Molecular Cell, Vol. 5, 49–57, January, 2000, Copyright 2000 by Cell Press
Signal Transduction by a Death Signal Peptide: Uncovering the Mechanism of Bacterial Killing by Penicillin Rodger Novak,† Emmanuelle Charpentier,† Johann S. Braun, and Elaine Tuomanen* St. Jude Children’s Research Hospital Department for Infectious Diseases 332 North Lauderdale Street Memphis, Tennessee 38105
Summary The binding of bactericidal antibiotics like penicillins, cephalosporins, and glycopeptides to their bacterial targets stops bacterial growth but does not directly cause cell death. A second process arising from the bacteria itself is necessary to trigger endogenous suicidal enzymes that dissolve the cell wall during autolysis. The signal and the trigger pathway for this event are completely unknown. Using S. pneumoniae as a model, we demonstrate that signal transduction via the two-component system VncR/S triggers multiple death pathways. We show that the signal sensed by VncR/S is a secreted peptide, Pep27, that initiates the cell death program. These data depict a novel model for the control of bacterial cell death. Introduction Bacteria are stabilized by the peptidoglycan that completely encloses the cell in a cytoskeleton (Weidel and Pelzer, 1964). Maintenance of this covalently closed network requires enzymes capable of cleaving the wall during bacterial growth and cell separation. However, cell wall hydrolases can also act as autolysins, completely degrading the cell wall and thereby representing suicidal enzymes (Tomasz, 1983). This life-and-death dichotomy of function emphasizes the need for efficient and strict regulation of hydrolytic activity, a paradigm conceptually similar to that of caspases in the process of eukaryotic apoptosis. Antibiotics, like penicillin, deregulate autolysin control, underscoring its medical importance (Tomasz and Holtje, 1977). It is unknown how the binding of antibiotics to bacterial targets, such as the ligation of cell wall synthetic enzymes by penicillin, leads to death by deregulation of extracellular autolytic enzymes. During normal growth, autolysin activity is believed to be subject to strong, prolonged downregulation as suggested by several facts. First, the expression of most hydrolases is constitutive throughout the cell cycle, but the enzyme(s) is only physiologically active during stationary phase lysis (Hakenbeck and Messer, 1977; Ronda et al., 1987). Autolysin activity during the exponential phase remains curtailed even when the gene is constitutively expressed * To whom correspondence should be addressed (e-mail: elaine.
[email protected]). † Present address: Skirball Institute of Biomolecular Medicine, Department for Molecular Pathology, New York University Medical Center, New York, New York 10016.
from a plasmid (Ronda et al., 1987). This indicates that regulation of autolysin activity is independent of transcription of the autolysin itself. Second, cell wall hydrolases are continuously present on the cell surface and, since triggering of wall hydrolysis does not require the synthesis of new enzyme (Kitano and Tomasz, 1979), these surface-located enzymes must be prevented from potential hydrolytic activity. The most striking example of physiological downregulation of autolysis is the stringent response that occurs during deprivation of an essential nutrient (Cashel et al., 1996). Starved bacteria bind antibiotic normally but do not lyse or die. Upon starvation, bacteria rapidly accumulate guanosine 3⬘, 5⬘-bispyrophosphate (ppGpp), which is synthesized by ppGpp synthetase I encoded by the relA gene (Metzger et al., 1988; Schreiber et al., 1991; Svitil et al., 1993). ppGpp in turn coordinately shuts down the synthesis of macromolecules such as DNA, phospholipids (Sokawa et al., 1968), and cell wall peptidoglycan (Ishiguro and Ramey, 1976). In this setting, antibiotic-induced lysis is blocked by an as yet uncharacterized defect in autolysin activation. This protection from death, termed phenotypic tolerance, is a property of all nongrowing bacteria and forms the basis of antibiotic selection for auxotrophs (Hobby et al., 1942; Tuomanen, 1986). Phenotypic tolerance is an important source of residual bacteria surviving antibiotic therapy of infections in vivo, leading to failure of therapy and promotion of acquisition of resistance (Handwerger and Tomasz, 1985; Tuomanen et al., 1986, 1988; Novak et al., 1999). In contrast to phenotypic tolerance, which is a response of all bacteria to poor growth conditions, tolerance to antibiotics can also result from genetic mutation. The simplest example of tolerance is the loss of function of the autolysin LytA, without which pneumococci fail to lyse and die (Tomasz et al., 1970). For reasons that are not clear, no clinical isolates have been found bearing loss-of-function mutation of the autolysin gene. However, up to 15% of clinical isolates of pneumococci are genetically tolerant (Tuomanen et al., 1986). Clinical tolerance appears to arise by genetic alteration at the level of regulation of autolysin activity (Tuomanen et al., 1988). What constitutes that regulatory process is unknown. To detect genes that are part of the trigger pathway of the pneumococcal autolysin, a library of loss-of-function mutants was created (Pearce et al., 1993) and screened for penicillin tolerance (Williamson and Tomasz, 1980). The screen identified 17 mutants that had an active autolysin but failed to die in the presence of penicillin. One mutant, SPSJ01, also failed to die in the presence of vancomycin and all other antibiotics tested (Novak et al., 1999). This breadth of tolerance is reminiscent of the stringent response mechanism. Analysis of the affected gene revealed a loss-of-function mutation of a two-component regulatory system, VncS–VncR. For signal transduction across the cell membrane, bacteria extensively use two-component systems, which have an input-sensing domain (histidine kinase) and an output effector domain (response regulator). Absence of the
Molecular Cell 50
Figure 1. Schematic of the vex, pep, and vnc Loci Organization of the pneumococcal gene loci encoding the putative ABC transporter Vex, the peptide Pep27, and the two-component regulatory system VncR–VncS. Putative promoters of the ABC transporter Vex (indicated by Pvex1 and Pvex3) are at positions ⫺26 and 1930.
