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We have found two murein hydrolases (LytB and LytC) tightly bound to the cell envelope that have completely changed the domain building plan previously reported for the murein hydrolases of Streptococcus pneumoniae. The active center of LytB and LytC is located at the C-terminal, whereas the binding domain is at the N-terminal. LytC has been characterized as the first lysozyme of S. pneumoniae and behaves as an autolysin at 30 degrees C. LytB appears as the main hydrolase responsible for cell separation since inactivation of lytB leads to the formation of long chains of more than 100 cells. These findings indicate that genetic adaptation of mobile domains is extremely efficient in pneumococcus.

We have localized, cloned and characterized the genes coding for the lytic system of the pneumococcal phage Dp-1. The lytic enzyme of this phage (Pal), previously identified as an N-acetyl-muramoyl-L-alanine amidase, shows a modular organization similar to that described for the lytic enzymes of Streptococcus pneumoniae and its bacteriophages. The construction of chimeric enzymes between pneumococcus and bacteria (or phages) that belong to different Gram-positive families has shown that the interchange of functional domains switches enzyme specificity. Interestingly, Pal appears to be a natural chimeric enzyme of intergeneric origin, that is the N-terminal domain was highly similar to that of the murein hydrolase coded by a gene found in the phage BK5-T that infects Lactococcus lactis, whereas the C-terminal domain was homologous to those found in the lytic enzymes of the pneumococcal system that is responsible for the binding to the choline residues present in the cell wall substrate. Biochemical analysis of Pal revealed that this enzyme shares important properties with those of the major LytA101 autolysin found in an atypical, clinical pneumococcal isolate. These peculiar characteristics have been ascribed to a modified C-terminal domain. The natural chimeric enzyme described here provides further support for the theory of modular evolution of proteins and its characteristics also furnish interesting clues on the molecular mechanisms involved in the more invasive types of atypical pneumococci.

The secondary and tertiary structures of the choline-dependent major pneumococcal autolysin LytA amidase and of its COOH-terminal domain, C-LytA, have been investigated by circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy. Deconvolution analysis shows that the far-UV CD spectrum of both proteins is governed by chiral contributions, ascribed to aromatic residue clusters contained in the COOH-terminal module. The secondary structure of LytA, determined from the FTIR spectral features of the amide I' band, results in 19% of alpha-helix and tight loops, 47% of beta-sheets, 23% of turns, and 11% of irregular structures. Similar values are obtained for C-LytA. The addition of choline significantly modifies the far- and near-UV CD spectra of LytA and C-LytA. These changes are attributed to alterations in the environment of their aromatic clusters, since the FTIR spectra indicate that the secondary structure is essentially unaffected. CD choline titration curves at different wavelengths show the existence of two types of binding sites/subunit. Data analysis assuming protein dimerization upon saturation of the high affinity sites reveals positive cooperativity between the low affinity sites. Thermal denaturation of both proteins occurs with the formation of unfolding intermediates and the presence of residual secondary structure in the final denatured state. The irreversibility of the thermal denaturation of LytA and C-LytA results from the collapse of the polypeptide chain into intermolecular extended structures. At saturating concentrations, choline prevents the formation of these structures in the isolated COOH-terminal module.

A chimeric trifunctional pneumococcal peptidoglycan hydrolase (CHL) has been constructed by fusing a choline-binding domain with two catalytic modules that provide lysozyme and amidase activity. The chimeric enzymes behaves as a choline-dependent enzyme and its activity is comparable to that of the parent enzymes. Site-directed mutagenesis of CHL produced a mutated enzyme [D9A,36A]CHL) that only exhibited an amidase activity. Comparative biochemical analyses of CHL and [D9A, E36A]CHL strongly suggest that the lysozyme catalytic module confers 88% of the total activity of CHL, whereas 12% of the activity can be ascribed to the amidase module. Both enzymatic activities are affected by the process of activation or conversion induced by choline suggesting that the conversion process is produced by a conformational change in the choline-binding domain. Our findings demonstrate that the three modules can acquire the proper folding conformation in the-three domain chimeric CHL enzyme. This experimental evidence supports the modular theory of protein evolution, and demonstrates that modular assembly of functional domains can be a rational approach to construct fully active chimeric enzymes with novel biological or biotechnological properties.

