Max F. Perutz Laboratories, Department of Microbiology and Immunobiology, University Departments at the Vienna Biocenter, Dr Bohrgasse 9/4, 1030 Vienna, Austria
Correspondence
Udo Bläsi
udo.blaesi{at}univie.ac.at
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ABSTRACT |
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INTRODUCTION |
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The best-studied holin is that of bacteriophage , which serves as a paradigm for the dual start motif holins. The
S gene comprises 107 codons and encodes two polypeptides. The holin inhibitor S107 is identical in sequence to the actual holin S105 except for an N-terminal extension comprising a Met and a Lys residue (Bläsi & Young, 1996
). S107 molecules form hetero-oligomers with S105 (Gründling et al., 2000
), which are non-functional in terms of formation of a membrane lesion until holin concentrations build to a critical level that leads to formation of an oligomeric complex, which disrupts and thus depolarizes the membrane (Gründling et al., 2001
). The basis for S107 inhibition of S105 is the additional positive charge at the N-terminus of S107, which prevents or slows down the translocation of the N-terminus of S107 across the inner membrane relative to that of S105 (Graschopf & Bläsi, 1999
). In this way the S107 inhibitor seems to function by titrating out the lysis effector S105 in a stoichiometric fashion (Gründling et al., 2000
). Upon depolarization of the membrane, transit of the N-terminus of S107 to the periplasm results in the functional assembly of S proteins and the S107 inhibitor participates in hole formation (Graschopf & Bläsi, 1999
).
The second group are ssDNA phages, exemplified by X174 and the ssRNA phages Q
and MS2 (Young, 1992
). In each of these cases, only a single gene is required for lysis: E in
X174, L in MS2 and A2 in Q
. They encode neither an endolysin nor a holin. Phage progeny release seems to be achieved by the interaction of phage-encoded proteins with host enzymes which are involved in bacterial peptidoglycan biosynthesis. This leads to an imbalance in or inhibition of peptidoglycan synthesis, which results in lysis and release of phage progeny (Bernhart et al., 2002
).
We have recently determined the genome sequence of the S. aureus lytic phage P68 (GenBank accession number AF513032), and have identified three putative lysis genes (Vybiral et al., 2003). A putative endolysin gene, lys16, was identified within the structural genes of P68. Embedded in the 1 register at the distal end of lys16, a putative holin gene, hol15, was further recognized. In addition, a second putative holin gene, hol12, encoding a predicted protein with similarity to the holin proteins of the Bacillus subtilis phage
105 and of Streptococcus phage Cp-1 was identified at the end of the structural genes (Vybiral et al., 2003
). Due to the lack of highly repressible expression systems for S. aureus, in this study we functionally analysed the P68 lysis genes in Escherichia coli
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METHODS |
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Amplification of gene hol15 with oligonucleotides A28 and B28 (Table 2) generated a 276 bp PCR fragment. The forward primer B28 included an artificial Shine and Dalgarno (SD) motif (see Table 2
) to direct translation of hol15. The PCR product was cloned into the SmaI site of plasmid pUC18, rendering hol15 under transcriptional control of the lacpo in plasmid pUCHOL15 (Fig. 1c
). In addition, this 276 bp PCR product was cloned into the SmaI site of plasmid pDOC55 to control transcription of hol15 by the
pL promoter in plasmid pDHOL15.
The oligonucleotide pair C28/D28 (Table 2) was used to generate a PCR fragment containing the hol12 gene. The 420 bp fragment including the hol12 gene was cloned into the SmaI site of plasmid pUC18 and of plasmid pK184, which resulted in plasmids pUCHOL12 (Fig. 1c
) and pKHOL12, respectively. In the latter plasmids transcription of hol12 is controlled by the lacpo. In addition, this 420 bp PCR product was cloned into the SmaI site of plasmid pDOC55, which gave rise to plasmid pDHOL12.
To construct plasmids containing the lys16-hol12 or lys16-hol15 genes, respectively, plasmid pDLYS16 was cleaved with XbaI and XhoI, and the resulting fragment containing the lys16 gene was cloned into the XbaI and SalI sites of plasmid pUCHOL12 and pUCHOL15, resulting in plasmids pUCHOLY1216 and pUCHOLY1516, respectively (Fig. 1c).
The oligonucleotide pair E28/A28 (Table 2) was used to construct a plasmid bearing the hol15 gene in its natural setting, embedded within the lys16 gene. The resulting 788 bp PCR fragment was cloned into the EcoRI site of plasmid pUHE20 (Table 1
), resulting in plasmid pUHHOLY1615 (Fig. 1c
).