VncS–VncR signal transduction resulted in loss of autolysin triggering. We proposed that the two-component system VncS–VncR represented an early element in the autolytic trigger pathway: a phosphorylated form of VncR represses the activation of autolysin, whereas a stimulus sensed by the histidine kinase VncS dephosphorylates VncR and enables triggering of autolysis (Novak et al., 1999). This implies that VncS–VncR functions as a relay station reacting to external signals that trigger lysis either physiologically in stationary phase or after the binding of antibiotics to their targets. We report analysis of the gene locus vncR-vncS, revealing the capability of encoding a peptide, Pep27, exported by a dedicated ABC transporter, Vex. We show that Pep27 is a death signal peptide enabling multiple death mechanisms, including antibiotic-induced autolysis. Results The Peptide Pep27 Induces Growth Inhibition and Cell Death Analysis of the gene cluster upstream of the two-component system, vncS-vncR, revealed an 84 bp open reading frame potentially encoding a 27–amino acid peptide, Pep27, and three genes encoding a putative ABC (ATPbinding cassette) transporter, Vex (Figure 1) (Higgins, 1992; Linton and Higgins, 1998). To address whether transcription of pep27 leads to a translational product, the linear peptide—MRKEFHNVLSSGQLLADKRPARD YNRK—was synthesized and used to raise specific antibodies. Western blot analysis of the cytoplasmic fraction and the supernatant of wild-type strain R6 demonstrated a 3 kDa product reactive with the Pep27-specific antiserum (Figure 2A). To determine the biological activities of Pep27, cultures of the wild-type R6 were exposed to the synthetic Pep27 and turbidity and viability were followed. Pep27 at 0.1 mM induced potent loss of viability of ⬎2 logs over 1 hr, a value comparable to bactericidal antibiotics penicillin or vancomycin (Figure 3A). Titration experiments using concentrations of Pep27 ranging from 0.05 to 200 M demonstrated that growth inhibition was dose dependent (Figure 3B). This behavior is distinct from antibiotics, which have a clear cut minimum inhibitory concentration (MIC). At a concentration of 0.05 M, the inhibition of growth was marginal, whereas treatment with 200 M synthetic peptide resulted in a complete growth arrest. At concentrations greater than 50 M, the loss of viability for R6 was ⱖ2 log bacteria over 4 hr. To ensure the specificity of the biological effect of Pep27, different peptide structural variants were synthesized and analyzed. Two 14-mer peptides representing the C or N termini or a truncated form lacking the five
C-terminal amino acids failed to induce growth arrest or killing (data not shown). In addition, the synthetic Pep27 was not antibacterial against different streptococcal species and Staphylococcus aureus (data not shown). Pep27 Is Capable of Triggering Different Death Pathways To identify the relationship between Pep27 and the only known death effector, the autolysin LytA, the effect of the peptide on the pneumococcal mutant Lyt-4-4, deficient in the major autolysin, was investigated. Compared to the diplococcal morphology of wild-type pneumococci, Lyt-4-4 grows in chains of up to 20–30 pneumococci. Addition of the peptide to the chains of the mutant Lyt-4-4 resulted in reversion to diplococci (Figure 4). In addition, the mutant Lyt-4-4 was killed by Pep27 but not as efficiently as wild-type R6 (data not shown). The difference in the rate of killing of R6 versus Lyt-4-4 indicates that LytA participates in peptide-induced killing. However, these data also support the contention that, in addition to LytA, alternative death pathways exist (Moreillon et al., 1990) and that these events can also be activated by Pep27. To investigate if Pep27 was not only sufficient for triggering death but was also necessary, a mutant deficient in Pep27 was investigated for antibiotic tolerance. Since efforts to create a null mutation in the small 84 bp gene
Figure 2. Secretion of Pep27 (A) Subcellular localization of Pep27. Immunoblot analysis of Pep27 using polyclonal anti-Pep27 antibodies (1:2000). Pep27 was detected at 3 kDa in the cytoplasm and the supernatant of R6. Pep27 was nondetectable in the supernatant of the loss-of-function mutants vex1 or vex3. (B) Immunoblot analysis of RecA. The cytoplasmic protein RecA was nondetectable in the supernatants of the parent strain R6 and the loss-of-function mutants vex1 and vex3.
Death Signal Triggers Bacterial Autolytic Pathways 51
Figure 4. Activity of Pep27 in the Absence of Autolysin Effect of synthetic Pep27 (100 M) on the morphology of the autolysin-deficient strain Lyt-4-4 (A) without peptide and (B) with peptide. Lyt-4-4 grows in chains with a length of up to 30–40 pneumococci.
Figure 3. Bactericidal Activity of Pep27 (A) Effect of the synthetic peptide Pep27 on viability: parent strain R6, loss-of-function mutant vncS, mutant vncR, mutant vex1, and mutant vex3. Cultures in the early exponential phase of growth (106 cfu/ml) were treated with 0.1 mM of Pep27, and viability was determined at 1 hr. (B) Dose-dependent effect of Pep27 on the growth of R6.
pep27 were unsuccessful due to its small size, insertion duplication mutagenesis of vex3 was performed so as to create a polar effect on pep27. Lack of Pep27 translation was confirmed by Western blot analysis (Figure 2A). After addition of 10 ⫻ MIC of vancomycin, the vex3/ pep27 mutant displayed tolerance, undergoing ⬍2 log kill in 4 hr in contrast to 4 log kill of the wild-type (Figure 5A). Tolerance extended to penicillin and cephalosporins (data not shown), suggesting that the peptide Pep27 was required for the initiation of a broad range of pneumococcal death responses. Expression and intrinsic activity of the autolysin LytA were confirmed in the vex3/ pep27-deficient mutant. Western blot analysis for LytA showed the expected 36 kDa band; an additional 54 kDa large band was also noted (Figure 5B). Autolysin from the vex3/pep27-deficient mutant was as efficient as wild-type at reconstituting lysis of the autolysin-deficient strain Lyt-4-4 (Figure 5C). Pep27 and the Stringent Response Mechanism The stringent response arrests antibiotic-induced death of nongrowing bacteria. To clarify the relationship of the
stringent response to Pep27 activity, protein synthesis of R6 was inhibited by leucine deprivation, and the bactericidal effect of Pep27 was monitored. Under these conditions, neither addition of 10 ⫻ the MIC of penicillin or vancomycin nor the addition of 200 M Pep27 resulted in lysis or death of R6. However, a combination of a cell wall synthesis–inhibiting antibiotic with Pep27 resulted in significant lysis of the cells (Figure 6). The minimum concentration of penicillin required to cause autolysis was equal to its MIC (0.1 mg/ml). This suggests that Pep27 releases the block to lysis engendered by the stringent response. Further, this cooperativity suggests that the stringent response interrupts two steps in the autolytic pathway, one circumvented by antibiotics and one by exogenous Pep27. Organization and Regulation of the Gene Locus The putative ABC transporter Vex is predicted to have a fused ABC-ABC organization with heterodimeric transmembrane domains (TMDs). The genes vex1 and vex3 encode putative hydrophobic transmembrane proteins, each consisting of four TMDs. vex1 and vex3 flank the gene vex2, which encodes an ATP-binding cassette (ABC) protein including the Walker A motif GX4GK(S/T) (Walker et al., 1982) at amino acid positions 41–46 and the Walker B motif (R/K)X6–8hyd4D (hyd, hydrophobic residues) (Ames et al., 1990; Hyde et al., 1990) at amino acid positions 142–156. An ABC signature sequence (L/Y)SGG(Q/M) (Higgins, 1992), at positions 130–134, is most likely essential for the integrity of the folded ABC protein (Hung et al., 1998). The fourth motif is a conserved histidine located 34 amino acids downstream of the aspartic acid of the Walker B motif, preceded by four hydrophobic residues and followed by a charged residue. ABC proteins often, but not exclusively, generate the energy of multicomponent membrane-bound transporters. They mediate the transport of a diversity
Molecular Cell 52
Figure 6. Bactericidal Activity of Pep27 during the Stringent Response Activity of Pep27 in nongrowing bacteria. Inhibition of protein synthesis of R6 (diamond) by leucine deprivation results in growth arrest. The effect of addition of 10 ⫻ MIC of penicillin (circle) (0.1 g/ml), 100 M peptide Pep27 (square), and a combination of penicillin and Pep27 (triangle) to the leucine-deprived R6 was monitored over 6 hr.
