The two-step lysis system of pneumococcal bacteriophage EJ-1 is functional in Gram-negative bacteria: triggering of the major pneumococcal autolysin in Escherichia coli

The holin function Ejh of the pneumococcal bacteriophage EJ-1 has been characterized. It shows structural features similar to, and functionally complemented, the prototype member of the holin family. In Escherichia coli and Pseudomonas putida the Ejh product caused cellular death, and changes in cell morphology could be accounted for by lesions in the cytoplasmic membrane. Expression of ejh resulted in the inhibition of growth in a variety of phylogenetically distant bacterial genera, suggesting a broad spectrum of action. Concomitant expression of the ejh and ejl (encodes a lysin) genes led to lysis of E. coli and P. putida cells. Remarkably, the Ejl lysin was able to attack murein from bacteria lacking choline in their sacculi, which suggests that pneumococcal lysins have a broader substrate specificity than previously assumed. Furthermore, the Ejh holin was able to trigger activity of the major pneumococcal autolysin cloned and expressed in E. coli , and this raised new questions about the regulation of this model autolysin. A new function for holins in systems where the phage lysin is supposed to be associated with the membrane is proposed.     

The murein hydrolase of the bacteriophage phi3626 dual lysis system is active against all tested Clostridium perfringens strains

Clostridium perfringens commonly occurs in food and feed, can produce an enterotoxin frequently implicated in food-borne disease, and has a substantial negative impact on the poultry industry. As a step towards new approaches for control of this organism, we investigated the cell wall lysis system of C. perfringens bacteriophage phi3626, whose dual lysis gene cassette consists of a holin gene and an endolysin gene. Hol3626 has two membrane-spanning domains (MSDs) and is a group II holin. A positively charged beta turn between the two MSDs suggests that both the amino terminus and the carboxy terminus of Hol3626 might be located outside the cell membrane, a very unusual holin topology. Holin function was experimentally demonstrated by using the ability of the holin to complement a deletion of the heterologous phage lambda S holin in lambdadeltaSthf. The endolysin gene ply3626 was cloned in Escherichia coli. However, protein synthesis occurred only when bacteria were supplemented with rare tRNA(Arg) and tRNA(Ile) genes. Formation of inclusion bodies could be avoided by drastically lowering the expression level. Amino-terminal modification by a six-histidine tag did not affect enzyme activity and enabled purification by metal chelate affinity chromatography. Ply3626 has an N-terminal amidase domain and a unique C-terminal portion, which might be responsible for the specific lytic range of the enzyme. All 48 tested strains of C. perfringens were sensitive to the murein hydrolase, whereas other clostridia and bacteria belonging to other genera were generally not affected. This highly specific activity towards C. perfringens might be useful for novel biocontrol measures in food, feed, and complex microbial communities.     

Role ofS gene product of bacteriophage lambda in host cell lysis

Studies with the induced lysogens of λS ⁺R⁺, λS^-R⁺, λS⁺R^- and λS-R^- phages have shown that while theS gene product is essential for the action of intracellularR gene product to release the periplasmic alkaline phosphatase in the presence of EDTA, the latter gene product can bring about this effect while acting onEscherichia coli cells from outside, in the absence of functionalS gene product; chloroform, could help the intracellularR gene product in effecting bacterial lysis in the absence ofS gene product. These result support the premise that theS gene product facilitates theR gene product in crossing the cytoplasmic membrane into the periplasmic space such that the latter can act on the peptidoglycan layer of the host cell thus causing both the release of alkaline phosphatase and cell lysis. An erratum to this article is available at .     

Biochemical characterization of a murein hydrolase induced by bacteriophage Dp-1 in Streptococcus pneumoniae: comparative study between bacteriophage-associated lysin and the host amidase.

