Cell lysis by induction of cloned lambda lysis genes

Garrett, Jinnie M.; Fusselman, Robert; Hise, Joseph; Chiou, Linda; Smith-Grillo, Dana; Schulz, John D; Young, Ry

doi:10.1007/bf00269678

Cited by 71 publications

(62 citation statements)

References 13 publications

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“…The same injury then would allow the release of autolytic enzymes to the cell wall and initiate lysis of the bacteria. This model is formally analogous to models proposed for the mechanism of lysis by bacteriophages in which the release of phage progeny is preceded by membrane injury through the insertion of a holin protein into the bacterial membrane (32,33). Our model also is analogous to the proposed mode of action of nisin at the level of lipid II (34).…”

Section: Discussionsupporting

confidence: 52%

The murMN operon: A functional link between antibiotic resistance and antibiotic tolerance in Streptococcus pneumoniae

Filipe

Severina²,

Tomasz³

2002

Proc. Natl. Acad. Sci. U.S.A.

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Inactivation of the recently identified murMN operon in penicillinresistant strains of Streptococcus pneumoniae was shown already to cause two major effects: elimination of branched-structured muropeptides from the cell wall and complete loss of penicillin resistance. We now show that cells with inactivated murMN also have a third phenotype: an increased susceptibility to lysis when exposed to low concentrations of fosfomycin, D-cycloserine, vancomycin, and nisin, indicating a wide-spectrum hypersensitivity to inhibitors of both early and late stages of cell wall biosynthesis. Mutants of murMN also lysed faster than the parental strain when treated with the detergent deoxycholate. Several different alleles of murM cloned in plasmid pLS578 and introduced into a murM deletion mutant of the penicillin-resistant strain Pen6 were able to reconstitute each one of the three mutant phenotypes: the highly branched cell wall structure, original high level of penicillin resistance, and normal sensitivity to lysis. In a penicillin-susceptible strain the same experiments caused increased concentration of cell wall branched peptides and suppression of sensitivity to antibiotic induced lysis. The observations suggest that the murMN operon plays a key role in the regulation of a stress-response pathway that can be triggered by perturbation of cell wall biosynthesis in S. pneumoniae. S everal penicillin-resistant clones of Streptococcus pneumoniaeproduce abnormal cell walls in which a high proportion of the stem peptide lysine residues carry short (seryl alanine or alanyl alanine) branches (1). The genetic determinants involved with the branching process, the murMN operon, were identified recently (2). Penicillin-resistant isolates with highly branched cell walls were shown to carry murM alleles that contained divergent sequences as compared with the murM genes of penicillin-susceptible pneumococci (3). The two proteins, MurM (407 amino acids) and MurN (411 amino acids), are involved with the addition of the first and second amino acid residues, respectively, to the cell wall precursor stem peptide at the lipid II stage of biosynthesis (4, 5). Inactivation of murMN by insertion-duplication mutagenesis did not interfere with growth of the bacteria but caused two phenotypes: elimination of branched peptides from the cell wall and complete loss of penicillin resistance (2). The purpose of the studies described in this communication was to follow up these earlier findings by the use of a deletion mutant of murM and complementation experiments. The surprising observation made was that inactivation of murM also caused a third phenotype: increased sensitivity of the bacteria to lysis by cell wall inhibitors. Experimental ProceduresStrains and Growth Conditions. All strains and plasmids used in this study are listed in Table 1. S. pneumoniae strains were grown in a casein-based semisynthetic medium (C ϩ Y) at 37°C without aeration as described (11). S. pneumoniae strains containing pLS578 (10) or its derivatives were grown in the presen...

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Section: Discussionsupporting

confidence: 52%

The murMN operon: A functional link between antibiotic resistance and antibiotic tolerance in Streptococcus pneumoniae

Filipe

Severina²,

Tomasz³

2002

Proc. Natl. Acad. Sci. U.S.A.