Figure 5. Requirements for Bactericidal Activity of Pep27 (A) Effect of loss of function of vex3 and vncS on bactericidal activity of vancomycin. Parent strain R6 (square), vex3 mutant (circle), and vncS mutant (diamond). Cultures in the early exponential phase of growth (107 cfu/ml) were treated with 10 ⫻ MIC of vancomycin (5 g/ml), and bacterial viability was followed for 6 hr. (B) Western blot analysis of autolysin preparations of parent strain R6, and the loss-of-function mutants vncS, vncR, vex3, and vex1. A polyclonal anti-autolysin (LytA) antibody (1:1000) was used. (C) Functional assay of autolytic activity. Autolysin preparations of parent R6 (square), loss-of-function mutant vex3 (diamond), and vex1 (circle) were added to cultures of the autolysis-defective strain Lyt-4-4 at an OD620nm of 0.3. 10 ⫻ MIC of penicillin (0.1 g/ml) was added to all samples, and lysis was followed over 6 hr. Lyt-4-4 served as a negative control (triangle) (addition of penicillin without autolysin).
of substrates, including ions, sugars, and peptides (Hiles et al., 1987; Russell et al., 1992; Jenkinson et al., 1996). The majority of ABC transporters are associated with periplasmic-binding proteins, which provide the primary substrate-binding site for uptake of solute into cells (Linton and Higgins, 1998). The absence of such a protein
in the putative Vex ABC transporter indicates that it is expected to be involved in substrate export. Northern blot analysis using a 435 bp probe specific for vex1 revealed a single transcript of 2 kb, indicating that vex1 and vex2 are transcribed from one promoter located upstream of vex1 (data not shown). Using a 900 bp probe specific for vex3, a 1.7 kb large transcript was obtained, a size exceeding the 1.4 kb of vex3. To confirm the possibility that the open reading frame pep27 downstream of vex3 was cotranscribed from a promoter upstream of vex3, a 0.3 kb probe specific for the intergenic region between vex3 and vncR was used in Northern blot analysis (data not shown). The resulting 1.7 kb transcript matched exactly the size of the transcript obtained with the probe specific for vex3, indicating transcription across the intergenic junction between vex3 and pep27. Two putative stem loop structures directly downstream of pep27 indicated strong termination of transcription. Genes involved in export of peptides are usually found adjacent to the structural gene they transport and share regulation (Kolter and Moreno, 1992). Analysis of the loss-of-function mutant of vex3/pep27, which is polar on the pep27 gene, resulted in loss of anti-Pep27 reactive species in both the cytoplasm and the supernatant (Figure 2A). Western blot analysis of a mutant defective in the first putative transmembrane protein, vex1, also showed no Pep27 in the supernatant (Figure 2A). This finding, as well as lack of a signal sequence of Pep27, is consistent with the hypothesis that Vex transports Pep27 outside the bacterial cell. Western blot analysis of these same samples using anti-RecA antibodies failed to detect the cytoplasmic protein RecA in the supernatant of the parent strain R6 as well as the vex1 and vex3–deficient mutants (Figure 2B). This made it unlikely that the difference in the detection of Pep27 in the supernatants was due to a higher autolysis rate of R6 compared to the vex1-deficient mutant. The proximity of the two-component system vncR/S to the ABC transporter genes and pep27 suggested possible
Death Signal Triggers Bacterial Autolytic Pathways 53
regulation of this locus by the two-component system. The VncR/S system is essential for stationary phase autolysis, perhaps in response to an unknown cell density signal (Novak et al., 1999). To investigate whether the response regulator VncR and Pep27 are regulated in a cell density–dependent manner, the wild-type strain R6 was subjected to Northern blot analysis at different growth stages. The level of transcription of pep27 was constitutive. Cells harvested from the early- and midexponential growth showed almost no transcription of vncR, whereas bacteria obtained from the stationary growth stage demonstrated a clearly increased transcription of vncR (data not shown). This raised the possibility that responsiveness to the peptide required the two-component system. Consistent with this notion, treatment of the vncS-deficient mutant with Pep27 did not result in growth inhibition or cell death (Figure 3A). The antimicrobial peptide cecropin B had similar bactericidal effects on R6 and the mutant vncS, indicating a specific defect in bactericidal activity of Pep27 in the vncS mutant. It is possible, therefore, that VncS functions as a receptor for Pep27. Triggering of Multiple Death Pathways by VncS Mutations of vncS have been shown to lead to loss of killing by penicillin, vancomycin, and a variety of other antibiotics (Novak et al., 1999). This tolerance phenotype indicates that VncR/S may participate in the triggering pathway for the major pneumococcal autolysin, LytA, and other potential death effectors. A model has been proposed that delineates VncS as a kinase/phosphatase controlling the level of phosphorylation of the response regulator VncR (Novak et al., 1999). Dephosphorylated VncR may enable triggering of LytA, for example, during stationary phase or following antibiotics. To address the question of whether the kinase/phosphatase VncS was linked to the major pneumococcal autolytic pathway, VncS was overexpressed from a plasmid in a vncS-defective mutant (RNET01). The construct placed vncS under the control of the promoter comA, which is inducible 40-fold with the competence-stimulating peptide (CSP). After addition of 10 g/ml of CSP to RNET01, the strain grew in long chains of up to 200 pneumococci (Figure 7). This morphological feature is characteristic for loss of function of both the major autolysin, LytA, and the glucosaminidase, LytB (Garcia et al., 1999). To establish a possible correlation between overexpression of the vncS gene and cell death, we exposed RNET01 and R6 to vancomycin. Upon exposure to 10 ⫻ MIC (5 g/ml), the strain RNET01 stopped growing but showed almost no loss in viability (1 log loss in viability over 4 hr). In contrast, the parent strain R6 started to die almost instantly (4 log loss in viability over 4 hr). This suggests that the histidine/phosphatase VncS is likely to regulate LytA and other as yet undefined death pathways, possibly including LytB. Western blot analysis demonstrated that overexpression of the histidine kinase vncS in RNET01 had no impact on the translation of LytA (data not shown). Discussion The mode of action of -lactams is far more complex than initially assumed. The finding that penicillin inhibits
Figure 7. The Relationship of VncS Expression to Autolysis Effect of overproduction of VncS on morphology and autolysis. (A) The strain ⌬vncS(pMU1328comAP-vncS) grows like the parent strain R6: diplococci or short chains with up to 6–8 pneumococci (magnification 1000⫻). (B) Induction of transcription of vncS leads to chain formation in the ⌬vncS(pMU1328comAP-vncS) strain (magnification 1000⫻).