A phage-associated lysin recently isolated and purified from Streptococcus pneumoniae infected with bacteriophage Dp-1 has been biochemically characterized as an endo-N-acetyl-muramyl-L-alanine amidase. The purified peptides obtained after treatment of the cell wall with phage-associated lysin are composed of glutamic acid, alanine, lysine, glycine, serine, and aspartic acid in the molar ratios of 1.0:1.6:1.0:1.0:0.8:0.6. The N-terminal amino acid of this peptide has been characterized as alanine. This amidase and the inactive form of the amidase (E form) previously purified (J.V. Höltje and A. Tomasz, J. Biol. Chem. 251:4199-4207, 1976) from S. pneumoniae differ in their molecular weights, as well as in their capacity to be stimulated by reducing agents, and do not cross-react immunologically, although anti-phage-associated lysin serum was able to recognize and inhibit both phage-associated lysin and the active form (C form) of the host amidase.     

Analysis of murein and murein precursors during antibiotic-induced lysis of Escherichia coli.

Lysis of Escherichia coli induced by either D-cycloserine, moenomycin, or penicillin G was monitored by studying murein metabolism. The levels of the soluble murein precursor UDP-N-acetylmuramyl-L-alanyl-D-glutamyl-m-diaminopimelyl-D-alanyl- D-alanine (UDP-MurNAc-pentapeptide) and the carrier-linked MurNAc-(pentapeptide)-pyrophosphoryl-undecaprenol as well as N-acetylglucosamine-beta-1,4-MurNAc-(pentapeptide)-pyrophosphoryl- undecaprenol varied in a specific way. In the presence of penicillin, which is known to interfere with the cross-linking of murein, the concentration of the lipid-linked precursors unexpectedly decreased before the onset of lysis, although the level of UDP-MurNAc-pentapeptide remained normal. In the case of moenomycin, which specifically blocks the formation of the murein polysaccharide strands, the lipid-linked precursors as well as UDP-MurNAc-pentapeptide accumulated as was expected. D-Cycloserine, which inhibits the biosynthesis of UDP-MurNAc-pentapeptide, consequently caused a decrease in all three precursors. The muropeptide composition of the murein showed general changes such as an increase in the unusual DL-cross bridge between two neighboring meso-diaminopimelic acid residues and, as a result of uncontrolled DL- and DD-carboxypeptidase activity, an increase in tripeptidyl and a decrease in tetrapeptidyl and pentapeptidyl moieties. The average length of the glycan strands decreased. When the glycan strands were fractionated according to length, a dramatic increase in the amount of single disaccharide units was observed not only in the presence of penicillin but also in the presence of moenomycin. This result is explained by the action of an exo-muramidase, such as the lytic transglycosylases present in E. coli. It is proposed that antibiotic-induced bacteriolysis is the result of a zipperlike splitting of the murein net by exo-muramidases locally restricted to the equatorial zone of the cell.     

Purification and polar localization of pneumococcal LytB,a putative endo-beta-N-acetylglucosaminidase: the chain-dispersing murein hydrolase

The DNA region encoding the mature form of a pneumococcal murein hydrolase (LytB) was cloned and expressed in Escherichia coli. LytB was purified by affinity chromatography, and its activity was suggested to be the first identified endo-beta-N-acetylglucosaminidase of Streptococcus pneumoniae. LytB can remove a maximum of only 25% of the radioactivity from [(3)H]choline-labeled pneumococcal cell walls in in vitro assays. Inactivation of the lytB gene of wild-type strain R6 (R6B mutant) led to the formation of long chains but did not affect either total cell wall hydrolytic activity at the stationary phase of growth or development of genetic competence. Longer chains were formed when the lytB mutation was introduced into the M31 strain (M31B mutant), which harbors a complete deletion of lytA, which codes for the major autolysin. Furthermore, the use of this mutant revealed that LytB is the first nonautolytic murein hydrolase of pneumococcus. Purified LytB added to pneumococcal cultures of R6B or M31B was capable of dispersing, in a dose-dependent manner, the long chains characteristic of these mutants into diplococci or short chains, the typical morphology of R6 and M31 strains, respectively. In vitro acetylation of purified pneumococcal cell walls did not affect the activity of LytB, whereas that of the LytA amidase was drastically reduced. On the other hand, the use of a translational fusion between the gene (gfp) coding for the green fluorescent protein (GFP) and lytB supports the notion that LytB accumulates in the cell poles of either the wild-type R6, lytB mutants, or ethanolamine-containing cells (EA cells). The GFP-LytB fusion protein was also able to unchain the lytB mutants but not the EA cells. In contrast, translational fusion protein GFP-LytA preferentially bound to the equatorial regions of choline-containing cells but did not affect their average chain length. These observations suggest the existence of specific receptors for LytB that are positioned at the polar region on the pneumococcal surface, allowing localized peptidoglycan hydrolysis and separation of the daughter cells.     

