Concepción Ronda

Species and Type Phages of Lactococcal Bacteriophages

Lactococcal phages are classified according to morphology and DNA homology. Phages are differentiated into 12 phage species, and type phages of each species are proposed. Members and possible members of each species are named. Available data on type phages are tabulated including morphology, DNA characteristics and phage protein bands.

Cloning and expression of the pneumococcal autolysin gene in Escherichia coli

SummaryA 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.

Interchange of functional domains switches enzyme specificity: construction of a chimeric pneumococcal‐clostridial cell wall lytic enzyme

Bacterial autolysins are endogenous enzymes that specifically cleave covalent bonds in the cell wall. These enzymes show both substrate and bond specificities. The former is related to their interaction with the insoluble substrate whereas the latter determine their site of action. The bond specificity allows their classification as muramidases (lysozymes), glucosaminldases, amidases, and endopeptidases. To demonstrate that the autolysin (LYC muramidase) of Clostridium acetobutylicum ATCC824 presents a domainal organization, a chimeric gene (clc) containing the regions coding for the catalytic domain of the LYC muramidase and the choline‐binding domain of the pneumococcal phage CPL1 muramidase has been constructed by in vitro recombination of the corresponding gene fragments. This chimeric construction codes for a choline‐binding protein (CLC) that has been purified using affinity chromatography on DEAE‐cellulose. Several biochemical tests demonstrate that this rearrangement of domains has generated an enzyme with a choline‐dependent muramidase activity on pneumococcal cell walls. Since the parental LYC muramidase was cholineindependent and unable to degrade pneumococcal cell walls, the formation of this active chimeric enzyme by exchanging protein domains between two enzymes that specifically hydrolyse cell walls of bacteria belonging to different genera shows that a switch on substrate specificity has been achieved. The chimeric CLC muramidase behaved as an autolytic enzyme when it was adsorbed onto a live autolysin‐defective mutant of Streptococcus pneumoniae. The construction described here provides experimental support for the theory of modular evolution which assumes that novel proteins have evolved by the assembly of preexisting polypeptide units.

Characterization of genetic transformation in Streptococcus oralis NCTC 11427: Expression of the pneumococcal amidase in S. oralis using a new shuttle vector

SummaryWe have worked out conditions for the study of competence development and genetic transformation in Streptococcus oralis NCTC 11427 (type strain), a species that contains choline in the cell wall. The peak of competence was found at the early exponential phase of growth and the optimal conditions for transformation were achieved with shuttle plasmids prepared from S. pneumoniae or from Escherichia coli serving as donor DNA. Transformation with dye-bouyant density gradient purified plasmid preparations followed first-order kinetics. The pneumococcal amidase can be expressed in S. oralis harbouring a plasmid carrying the lytA gene. This enzyme lysed the cell wall of the transformed cell in the presence of detergents.

Role of the C-terminal domain of the lysozyme of Clostridium acetobutylicum ATCC 824 in a chimeric pneumococcal-clostridial cell wall lytic enzyme.

An active chimeric cell wall lytic enzyme has been constructed by domain substitution between the major autolysins of Clostridium acetobutylicum ATCC 824 and Streptococcus pneumoniae. The chimeric enzyme, built up by the fusion of the N‐terminal domain of the pneumococcal LYTA amidase and the C‐terminal domain of the clostridial LYC lysozyme, exhibited an amidase activity capable of hydrolyzing choline‐containing clostridial cell walls with an efficiency 250‐times higher than when tested on pneumococcal cell walls. This experimental approach demonstrates the basic role of the C‐terminal domain of the LYC lysozyme in substrate recognition and provides additional support to our hypothesis of modular evolution of these lytic enzymes. Moreover, the construction described here confirmed the role of the C‐terminal domains of the modular cell wall lytic enzymes on the optimal pH for catalytic activity. To our knowledge, this is the first example of the construction of an active chimeric lytic enzyme by fusing genes that lack nucleotide homology and are derived from different bacterial genera.