Like the holin gene S of bacteriophage (Bläsi & Young, 1996
), hol15 contains two putative start codons, Met1 and Met3 (Fig. 1a, b
). To test whether the polypeptides originating from either start codon function as a holin and as an anti-holin two hol15 alleles were created (see Fig. 1a, b
). The oligonucleotide pair C29/D29 (Table 2
) was used to generate a PCR fragment where AUG3 of hol15 was replaced by a leucine (CUG) codon. The corresponding 298 bp fragment, encoding the hol15-92 allele (starts at Met codon 1) (Fig. 1a, b
), was cloned into the XbaI and EcoRI sites of plasmid pUC19, resulting in plasmid pUCHOL15-Met1. The oligonucleotide pair B29/D29 (Table 2
) was used to generate a PCR fragment where the Met1 and Lys2 codons of hol15 were deleted. This 292 bp fragment, encoding the hol-90 allele (starts at Met codon 3) (Fig. 1a, b
) was inserted into the XbaI and EcoRI sites of plasmid pUC19, resulting in plasmid pUCHOL15-Met3. It should be noted that translation of both hol15 alleles is directed by the same SD motif and that the 5'-untranslated region upstream of the start codon of either hol15 allele is identical in both pUCHOL15-Met1 and pUCHOL15-Met3.
Zymogram assays.
SDS-PAGE was performed as described by Laemmli (1970) and the zymogram assay for detection of the Lys16 muralytic activity was carried out as described by Potvin et al. (1988)
and Lepeuple et al. (1998)
. Briefly, protein Lys16 synthesis was induced in E. coli MC4100F'(pUCLYS16) grown in LB medium at 37 °C upon addition of IPTG to a final concentration of 1 mM. After further incubation for 3 h at 37 °C, the cells were harvested by centrifugation, resuspended in protein sample buffer (62·5 mM Tris/HCl, pH 6·8, 2·3 % SDS, 1 % mercaptoethanol, 10 % glycerol, 0·01 % bromophenol blue) and heated for 10 min at 100 °C. The samples were separated on a 10 % SDS-polyacrylamide gel containing 0·2 % of the autoclaved S. aureus 68 or E. coli TOP10 cells. After electrophoresis, the zymograms were washed for 30 min with distilled water at room temperature, then transferred into buffer containing 25 mM Tris/HCl (pH 7·5) and 0·1 % Triton X-100, and further incubated for 16 h at 37 °C. The zymograms were rinsed with distilled water, stained with 0·1 % methylene blue in 0·001 % KOH for 2 h at room temperature, and then de-stained with distilled water. Peptidoglycan hydrolase activity was detected as a clear zone on a dark blue background.
In vitro translation.
The genetic information encoded by plasmids pUCHOL12, pUCHOLY1216, pUCHOL15, pUCHOLY1516 and pUHHOLY1615 was expressed in a coupled in vitro transcriptiontranslation system (Promega). The proteins were labelled with [35S]methionine according to the manufacturer's instructions; 1 µg of the respective plasmid DNA was used as a template in each reaction. After 45 min incubation, aliquots were withdrawn and the reactions were stopped by addition of 3 vols ice-cold 10 % trichloroacetic acid and placing on ice for 15 min, followed by centrifugation at 6000 g. The resulting pellets were washed once with 90 % acetone, dried under vacuum for 5 min, and then resuspended in 25 µl protein sample buffer. The samples were separated on a 10 % SDS-polyacrylamide gel without heating, or after heating at 100 °C for 2, 5 or 10 min. The translation products were visualized by autoradiography using a PhosphoImager (Molecular Dynamics).
Antimicrobial activity of Lys16 against clinical isolates of S. aureus.
Lys16-containing lysate was prepared from 25 ml of E. coli MC4100F'(pUCLYS16) upon induction of lys16 with 1 mM IPTG at an OD600 of 0·25. At an OD600 of 0·6, the cells were harvested and disrupted by sonication and the cell debris was removed by centrifugation; 12·5 ml samples of these lysates were added to 50 ml growing cells (OD600 0·5) of 15 S. aureus strains isolated from patients of the Vienna General Hospital.
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RESULTS |
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Do both hol12 and hol15 function as holins?