Genetic and physiological control of host cell lysis by bacteriophage lambda.

The timing of host cell lysis at the end of the lytic cycle of phage lambda is under complex control. The lambda S protein stimulates lysis. Another physiological system, the lysis regulator, inhibitis lysis from occurring prematurely. The effects of a series of phage and bacterial mutations on these controls are described. They show that the lambda rex gene plays a role in regulating lysis under suboptimal growth conditions. In certain mutant cells, and especially under anaerobic culture conditions, the rex gene aids in the scheduling of host cell lysis. The data also suggest that the lysis regulator may control the transition of the lambda S protein from an inactive to an active state.     

Carboxy-terminal deletion analysis of the major pneumococcal autolysin.

Autolysins are endogenous enzymes that specifically degrade the covalent bonds of the cell walls and eventually can induce bacterial lysis. One of the best-characterized autolysins, the major pneumococcal LytA amidase, has evolved by the fusion of two domains, the N-terminal catalytic domain and the C-terminal domain responsible for the binding to cell walls. The precise biochemical role played by the six repeat units that form the C-terminal domain of the LytA amidase has been investigated by producing serial deletions. Biochemical analyses of the truncated mutants revealed that the LytA amidase must contain at least four units to efficiently recognize the choline residues of pneumococcal cell walls. The loss of an additional unit dramatically reduces its hydrolytic activity as well as the binding affinity, suggesting that the catalytic efficiency of this enzyme can be considerably improved by keeping the protein attached to the cell wall substrate. Truncated proteins lacking one or two repeat units were more sensitive to the inhibition by free choline than the wild-type enzyme, whereas the N-terminal catalytic domain was insensitive to this inhibition. In addition, the truncated proteins were inhibited by deoxycholate (DOC), and the expression of a LytA amidase lacking the last 11 amino acids in Streptococcus pneumoniae M31, a strain having a deletion in the lytA gene, conferred to the cells an atypical phenotype (Lyt+ DOC-) (cells autolysed at the end of the stationary phase but were not sensitive to lysis induced by DOC), which has been previously observed in some clinical isolates of pneumococci. Our results are in agreement with the existence of several choline-binding sites and suggest that the stepwise acquisition of the repeat units and the tail could be considered an evolutionary advantage for the enzyme, since the presence of these motifs increases its hydrolytic activity.     

Role of the major pneumococcal autolysin in the atypical response of a clinical isolate of Streptococcus pneumoniae.

The autolytic enzyme (an N-acetylmuramyl-L-alanine amidase) of a clinical isolate, strain 101/87, which is classified as an atypical pneumococcus, has been studied for the first time. The lytA101 gene coding for this amidase (LYTA101) has been cloned, sequenced, and expressed in Escherichia coli. The LYTA101 amidase has been purified and shown to be similar to the main autolytic enzyme (LYTA) present in the wild-type strain of Streptococcus pneumoniae, although it exhibits a lower specific activity, a higher sensitivity to inhibition by free choline, and a modified thermosensitivity with respect to LYTA. Most important, in contrast with the LYTA amidase, the activity of the LYTA101 amidase was inhibited by sodium deoxycholate. This property is most probably responsible of the deoxycholate-insensitive phenotype shown by strain 101/87. Phenotypic curing of strain 101/87 by externally adding purified LYTA or LYTA101 amidase restored in this strain some typical characteristics of the wild-type strain of pneumococcus (e.g., formation of diplo cells and sensitization to lysis by sodium deoxycholate), although the amount of the LYTA101 amidase required to restore these properties was much higher than in the case of the LYTA amidase. Our results indicate that modifications in the primary structure or in the mechanisms that control the activity of cell wall lytic enzymes seem to be responsible for the characteristics exhibited by some strains of S. pneumoniae that have been classically misclassified and should be now considered atypical pneumococcal strains.     