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“…Phage has four lysis genes, S, R, Rz, and Rz1, clustered in the lysis cassette and transcribed from the single late gene promoter, pRЈ (7,17,20,21,30). Under standard laboratory conditions, only S and R are required for host cell lysis (11,28). R, the endolysin gene, encodes a transglycosylase, which accumulates fully folded in the cytoplasm (3,10).…”

mentioning

confidence: 99%

Genetic and Biochemical Analysis of Dimer and Oligomer Interactions of the λ S Holin

2000

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Bacteriophage uses a holin-endolysin system for host cell lysis. R, the endolysin, has muralytic activity. S, the holin, is a small membrane protein that permeabilizes the inner membrane at a precisely scheduled time after infection and allows the endolysin access to its substrate, resulting in host cell lysis. S has a single cysteine at position 51 that can be replaced by a serine without loss of the holin function. A collection of 27 single-cysteine products of alleles created from S C51S were tested for holin function. Most of the singlecysteine variants retained the ability to support lysis. Mutations with the most defective phenotype clustered in the first two hydrophobic transmembrane domains. Several lines of evidence indicate that S forms an oligomeric structure in the inner membrane. Here we show that oligomerization does not depend on disulfide bridge formation, since the cysteineless S C51S (i) is functional as a holin and (ii) shows the same oligomerization pattern as the parental S protein. In contrast, the lysis-defective S A52V mutant dimerizes but does not form cross-linkable oligomers. Again, dimerization does not depend on the natural cysteine, since the cysteineless lysis-defective S A52V/C51S is found in dimers after treatment of the membrane with a cross-linking agent. Furthermore, under oxidative conditions, dimerization via the natural cysteine is very efficient for S A52V . Both S A52V (dominant negative) and S A48V (antidominant) interact with the parental S protein, as judged by oxidative disulfide bridge formation. Thus, productive and unproductive heterodimer formation between the parental protein and the mutants S A52V and S A48V , respectively, may account for the dominant and antidominant lysis phenotypes. Examination of oxidative dimer formation between S variants with single cysteines in the hydrophobic core of the second membrane-spanning domain revealed that positions 48 and 51 are on a dimer interface. These results are discussed in terms of a three-step model leading to S-dependent hole formation in the inner membrane.

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“…Only S and R are absolutely required for host cell lysis under standard laboratory conditions (14,15). Rz and Rz1 are required for lysis only when the outer membrane is stabilized by millimolar concentrations of divalent cations (40,42).…”

mentioning

confidence: 99%

Dimerization between the Holin and Holin Inhibitor of Phage λ

et al. 2000

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Holins are integral membrane proteins that control the access of phage-encoded muralytic enzymes, or endolysins, to the cell wall by the sudden formation of an uncharacterized homo-oligomeric lesion, or hole, in the membrane, at a precisely defined time. The timing of -infected cell lysis depends solely on the 107 codon S gene, which encodes two proteins, S105 and S107, which are the holin and holin inhibitor, respectively. Here we report the results of biochemical and genetic studies on the interaction between the holin and the holin inhibitor. A unique cysteine at position 51, in the middle of the second transmembrane domain, is shown to cause the formation of disulfide-linked dimers during detergent membrane extraction. Forced oxidation of membranes containing S molecules also results in the formation of covalently linked dimers. This technique is used to demonstrate efficient dimeric interactions between S105 and S107. These results, coupled with the previous finding that the timing of lysis depends on the excess of the amount of S105 over S107, suggest a model in which the inhibitor functions by titrating out the effector in a stoichiometric fashion. This provides a basis for understanding two evolutionary advantages provided by the inhibitor system, in which the production of the inhibitor not only causes a delay in the timing of lysis, allowing the assembly of more virions, but also increases effective hole formation after triggering.

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Cell lysis by induction of cloned lambda lysis genes

Cited by 71 publications

References 13 publications

The murMN operon: A functional link between antibiotic resistance and antibiotic tolerance in Streptococcus pneumoniae

The murMN operon: A functional link between antibiotic resistance and antibiotic tolerance in Streptococcus pneumoniae

Genetic and Biochemical Analysis of Dimer and Oligomer Interactions of the λ S Holin

Dimerization between the Holin and Holin Inhibitor of Phage λ

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