As genuine holins cause lethal lesions in the cytoplasmic membrane of bacterial cells, we next tested whether the expression of either hol12 or hol15 resulted in a loss of viability of E. coli. The expression of either plasmid-borne hol12 or hol15 gene was followed by growth arrest (Fig. 6a) and killing of more than 99 % of the cells (Fig. 6b
) approximately 60 min after induction in E. coli MC4100F' harbouring pUCHOL12 or pUCHOL15. This suggested that both proteins cause comparable membrane damage.
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In contrast, the concomitant expression of hol12 and lys16 from plasmid pUCHOLY1216 was sufficient to cause efficient cell lysis (Fig. 6a). Similarly, the concomitant expression of hol15 and lys16 from plasmid pUCHOLY1516 together with hol12 from the compatible plasmid pKHOL12 resulted in cell lysis, and the lysis profile was indistinguishable from that obtained upon induction of both lys16 and hol12 from plasmid pUCHOLY1216 (Fig. 6a
). These studies showed that Hol12 functions as a genuine holin, whereas the membrane damage caused by Hol15 appeared not to be sufficient for Lys16 release to the periplasm unless the membrane potential is abolished.
To verify these results, we next asked whether Hol12 and Hol15 differ with regard to the release of the 28 kDa endolysin 15 of B. subtilis phage
29. These experiments paralleled the experiments shown in Fig. 6
in that the concomitant expression of hol12 and
29 gene 15 in strain MC4100F'(pUCHOL12; pKB29-15) resulted in efficient lysis, whereas the simultaneous expression of P68 hol15 and
29 gene 15 resulted in retardation of growth (Fig. 7a
). However, as observed upon simultaneous synthesis of the P68 endolysin Lys16 and Hol15 (Fig. 6a
), the addition of DNP 25 min after induction of genes P68 hol15 and
29 gene 15 resulted in cell lysis (Fig. 7a
).
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Evidence for a dual start motif in the hol15 gene
The hol15 gene has a dual start motif beginning with the codons Met1-Lys2-Met3 (Fig. 1a, b). Like the
S paradigm (Bläsi & Young, 1996
), hol15 could therefore potentially direct the synthesis of a lysis inhibitor comprising 92 amino acids (Hol15-92; anti-holin) and a lysis effector comprising 90 amino acids (Hol15-90; actual holin), repectively. Due to the absence of an apparent SD motif upstream of AUG1 of hol15 at the phage P68 lys16/hol15 overlap (Fig. 1a
), an artificial SD sequence was placed at an appropriate distance to AUG1 of hol15 in all the plasmids, except pUHHOLY1615, used for the experiments described above. Similar experiments with the
S holin gene have shown that such a genetic set-up results in translation initiation almost exclusively at AUG1 of the S gene (Bläsi et al., 1989
; Steiner & Bläsi, 1993
), and thus in synthesis of the lysis inhibitor. To test whether the data described above can be rationalized with this possibility two plasmids were constructed. As described in Methods, the plasmids pUCHOL15-Met1 and pUCHOL15-Met3 were designed for expression of the hol15-92 and the hol15-90 allele, respectively, and were thus expected to direct synthesis of the putative Hol15-92 lysis inhibitor (starting at Met1) and Hol15-90 lysis effector (starting at Met3), respectively. The induction of either hol15 allele was followed by growth arrest, approximately 60 min after induction in E. coli MC4100F' harbouring the respective plasmids (Fig. 8
). The effects on cell growth and viability after concomitant expression of hol15-92 from plasmid pUCHOL15-Met1 together with the lys16 gene from the compatible plasmid pKLYS16 were similar to those obtained by the sole expression of the hol15-92 gene from plasmid pUCHOL15-Met1, and Lys16-dependent lysis was not observed (Fig. 8a
). However, depolarization of the membrane by addition of DNP to strain MC4100F'(pUCHOL15-Met1; pKLYS16) 25 min after induction triggered cell lysis (Fig. 8a
). In contrast, the concomitant expression of hol15-90 from plasmid pUCHOL15-Met3 together with the lys16 gene from plasmid pKLYS16 resulted in cell lysis (Fig. 8b
). These experiments taken with the results described above are consistent with the idea that the P68 hol15 gene has a dual start motif, which can direct synthesis of an anti-holin (Hol15-92) and of the lysis effector Hol15-90.