cell wall cross-linking led to the early model that mechanically weakened cell wall was eventually ruptured by osmotic pressure. More recently, an indirect role for penicillin in lysis was established by the fact that timely addition of penicillinase to a penicillin-inhibited culture could reinitiate culture growth (Forsberg and Rogers, 1971). Furthermore, pneumococci lacking the major cell wall hydrolase (amidase, LytA) stopped growing but did not undergo lysis or cell death upon exposure to penicillin (Tomasz et al., 1970). These two findings forced a revised model that binding of penicillin and the consequent arrest of cell wall synthesis was not sufficient for cell death. Inhibition of growth and loss of viability by lysis were clearly mechanistically separable. The subsequent models for the mode of action of penicillin acknowledged that many bacteria undergo rapid cell death by lysis while others die readily without accompanying autolysis (Horne and Tomasz, 1977; Handwerger and Tomasz, 1985; McDowell and Lemanski, 1988). Mutants in the death pathway, independent of lysis by LytA, could be selected by cyclic antibiotic pressure, but the genes involved remained uncharacterized (globally referred to as cid loci) (Moreillon and Tomasz, 1988; Moreillon et al., 1990). That autolysis-dependent and autolysis-independent killing mechanisms might interact was suggested by the reduced amidase activity in ⌬cid cells (Moreillon et al., 1990). On the basis of these observations, it was suggested that the irreversible effects of -lactams were caused by a set of secondary events dependent on whether or not the autolytic system was active (Tomasz and Holtje, 1977). The mechanism of the control of the autolytic system and how it was disturbed during penicillin treatment remained speculative (Tomasz, 1979). The studies presented here address the process by which Streptococcus pneumoniae and perhaps other
Molecular Cell 54
autolytic pathogens regulate their suicidal autolytic systems. The downregulation of autolysis leading to tolerance is of significance to clinical medicine. Failure to eradicate tolerant bacteria might lead to prolongation and even failure of therapy (Handwerger and Tomasz, 1985; Tuomanen et al., 1988; Meeson et al., 1990; Entenza et al., 1997; Novak et al., 1999). Furthermore, tolerance serves as a preferred background for importation of antibiotic resistance (Tomasz et al., 1970; Novak et al., 1999). Gram-positive bacteria often control important physiological processes by intercellular signals in the form of extracellular peptides (Dunny and Leonard, 1997). For example, competence for genetic transformation in S. pneumoniae appears abruptly in an exponential growing culture when it reaches a critical cell density. An autocatalytic extracellular peptide hormone, CSP, plays a critical role in this process (Havarstein et al., 1995). At low extracellular levels, CSP stimulates its own synthesis, and at higher levels, it stimulates the induction of competence (Morrison and Baker, 1979). CSP is exported by the ABC transporter comA and is most likely sensed by comD, a histidine kinase (Pestova et al., 1996). In other gram-positive bacteria, autoinducers are also peptides that are actively secreted and function as ligands for signal transduction receptors (Dunny and Leonard, 1997). Autoinduction is usually activated at a critical concentration of the specific autoinducer and leads to a population-wide response. This molecular mechanism employed by bacteria to monitor cell density is termed quorum sensing (Fuqua et al., 1994; Kleerebezem et al., 1997). A model for the pneumococcal autolytic trigger pathway must account for the following observations. (1) Signal transduction by VncS is required for autolytic and nonautolytic death events. (2) Overexpression of the kinase/phosphatase VncS blocks lysis and daughter cell separation to an extent equivalent to a double knockout of the autolysins LytA and LytB, indicating that VncS is a negative regulator. (3) Transcription of VncR is upregulated during late logarithmic stage when pneumococci are known to undergo spontaneous autolysis consistent with a quorum sensing paradigm. (4) pep27 is cotranscribed with a gene encoding a putative transmembrane protein that is part of the ABC transporter, Vex. Vex is required for appearance of Pep27 in the supernatant. The locus for the peptide/transporter complex is located directly upstream of the locus encoding the two-component system, VncR/S. (5) Transcription of pep27 remains constitutive during growth. (6) The synthetic form of the peptide Pep27 functions as a death effector (with and without LytA). (7) The peptide uncouples the stringent response, the most powerful downregulator of autolysis known in bacteria. A combination of a cell wall active antibiotic with the peptide relaxes the stringent response. (8) Several findings suggest that Pep27 functions as a signal necessary for the triggering of death pathways in S. pneumoniae. Pep27 is able to stop growth and induce cell death in LytA⫹ and LytA-deficient strains. The fact that Pep27 promotes cell death in both backgrounds suggests that the histidine kinase/phosphatase VncS regulates the LytA-dependent and at least one additional death pathway in pneumococcus. These in vitro findings are supported by preliminary data obtained in vivo with the rabbit meningitis model. A single
intrathecal injection of 5 mg/kg Pep27 caused ⬎2 log kill of a clinical pneumococcal isolate over 8 hr, a result commensurate with penicillin. The following model integrates the biology of the peptide Pep27 with the signal transduction pathway initiated by the two-component regulatory system VncR/S. During logarithmic phase, we hypothesize that the response regulator VncR is phosphorylated and binds chromosomal DNA, repressing the autolytic and other death systems (Novak et al., 1999). Under these conditions, the pep27 gene is constitutively transcribed at low levels, and Pep27 gradually accumulates in the extracellular compartment. During stationary phase, which is characterized by autolysis, the concentration of Pep27 reaches a critical concentration sensed by the histidine kinase VncS. Under these conditions, VncS works predominantly as a phosphatase, leading to dephosphorylation of the response regulator VncR. The dephosphorylated response regulator detaches from its DNA-binding site, initiating a change in target gene transcription, finally triggering the autolytic event. Several mechanisms of action can be contemplated for Pep27. The biological effect of Pep27 might be receptor or nonreceptor mediated. Cationic peptides, like antibiotics and bacteriocins, do not use receptors (Kolter and Moreno, 1992; Piers et al., 1994; Gough et al., 1996). Cationic peptides have a net positive charge of at least ⫹2, and they have a defined