Sequence of the Streptococcus pneumoniae bacteriophage HB-3 amidase reveals high homology with the major host autolysin.

We have sequenced a DNA fragment containing the pneumococcal bacteriophage HB-3 hbl gene, which codes for the phage lytic amidase. A remarkable nucleotide similarity (87.1%) between the lytA gene, coding for the pneumococcal amidase, the major autolysin of Streptococcus pneumoniae, and the hbl gene was found. This similarity completely disappeared outside the open reading frames coding for both amidases. The hbl gene transformed amidase-deficient strains of S. pneumoniae to the wild-type phenotype, and Southern blotting experiments provided evidence for recombination between donor and recipient genes. A comprehensive evaluation of these and previous results on the peptidoglycan hydrolases of S. pneumoniae and its bacteriophages suggested that recombination mechanisms participate in the evolution of the genes coding for these enzymes.     

Selective lysis of cultures and cell walls of penicillin-resistant but not penicillin-susceptible Streptococcus pneumoniae strains by a murein hydrolase complex.

A murein hydrolase complex selectively lysed cultures of penicillin-resistant pneumococci and their cell walls in which the majority of muropeptide subunits were indirectly cross-linked through oligopeptide substituents (alanyl-alanine or alanyl-serine) on the epsilon-amino group of the stem peptide lysine residues. Walls of penicillin-susceptible strains were not hydrolyzed.     

Cloning and expression of the pneumococcal autolysin gene in Escherichia coli

Summary A 7.5 kb BclI-fragment of Streptococcs pneumoniae DNA has been cloned in Escherichia coli HB101 using pBR322 as a vector. The new plasmid (pGL30) of 12.0 kb expresses a protein that has been characterized by biochemical, immunological and genetic methods as the inactive form (E-form) of the pneumococcal N-acetyl-muramyl-l-alanyl amidase (EC 3.5.1.28). Our results demonstrate that the E-form is the primary product of the lyt gene of S. pneumoniae. The inactive E-form can be converted to the active C-form in vitro by incubation of the E-form enzyme with choline-containing pneumococcal cell walls at low temperature in a similar way to enzyme production in the homologous system. The production of this protein in E. coli HB101 was 500-fold higher than in the homologous host. E. coli CSR603 containing pGL30 and labeled with [³⁵S]methionine synthesized a 35 kd protein. pGL30 can transform at high frequency an autolysin-defective mutant of S. pneumoniae to the lyt⁺ phenotype.     

A murein hydrolase is the specific target of bulgecin in Escherichia coli.

A deletion in the structural gene for the soluble lytic transglycosylase, the predominant murein hydrolase in the soluble fraction of Escherichia coli, has been constructed. The mutant grows normally but exhibits increased sensitivity toward mecillinam, a beta-lactam specific for penicillin-binding protein 2. In the presence of furazlocillin or other beta-lactams with a specificity for penicillin-binding protein 3 which normally cause filamentation, bulges were formed prior to rapid bacteriolysis. Similar morphological alterations are known to develop in wild type E. coli cells when furazlocillin is combined with bulgecin, an antibiotic of unusual glucosaminyl structure. It turned out that bulgecin specifically inhibits the Sl-transglycosylase in a noncompetitive manner. Since bulgecin shows some structural analogy to the murein subunits we postulate that the soluble lytic transglycosylase, in addition to its active site, has a recognition site for specific murein structures. The possibility of an allosteric modulation of the activity of the enzyme by changes in the structure of the murein sacculus is discussed.     

Searching for autolysin functions. Characterization of a pneumococcal mutant deleted in the lytA gene

The first mutant of Streptococcus pneumoniae showing a complete deletion in the lytA gene coding for the N-acetylmuramyl-L-alanine amidase has been isolated and characterized. This amidase was previously the only autolysin detected in this species. This mutant shows a normal growth rate and can be transformed using either chromosomal or plasmid DNA. The most remarkable biological consequences of the absence of the amidase are the formation of small chains (six to eight cells) and the absence of lysis in the stationary phase of growth. In addition, this mutant exhibits a tolerant response against the beta-lactam antibiotics.