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DISCUSSION |
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The P68 endolysin Lys16 shares the typical signature(s) of a D-alanyl-glycyl endopeptidase domain with the endolysins of several S. aureus phages (Navarre et al., 1999), whereas an amidase domain seems to be absent (see Fig. 3a, b
). Nonetheless, the enzyme was able to hydrolyse E. coli murein (see Figs 4 and 5
). Due to the differences in the peptidoglycan cross-bridges between E. coli and S. aureus this cannot be reconciled with an exclusive D-alanyl-glycyl endopeptidase activity of Lys16. It therefore remains to be elucidated whether the enzyme is indeed devoid of an N-acetylmuramoyl-L-alanine amidase activity, which would be able to cleave also the backbone of E. coli murein at the nearly ubiquitously present MurNAc-L-alanine linkage. On the other hand the D-alanyl-glycyl endopeptidase activity of Lys16 could explain why three of the clinical S. aureus strains tested are resistant to Lys16. It has been shown that the amino-acid composition of the peptidoglycan cross-bridges can vary in S. aureus mutants (De Jonge et al., 1993
). Such alterations in the cross bridge(s) of the peptidoglycan of the clinical isolates could be at least one reason for the inability of Lys16 to lyse these strains. Comparable with the overexpression of the
R transglycosylase gene (Garrett & Young, 1982
) and the P22 19 lysozyme gene (Rennell & Poteete, 1985
), expression of P68 lys16 affected neither viability nor growth in the absence of either the hol12 or hol15-90 function (Figs 6 and 8
). Taken with the finding that addition of chloroform resulted in immediate lysis upon expression of lys16 (Fig. 5
), these data show that Lys16 depends on holin function for transit across the inner membrane.
Both Hol12 and Hol15 share characteristic traits of holins, i.e. two (Hol12) or three (Hol15) predicted transmembrane domains, which are followed at least in the case of Hol12 by a highly charged C-terminus (Fig. 1b). According to these primary features Hol15 and Hol12 classify as a class I and a class II holin, respectively (Young & Bläsi, 1995
). The co-expression of hol12 together with either P68 lys16,
29 endolysin gene 15 or the
R gene was followed by cell lysis (Figs 6 and 7
). In addition, the use of
111Sam cts as a standard test system for identifying holins (Wang et al., 2000
) classified Hol12 as a genuine holin (Fig. 7
). In contrast, expression of hol15 encoded by plasmids pUCHOL15 or pUCHOLY1516 did not result in the release of either P68 Lys16 or the
29 endolysin to the periplasm as long as the membrane was energized (Figs 6 and 7
). This behaviour of Hol15 is reminiscent of lysis inhibitor proteins or anti-holins encoded by dual start motif holin genes (Bläsi & Young, 1996
). As in the case of S107 (Bläsi et al., 1990
), Hol15-mediated lysis in the presence of endolysins could be triggered by the energy poison DNP. Since the basis for the functional conversion of a class I holin inhibitor into a functional holin apparently involves transit of its N-terminus across the inner membrane, which in turn is triggered by energy poisons (Graschopf & Bläsi, 1999
), these findings could suggest that the N-terminus of Hol15 has to traverse the lipid bilayer. Analogously to the
S gene, the hol15 gene has a dual start motif beginning with codons Met1-Lys2-Met3, which could result in the synthesis of Hol15-92 and Hol15-90, comprising 92 and 90 amino acids, respectively (Fig. 1b
). However, in contrast to the
S (Bläsi et al., 1989
; Chang et al., 1995
) or the phage 21 S (Barenboim et al., 1999
) genes, no distinct translational initiation signals, i.e. SD sequences, could be assigned for either potential start codon of the hol15 gene (Fig. 1a
; Vybiral et al., 2003
). The molecular mechanism underlying translation initiation of hol15 in the presence of concomitant upstream translation of lys16 therefore remains elusive. Clearly, more experiments are required to show whether translational starts in phage P68 mRNA occur at Met1 or Met3 or at both potential start codons. Again, it should be noted that, with the exception of plasmid pUHHOLY1615, the transcription of the wild-type hol15 gene from all plasmids used in this study resulted in mRNAs containing an artificial SD sequence at an optimal distance from AUG1 of hol15 (see Table 2
). From other studies (Bläsi et al., 1989
; Hartz et al., 1991
; Steiner & Bläsi, 1993
) it can be inferred that this genetic set-up results predominantly in translational starts at AUG1 of hol15, and consequently in the synthesis of Hol15-92 protein starting with Met1-Lys2-Met3, which in turn resembles known anti-holins (Young & Bläsi, 1995
; Young et al., 2000
). Hence, the release of endolysins by Hol15 upon addition of the energy poison DNP (Figs 6 and 7
) could be readily explained if Hol15-92 indeed serves as an anti-holin. The results presented in Fig. 8
corroborate this assumption. Only the concomitant synthesis of the Hol15-90 lysis effector and Lys16 resulted in lysis, whereas concomitant synthesis of the apparent lysis inhibitor Hol15-92 and Lys16 did not, unless the membrane was depolarized by addition of DNP (Fig. 8
).