three-dimensional structure (Hancock, 1997). Although Pep27 has a net charge of ⫹4 at neutral pH, and preliminary structural analysis shows an alpha-helical structure, evidence against Pep27 being a member of this class is the absence of an MIC and failure to kill the vncS-deficient mutant. A cationic peptide should not require a receptor. The fact that the antimicrobial peptide cecropin B had similar bactericidal effects on R6 and the histidine kinase–deficient mutant vncS suggested that the resistance of the vncS mutant toward Pep27 is not due to a general change in susceptibility toward cationic peptides. In addition, the synthetic Pep27 was not antibacterial against different streptococcal species and S. aureus. Taken together, the data in the present study provide important evidence that the two-component system VncR/S is in a signal transduction pathway involved in regulating death in S. pneumoniae. One of the death signals seems to be the peptide Pep27, which may act in a quorum sensing manner. After being exported and accumulating during late logarithmic and stationary phase, it is likely to be sensed by the histidine/phosphatase VncS, triggering different pathways leading to cell death. The RelA-dependent stringent response modulates the pathway in a manner yet to be detailed. It also remains to be established how inhibition of cell wall synthesis by antibiotics feeds into the death peptide pathway. Under antibiotic selection, bacteria circulating in the community have begun to modulate this signal transduction cascade so as to create mutants that are virulent but not killed by a broad spectrum of antibiotics.
Experimental Procedures Strains of Pneumococci and Growth Conditions S. pneumoniae strain R6 (Tiraby and Fox, 1973) was obtained from the Rockefeller University collection. The autolysin-deficient strain,
Death Signal Triggers Bacterial Autolytic Pathways 55
Lyt-4-4, was provided from the collection of Dr A. Tomasz, Rockefeller University. This strain is a stable point mutant created by chemical mutagenesis. S. pneumoniae was cultured on tryptic soy agar (TSA, Difco, Detroit, MI, USA) supplemented with sheep blood 3% (v/v). For growth in liquid culture, the bacteria were grown at 37⬚C without aeration in 5% CO2 using a semisynthetic casein hydrolysate medium supplemented with yeast extract (C⫹Y medium) (Lacks and Hotchkiss, 1960). For the selection and maintenance of pneumococci containing chromosomally integrated plasmids and the extrachromosomal construct pMU1328comAP-vncS, bacteria were grown in the presence of 1 g/ml erythromycin (Sigma, St. Louis, MO, USA). Recombinant DNA Methods DNA ligations, restriction endonuclease digestions, agarose gel electophoresis, and DNA amplification by PCR were performed according to standard techniques (Sambrook et al., 1990). Plasmid DNA preparation and purification were performed using kits from Qiagen (Qiagen, S. Clarita, CA, USA) and Promega/Wizard (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Transformation of E. coli with plasmid DNA was carried out with CaCl2-treated cells as described previously (Brown et al., 1979). Transformation of S. pneumoniae was performed according to standard protocols (Pearce et al., 1993). Insertional Inactivation of vex1 and vex3 To create the knockout mutants, the method of insertional duplication mutagenesis, which is a homology-directed insertion of foreign DNA, was used (Haldenwang et al., 1980; Mejean et al., 1981). For insertional duplication mutagenesis of vex1, an internal 435 bp fragment (bp 473–908) was amplified using total DNA of R6 as a template and evex1 (5⬘-ACGAAGAATTCGCTAAGAAGAACGGT-3⬘) and bvex1 (5⬘-ATTAAGGATCCAGCTATCAA-3⬘) as primers. An identical strategy was used to knock out the other genes. The insertional inactivation of vex3 was performed using the primers evex3 (5⬘-ATCAAG GGATCCACTGCCAAGGC-3⬘) and bvex3 (5⬘-AGAGGAGAATTC CCACTTCCTTGCG-3⬘). The resulting fragment was 900 bp long (bp 106–906). The amplified fragments were digested with BamHI and EcoRI and ligated to pJDC9 (Chen and Morrison, 1987). The resulting recombinant plasmids were then used to transform R6. Mutations were confirmed by PCR and sequencing (data not shown). Penicillin and Vancomycin Susceptibility and Autolysis Rates Autolysis rates of the strains were determined using 10 ml cultures of S. pneumoniae exposed to ten times the minimal inhibition concentration (MIC) of benzylpenicillin (0.1 g/ml) when the OD620nm reached 0.25–0.3. Autolysis rates were calculated as the first order rate constant K ⫽ ln (A0/A120) ⫻ min⫺1, where A0 represents the peak of absorbance reading at 620 nm, and A120 represents the reading after a further 120 min of incubation (Liu and Tomasz, 1985). The effect of penicillin and vancomycin treatment on the viability was determined by exposing 10 ml cultures in the early exponential phase of growth (OD620nm ⫽ 0.3, corresponding to 5 ⫻ 107 cfu/ml) to ten times the MIC of benzylpenicillin or vancomycin, respectively. After various times of exposure, 100 l portions were removed, serially diluted in C⫹Y, and in the case of penicillin, supplemented with 100 U of penicillinase (Sigma), and plated on tryptic-soy agar supplemented with 3% sheep blood (v/v). Antimicrobial Peptide Sensitivity Tests Survival and autolysis rates in the presence of antimicrobial peptides were determined by exposing the parent strain R6 and the vncSdeficient mutant to different concentrations (0.05–200 M) of Pep27 or cecropin B (American Peptide Company, Sunnyvale, CA, USA) at an OD620nm 0.05 ( 106 cfu/ml). For the disk diffusion assay, agar plates were seeded with a lawn of S. pneumoniae, Enterococcus faecalis, E. faecium, Streptococcus agalactiae, S. pyogenes, or S. aureus. The plates were incubated for 12 hr at 37⬚C with a disk soaked with the synthetic Pep27 (2 mM). The size of the halo was determined with a ruler. Subcellular Fractionation Pneumococci were separated in subcellular fractions by a modification of a previously described method (Hakenbeck et al., 1986). In
brief, bacteria were grown in 10 ml of C⫹Y medium to an OD620nm of 0.9 and isolated by centrifugation at 17,000 ⫻ g for 10 min. The supernatant was harvested and stored at ⫺70⬚C. Cell pellets were resuspended in 250 l of TEP (25 mM Tris–HCl [pH 8.0], 1 mM EDTA, 1 mM phenyl methyl sulphonyl fluoride). The suspension was sonicated for a total of 1 min with 15 s bursts. Cellular debris was removed by centrifugation at 17,000 ⫻ g for 10 min. The bacterial membranes and the cytoplasmic contents were separated by centrifugation at 100,000 ⫻ g for 4 hr. The supernatant from this final step contained the cytoplasmic fraction while the pellet contained the bacterial membranes. Samples from each fraction were evaluated for protein concentration and solubilized in SDS sample buffer for subsequent gel electrophoresis.