Isolation, characterization and physiological properties of an autolytic-deficient mutant of Streptococcus pneumoniae

SummaryA spontaneous mutation in the gene lyt encoding the pneumococcal autolysin has been characterized. This mutation, named lyt-32, which behaves as a high-efficiency marker in pneumococcal transformation, is a single base pair GC deletion causing the appearance of two consecutive termination codons in the amino terminal part of the sequence of the autolysin gene. The mutant lyt gene did not code for a polypeptide of relative molecular mass corresponding to the pneumococcal E form amidase in Escherichia coli maxicells. Pneumococcal cells containing the lyt-32 mutation (M32) were fully transformable, multiplied at a normal growth rate forming small chains and showed a tolerant response when treated with beta-lactam antibiotics. Strain M32 represents the first example of a mutant of Streptococcus pneumoniae completely lacking amidase as a consequence of an alteration in the structural gene coding for the pneumococcal autolysin.

Restriction cleavage maps of the DNAs of Streptococcus pneumoniae bacteriophages containing protein covalently bound to their 5′ ends

SummarySeveral pneumococcal bacteriophages showing a morphology similar to that previously described for Cp-1 (Ronda et al. 1981) have been isolated and purified from throat samples taken from healthy children. Three of these phages (Cp-5, Cp-7 and Cp-9) have been studied in detail and compared to Cp-1. The four phages differed in several respects, e.g. size, structural polypeptides, restriction enzyme cleavage patterns, etc. The DNA of Cp-5, Cp-7 and Cp-9 showed protease-sensitive transfecting activity. This, together with the results obtained by electrophoretic analyses as well as by isotopic labelling of these DNAs with [γ-32P] ATP and polynucleotide kinase indicated that all these new phages have a protein covalently linked to the 5′ ends of their DNAs as in the case of Cp-1 (García et al. 1983). Restriction enzyme cleavage maps of Cp-1, Cp-5, Cp-7 and Cp-9 have been constructed.

Inhibition of lysis by antibody against phage-associated lysin and requirement of choline residues in the cell wall for progeny phage release inStreptococcus pneumoniae

We have prepared an antiserum against a new lytic enzyme (PAL) that has recently been isolated and purified from a mutant ofStreptococcus pneumoniae infected with the bacteriophage Dp-1. This antiserum inhibited the activity of the PALin vitro and was also capable of blocking the lysis of an amidase-deficient transformant of pneumococcus infected with Dp-1. This is a direct illustration of the involvement of the PAL in the liberation of the phage progeney in amidase-deficient strains of pneumococcus. The myeloma protein TEPC-15, an antibody that specifically blocks phosphorylcholine residues, inhibited the degradation of purified choline cell walls by the PAL. Our results also show that the liberation of the phage progeny to the medium requires the presence of choline in the cell wall. These findings demonstrate the basic role of the choline residues for the activity of the PALin vivo andin vitro.

A Phage-associated Murein Hydrolase in Streptococcus pneumoniae Infected with Bacteriophage Dp-1

A phage-associated murein hydrolase activity capable of degrading pneumococcal cell walls was isolated and purified to homogeneity from the phage-induced lysate of an autolysis-defective pneumococcal mutant infected with the bacteriophage Dp-1. Some properties of the enzyme resembled those of the wild-type (host) pneumococcal murein hydrolase: cell walls prepared from ethanolamine-grown pneumococci were resistant to the enzyme; the activity was inhibited by the Forssman antigen and was sensitive to proteolytic enzymes. The phage-associated enzyme was not inhibited by antiserum prepared against the purified pneumococcal murein hydrolase; the activity was stimulated by reducing agents and was partially inhibited by cardiolipin. The subunit molecular weight of the phage-associated enzyme was somewhat smaller (31 000) than that of the pneumococcal hydrolase (35 000). This appears to be the first description of a phage-associated murein hydrolase activity in pneumococci.

Molecular characterization of an autolysin-defective mutant of Streptococcuspneumoniae

The mutant gene lyt-4 of the autolysin-defective mutant R6ly4-4 of Streptococcus pneumoniae, which synthesized a temperature-sensitive autolytic enzyme, has been cloned in Escherichia coli. The nucleotide defect of the lyt-4 mutation has been characterized as a CG to TA transition. This transition causes the appearance of a glutamic acid instead of a glycine in the amino acid sequence of the autolysin, altering the hydropathic profile of the protein. This alteration might explain the observed thermosensitivity of the mutated autolytic enzyme. The present work represents the first molecular characterization of a mutation in the structural gene of a bacterial autolysin.