In contrast to the inability of Hol15-92 to release the P68 Lys16 and 29 endolysin, expression of the hol15-92 allele caused a decrease in cell viability (Fig. 6
), and mediated export of the
transglycosylase (Fig. 7
). There appear to be several possible explanations for these results. First, the artificial SD sequence may direct Hol15-92 protein synthesis at high levels, which are sufficient to cause damage to the membrane as previously observed upon high-level synthesis of the
S107 lysis inhibitor (Steiner & Bläsi, 1993
). In support, the in vitro expression studies indicated that the hol15 gene is expressed at a much higher level from plasmid pUCHOL15 than from plasmid pUHHOLY1615 (Fig. 2
). Second, the lesion caused by Hol15 may be large enough to permit release of the
17·5 kDa
endolysin but not that of the larger endolysins of P68 and
29. However, since the
S-induced membrane lesion permits release of proteins larger than 480 kDa (Wang et al., 2003
), we consider this possibility less likely. We rather assume that the level of Hol15-92 induces only moderate damage to the membrane, which is sufficient to depolarize the membrane, i.e. to stop growth (Fig. 6
), but insufficient for P68 and
29 endolysin release. In contrast, the thermal induction of the
111 lysogen and the subsequent vegetative cycle could perturb the energy status of the membrane, and thereby trigger Hol15-92 to allow release of the endolysin. In fact, upon thermal induction of
111 the Hol15-92-mediated lysis and Hol12-mediated lysis profiles differ in that lysis onset mediated by Hol15 was delayed by 30 min, suggesting that Hol15 must accumulate to appreciable levels to achieve hole formation in the membrane under these conditions.
The in vitro transcriptiontranslation studies with plasmids containing either holin gene, hol12 or hol15, revealed an additional SDS-resistant 120 kDa protein band (Fig. 2
). As shown in Fig. 2(b)
, this band seems to represent Hol15 aggregates. It has been shown that phage
protein S forms an oligomeric structure in the inner membrane (Gründling et al., 2000
). Moreover, dimers, trimers, tetramers and pentamers of S protein had been previously observed upon cross-linking (Zagotta & Wilson, 1990
). Nevertheless, the appearance of SDS-resistant holin aggregates of the size observed with Hol12 (not shown) and Hol15 is unparalleled.
The overlap of ORF15 and ORF16 resembles the holinendolysin entity in S. aureus phage 187 (Loessner et al., 1999), except that in this case the holin gene overlaps with the 5' end of the endolysin gene. A similar genetic set-up as the P68 gene 16/15 overlap is potentially present in the Lactococcus lactis phage
vML3 (Shearman et al., 1994
). Here, the putative holin gene would be translated in the same reading frame as the upstream endolysin gene. However, in the majority of phages analysed to date the holin gene precedes the endolysin gene (Wang et al., 2000
). Nevertheless, in the Oenococcus oeni phages fOg44 (Parreira et al., 1999
) and 10MC (Gindreau & Lonvaud-Funel, 1999
) as well as in B. subtilis phage SPP1 (Alonso et al., 1997
), this gene organization is inverted (lys upstream of hol). Unusual holinendolysin arrangements are also found in other phages. For instance several phages of Streptococcus thermophilus appear to encode a class I and a class II holin upstream of the endolysin gene (Bruttin et al., 1997
; Sheehan et al., 1999
). A similar picture involving two endolysin genes and two putative holin genes has emerged from the study of the lysis genes of the B. subtilis PBSX phage (Krogh et al., 1998
). The lys16hol15 overlap and hol12 seem to be transcribed from different promoters (Vybiral et al., 2003
), which could suggest some sort of temporal regulation of lysis. However, the question why P68 encodes two holin functions may only be solved by functional knockouts of either holin gene in the phage context. Whatever the reason for the presence of two holin genes, since Hol15-92 apparently does not interfere with the holin function of Hol12 (see Fig. 6
) it is unlikely that there is a direct interaction between the two holins.
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Received 2 February 2005;
revised 10 March 2005;
accepted 14 March 2005.
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