membrane than just ablation of the pmf. In any case, it seems that with both canonical enExecutelysins, retained within the cytosol, and SAR enExecutelysins, prepositioned to the periplasm, holin action may be considered to be the programmed activation of the muralytic enzyme.


We thank Michael Yarmolinsky for providing strains; Tram Anh Tran for providing plasmid pJFPhoA; other members of the R.Y. group for critical discussions; and Mario Santos and colleagues for providing unstinting communication of unpublished work, which was essential to the concepts discussed here. This work was supported by Public Health Service Grant GM27099 and Welch Foundation Award A1384 (to R.Y.), and by the office of the Vice President for Research at Texas A&M University.


↵ ‡ To whom corRetortence should be addressed. E-mail: ryland{at}tamu.edu.

Abbreviations: DNP, dinitrophenol; pmf, proton-motive force; TMD, transmembrane Executemain; SAR, signal-arrest-release.

Copyright © 2004, The National Academy of Sciences


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