Peptide Synthesis Edman degradation was performed with a Perkin-Elmer Applied Biosystems 433A automatic sequencer. The synthetic peptide Pep27 was prepared by the Center for Biotechnology at St. Jude Children’s Research Hospital. HPLC analysis of the peptide revealed a purity grade of 90%. Mass spectrographic analysis of the synthetic peptide demonstrated a molecular mass identical to the predicted mass of Pep27.
Northern Blot Total RNA was prepared according to the manufacturer’s instructions (Qiagen). Approximately 10–20 g of total RNA was separated in a 1.2% formaldehyde gel. The gel was rinsed in 20 ⫻ SSC buffer, and RNA was transferred to nylon membranes (Hybond-N, Amersham) by capillary blotting (Sambrook et al., 1990). A 435 bp PCR fragment generated with primers evex1 and bvex1 was used as the vex1-specific probe. A 900 bp PCR fragment was generated with prime ␣-32P[dCTP] (Amersham). Hybridization under stringent conditions was performed according to standard protocols.
Preparation of Autolysin and Assay for Autolytic Activity Purification of pneumococcal autolysin as well as the biological assay for autolytic activity was performed on the basis of an established protocol (Mosser and Tomasz, 1970; Novak et al., 1998).
Immunoblotting The autolysin LytA and RecA were analyzed by running precasted 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and by Western blotting using Immobilon-P membranes (Millipore Corporation, Bedford, MA). The peptide Pep27 was analyzed by running precasted 16.5% Tris–Tricine gels (Bio-Rad, CA). The membranes were incubated with polyclonal rabbit antiPep27 antibody (1:2000), anti-autolysin antibody (1:1000), or antiRecA antibody (1:2000). The membranes were developed using goat anti-rabbit horseradish peroxidase (ECL Chemiluminescence Kit, Amersham, Buckinghamshire, England). To prepurify the peptide Pep27 for Western blot analysis, cells were removed by centrifugation (5000 ⫻ g for 5 min) at early stationary phase. The culture supernatants were dialyzed overnight at 4⬚C against C⫹Y medium. The medium in the dialysis bag was discarded. The dialyzed culture supernatants were passed through a 10 kDa cutoff filter (Amicon, Beverly, MA, USA) and concentrated to one tenth of the original volume at 4⬚C. Alternatively, the cultures were lyophilized after dialysis, suspended in PBS, and filtered through a Centricon 3 filter (Amicon).
A Rabbit Model of Pneumococcal Meningitis Pneumococcal meningitis was established according to a standard protocol (Tauber and Zwahlen, 1994). After withdrawal of CSF (300 l), 104 bacteria of a vancomycin-tolerant clinical serotype 23 isolate (RNET05) in 200 l saline was introduced intracisternally in all animals. The rabbits in group one received no treatment, group two received vancomycin (60 mg/kg) 6 hr after infection, group three received a combination of vancomycin (60 mg/kg) and Pep27 (4 mg/ kg) 6 hr after infection, and group four received Pep27 (4 mg/kg) 6 hr after infection. At 6 hr intervals, CSF (200 l) was withdrawn and tested for bacterial and leukocyte density.
Molecular Cell 56
Computer-Assisted Sequence Analysis Sequence analysis and alignments were conducted with the program DNA-Star and with the Genetics Computer Group sequence analysis software package. The computer program BLAST was used to search for amino acid sequences that were homologous to those of vex1, vex2, vex3, pep27, vncR, and vncS gene products (Altschul et al., 1990). Acknowledgments This work was supported in part by grants AI27913 and AI39482, Cancer Center Support CORE grant P30 CA 21765, and the American Lebanese Syrian Associated Charities. We acknowledge the excellent technical assistance of Juan Li. Received August 2, 1999; revised November 15, 1999. References Altschul, S.F., Gish, W., Miller, W., Myers, E.W., and Lipman, D.J. (1990). Basic local alignment search tool. J. Mol. Biol. 215, 403–410. Ames, G.F., Mimura, C.S., and Shyamala, V. (1990). Bacterial periplasmic permeases belong to a family of transport proteins operating from Escherichia coli to human: traffic ATPases. FEMS Microbiol. Rev. 6, 429–446. Brown, M.C.M., Weston, A., Saunders, J.R., and Humphreys, G.O. (1979). Transformation of E. coli C600 by plasmid DNA at different phases of growth. FEMS Microbiol. Lett. 5, 219–222. Cashel, M., Gentry, D.R., Hernandez, V.J., and Vinella, D. (1996). The stringent response. In Escherichia coli and Salmonella: cellular and molecular biology, F.C. Neidhardt, R. Curtis III, J.L. Ingraham, E.C.C. Lin, K.B. Low, B. Magasanik, W.S. Reznikoff, M. Riley, M. Schaechter, and H.E. Umbarger, eds. (Washington, D.C.: ASM Press). Chen, J.D., and Morrison, D.A. (1987). Cloning of Streptococcus pneumoniae DNA fragments in Escherichia coli requires vectors protected by strong transcriptional terminators. Gene 55, 179–187. Dunny, G.M., and Leonard, B.A.B. (1997). Cell-cell communication in gram-positive bacteria. Annu. Rev. Microbiol. 51, 527–564. Entenza, J.M., Caldelari, I., Glauser, M.P., Francioli, P., and Moreillon, P. (1997). Importance of genotypic tolerance in the treatment of experimental endocarditis due to Streptococcus gordonii. J. Infect. Dis. 175, 70–76. Forsberg, C., and Rogers, H.J. (1971). Autolytic enzymes in growth of bacteria. Nature 229, 272–273. Fuqua, W.C., Winans, S.C., and Greenberg, E.P. (1994). Quorum sensing in bacteria: the LuxR-LuxI family of cell density-responsive transcriptional regulators. J. Bacteriol. 176, 269–275. Garcia, P., Gonzalez, M.P., Garcia, E., Rubens, L., and Garcia, J.L. (1999). Lyt B, a novel pneumococcal murein hydrolase essential for cell separation. Mol. Microbiol. 31, 1275–1277. Gough, M., Hancock, R.E.W., and Kelly, N.M. (1996). Anti endotoxic potential of cationic peptide antimicrobials. Infect. Immun. 64, 4922– 4927. Hakenbeck, R., and Messer, W. (1977). Activity of murein hydrolases in synchronized cultures of E. coli. Eur. J. Biochem. 129, 1239–1244. Hakenbeck, R., Ellerbrok, H., Briese, T., Handwerger, S., and Tomasz, A. (1986). Penicillin-binding proteins of penicillin-susceptible and -resistant pneumococci: immunological relatedness of altered proteins and changes in peptides carrying the beta-lactam binding site. Antimicrob. Agents Chemother. 30, 553–558. Haldenwang, W.G., Banner, C.D., Ollington, J.F., Losick, R., Hoch, J.A., O’Connor, M.B., and Sonenshein, A.L. (1980). Mapping a cloned gene under sporulation control by insertion of a drug resistance marker into the Bacillus subtilis chromosome. J. Bacteriol. 142, 90–98.
unmodified heptadecapeptide pheromone induces competence for genetic transformation in Streptococcus pneumoniae. Proc. Natl. Acad. Sci. USA 92, 11140–11144. Higgins, C.F. (1992). ABC transporters: from microorganisms to man. Annu. Rev. Cell Biol. 8, 67–113. Hiles, I.D., Gallagher, M.P., Jamieson, D.J., and Higgins, C.F. (1987). Molecular characterization of the oligopeptide permease of Salmonella typhimurium. J. Mol. Biol. 195, 125–142. Hobby, G.L., Meyer, K., and Chaffee, E. (1942). Observations on the mechanism of action of penicillin. Proc. Soc. Exp. Biol. 50, 281–285. Horne, D., and Tomasz, A. (1977). Tolerant response of Streptococcus sanguis to beta-lactams and other cell wall inhibitors. Antimicrob. Agents Chemother. 11, 888–896. Hung, L.-W., Wang, I.X., Nikaido, K., Liu, P.-Q., Ames, G.F.-L., and Kim, S.-H. (1998). Crystal structure of the ATP-subunit of an ABC transporter. Nature 396, 703–707. Hyde, S.C., Emsley, P., Hartshorn, M.J., Mimmack, M.M., Gileadi, U., Pearce, S.R., Gallagher, M.P., Gill, D.R., Hubbard, R.E., and Higgins, C.F. (1990). Structural model of ATP-binding proteins associated with cystic fibrosis, multidrug resistance and bacterial transport. Nature 346, 362–365. Ishiguro, E.E., and Ramey, W.D. (1976). Stringent control of peptidoglycan biosynthesis in Escherichia coli K-12. J. Bacteriol. 127, 1119–1126. Jenkinson, H.F., Baker, R.A., and Tannock, G.W. (1996). A bindinglipoprotein-dependent oligopeptide transport system in Streptococcus gordonii essential for uptake of hexa- and heptapeptides. J. Bacteriol. 178, 68–77. Kitano, K., and Tomasz, A. (1979). Triggering of autolytic cell wall degradation in E. coli by betalactam antibiotics. Antimicrob. Agents Chemother. 16, 838–848. Kleerebezem, M., Quadri, L.E., Kuipers, O.P., and de Vos, W.M. (1997). Quorum sensing by peptide pheromones and two component signal-transduction systems in Gram-positive bacteria. Mol. Microbiol. 24, 895–904. Kolter, R., and Moreno, F. (1992). Genetics of ribosomally synthesized peptide antibiotics. Annu. Rev. Microbiol. 46, 141–165. Lacks, S., and Hotchkiss, R.D. (1960). A study of the genetic material determining an enzyme activity in pneumococcus. Biochem. Biophys. Acta 39, 508–517. Linton, K.J., and Higgins, C.F. (1998). The Escherichia coli ATPbinding cassette (ABC) proteins. Mol. Microbiol. 28, 5–13. Liu, H.H., and Tomasz, A. (1985). Penicillin tolerance in multiply drug-resistant natural isolates of Streptococcus pneumoniae. J. Infect. Dis. 152, 365–372. McDowell, T.D., and Lemanski, C.L. (1988). Absence of autolytic activity (peptidoglycan nicking) in penicillin-induced nonlytic death in group A streptococcus. J. Bacteriol. 170, 1783–1788. Meeson, J., McColm, A.A., Acred, P., and Greenwood, D. (1990). Differential response to benzylpenicillin in vivo of tolerant and nontolerant variants of Streptococcus sanguis II. J. Antimicrob. Chemother. 25, 103–109. Mejean, V., Claverys, J.P., Vasseghi, H., and Sicard, A.M. (1981). Rapid cloning of specific DNA fragments of Streptococcus pneumoniae by vector integration into the chromosome followed by endonucleolytic excision. Gene 15, 289–293. Metzger, S., Dror, I.B., Aizenman, E., Schreiber, G., Toone, M., Friesen, J.D., Cashel, M., and Glaser, G. (1988). The nucleotide sequence and characterization of the relA gene of Escherichia coli. J. Biol. Chem. 263, 15699–15704. Moreillon, P., and Tomasz, A. (1988). Penicillin resistance and defective lysis in clinical isolates of pneumococci: evidence for two kinds of antibiotic pressure operating in the clinical environment. J. Infect. Dis. 157, 1150–1157.
Handwerger, S., and Tomasz, A. (1985). Antibiotic tolerance among clinical isolates of bacteria. Rev. Infect. Dis. 7, 368–386.
Moreillon, P., Markiewicz, Z., Nachman, S., and Tomasz, A. (1990). Two bactericidal targets for penicillin in pneumococci: autolysisdependent and autolysis-independent killing mechanisms. Antimicrob. Agents Chemother. 34, 33–39.
Havarstein, L.S., Coomaraswamy, G., and Morrison, D.A. (1995). An
Morrison, D.A., and Baker, M.F. (1979). Competence for genetic
Hancock, R.E.W. (1997). Peptide antibiotics. Lancet 349, 418–422.
Death Signal Triggers Bacterial Autolytic Pathways 57
transformation in pneumococcus depends on synthesis of a small set of proteins. Nature 282, 215–217. Mosser, J.L., and Tomasz, A. (1970). Choline-containing teichoic acid as a structural component of pneumococcal cell wall and its role in sensitivity to lysis by an autolytic enzyme. J. Biol. Chem. 245, 287–298.
Walker, J.E., Saraste, M., Runswich, M.J., and Gay, N.J. (1982). Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases, and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J. 1, 945–951. Weidel, J., and Pelzer, H. (1964). Bag shaped macromolecules—a new look on bacterial cell walls. Adv. Enzymol. 26, 193–232.
Novak, R., Braun, J.S., Charpentier, E., and Tuomanen, E. (1998). Penicillin tolerance genes of Streptococcus pneumoniae: the ABCtype manganese complex Psa. Mol. Microbiol. 29, 1285–1296.
Williamson, R., and Tomasz, A. (1980). Antibiotic-tolerant mutants of Streptococcus pneumoniae that are not deficient in autolytic activity. J. Bacteriol. 144, 105–113.
Novak, R., Henriques, B., Charpentier, E., Normark, S., and Tuomanen, E. (1999). Emergence of vancomycin tolerance in Streptococcus pneumoniae. Nature 399, 590–593.
GenBank Accession Number
Pearce, B.J., Yin, Y.B., and Masure, H.R. (1993). Genetic identification of exported proteins in Streptococcus pneumoniae. Mol. Microbiol. 9, 1037–1050. Pestova, E.V., Havarstein, L.S., and Morrison, D.A. (1996). Regulation of competence for genetic transformability in Streptococcus pneumoniae by an auto-induced peptide pheromone and a two-component regulatory system. Mol. Microbiol. 21, 853–862. Piers, K.L., Brown, M.H., and Hancock, R.E.W. (1994). Improvement of outer membrane-permeabilization and lipopolysaccharide-binding activities of an antimicrobial cationic peptide by C-terminal modification. Antimicrob. Agents Chemother. 38, 2311–2316. Ronda, C., Garcia, J.L., Garcia, E., Sanchez-Puelles, J.M., and Lopez, R. (1987). Biological role of the pneumococcal amidase. Cloning of the lytA gene in Streptococcus pneumoniae. Eur. J. Biochem. 164, 621–624. Russell, R.R., Aduse, O.J., Sutcliffe, I.C., Tao, L., and Ferretti, J.J. (1992). A binding protein-dependent transport system in Streptococcus mutans responsible for multiple sugar metabolism. J. Biol. Chem. 267, 4631–4637. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1990). Molecular Cloning: A Laboratory Manual, Second Edition. (Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press). Schreiber, G., Metzger, S., Aizenbaum, E., Roza, S., Cashel, M., and Glaser, G. (1991). Overexpression of the relA gene in Escherichia coli. J. Biol. Chem. 266, 3760–3767. Sokawa, Y., Nakao, E., and Kaziro, Y. (1968). On the nature of the control by RC gene in E. coli: amino acid-dependent control of lipid synthesis. Biochem. Biophys. Res. Commun. 33, 108–112. Svitil, A.L., Cashel, M., and Zyskind, J.W. (1993). Guanosine tetraphosphate inhibits protein synthesis in vivo. A possible protective mechanism for starvation stress in Escherichia coli. J. Biol. Chem. 268, 2307–2311. Tauber, M.G., and Zwahlen, A. (1994). Animal models of meningitis. Methods Enzymol. 235, 92–106. Tiraby, G., and Fox, M.S. (1973). Marker discrimination in transformation and mutation of pneumococcus. Proc. Natl. Acad. Sci. USA. 70, 3541–3545. Tomasz, A. (1979). From penicillin-binding proteins to the lysis and death of bacteria: a 1979 view. Rev. Infect. Dis. 1, 434–467. Tomasz, A. (1983). Murein hydrolases—enzymes in search of a physiological function. In The Target of Penicillin, R. Hakenbeck, J. Holtje and L.H., eds. (Berlin: Walter de Gruyter), pp. 155–172. Tomasz, A., and Holtje, J.V. (1977). Murein hydrolases and the lytic and killing action of penicillin. In Microbiology, D. Schlesinger, ed. (Washington DC: American Society for Microbiology), pp. 209–215. Tomasz, A., Albino, A., and Zanati, E. (1970). Multiple antibiotic resistance in a bacterium with suppressed autolytic system. Nature 227, 138–140. Tuomanen, E. (1986). Phenotypic tolerance: the search for -lactam antibiotics that kill nongrowing bacteria. Rev. Infect. Dis. 8, Suppl. 3, 279–291. Tuomanen, E., Durack, D.T., and Tomasz, A. (1986). Antibiotic tolerance among clinical isolates of bacteria. Antimicrob. Agents Chemother. 30, 521–527. Tuomanen, E., Pollack, H., Parkinson, A., Davidson, M., Facklam, R., Rich, R., and Zak, O. (1988). Microbiological and clinical significance of a new property of defective lysis in clinical strains of pneumococci. J. Infect. Dis. 158, 36–43.
The DNA sequence of the 3.1 kb vex locus has been assigned the GenBank accession number AF140784.