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), glucosaminidases, 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 choline-independent 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.

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.

The first temperate bacteriophage (EJ-1) of Streptococcus pneumoniae with Myoviridae morphotype A1 isolated from a clinical atypical strain has been purified and characterized. This phage has a double-stranded linear genome about 42 kb long, but in contrast to the other pneumococcal temperate phages that have been characterized so far, EJ-1 does not contain any protein covalently linked to it. We have sequenced a fragment of EJ-1 DNA containing the ejl gene, encoding a cell wall lytic enzyme (EJL amidase). This gene has been cloned and expressed in Escherichia coli, and the EJL enzyme was purified and biochemically characterized as an N-acetylmuramyl-L-alanine amidase that shares many similarities with the major pneumococcal autolysin. The EJL amidase is a choline-dependent enzyme that needs the process of conversion to achieve full enzymatic activity, but in contrast to the wild-type pneumococcal LYTA amidase, this process was found to be reversible. Comparisons of the primary structure of this new lytic enzyme with that of the other cell wall lytic enzymes of S. pneumoniae and its bacteriophages characterized so far provided new insights as to the evolutionary relationships between phages and bacteria. The nucleotide sequences of the attachment site (attP) on the phage genome and one of the junctions created by the insertion of the prophage were determined. Interestingly, the attP site was located near the ejl gene, as previously observed for the pneumococcal temperate bacteriophage HB-3 (A. Romero, R. López, and P. García, J. Virol. 66:2860-2864, 1992). A stem-and-loop structure, some adjacent direct and inverted repeats, and two putative integration host factor-binding sites were found in the att sites.

The complete lyc gene encoding the autolytic lysozyme of Clostridium acetobutylicum ATCC 824 was reconstructed from two overlapping DNA fragments and cloned into a suitable plasmid enabling Escherichia coli to produce this lytic enzyme under the control of the lac promoter. A polypeptide with an apparent M(r) of 35,000, corresponding to that predicted from the nucleotide sequence, was observed by maxicell analysis of whole-cell extracts of E. coli harboring the clostridial gene. The enzyme yield was shown to depend on the pH of the culture medium, since the protein was unstable at alkaline pH. The expression of the lyc gene was not increased by using the E. coli strong promoter, lpp-lac, probably due to the limit imposed by the extreme differences in codon usage. Although the LYC lysozyme does not contain a cleavable signal peptide, most of the protein was found in the periplasmic fraction of E. coli suggesting that this enzyme was secreted through a specific mechanism, as already observed for other autolysins.

Pneumococcal peptidoglycan amidase (N-acetylmuramoyl-L-alanine amidase, EC 3.5.1.28) and phage CPL1 lysozyme degrade a common substrate (choline-containing pneumococcal cell walls); the former hydrolyzes the bond between muramic acid and alanine, whereas the latter breaks down the linkage between muramic acid and glucosamine. The amino acid sequences of their C-terminal domains are homologous. Chimeric genes were constructed by site-directed mutagenesis: a unique SnaBI restriction site in the cpl1 gene, coding for the phage lysozyme, was introduced at a location equivalent to the SnaBI site present in the lytA gene, which codes for the pneumococcal amidase. The resulting genes expressed lytic activities at levels similar to those of the parental genes. The gene products, which have been purified to electrophoretical homogeneity, exhibited unusual combined biochemical properties--e.g., by exchange of protein domains, we have switched the regulatory properties of these enzymes without altering their catalytic activities. Chimeric gene construction in Streptococcus pneumoniae and its bacteriophages is an excellent model to study the modular organization of genes and proteins and to help to establish evolutionary relationships between phage and bacteria. These constructions provide an experimental approach to the molecular processes involved in cassette recruitment during evolution and contribute support to the concept of bacteria as adaptable chimeras.

A promoter-probe plasmid, pLSE4, containing the promoterless lytA gene that encodes the major pneumococcal autolysin, was developed to isolate and characterize nucleotide sequences of Streptococcus pneumoniae involved in transcriptional regulation. This vector was derived from the broad-host-range plasmid pLS1 and is suitable for the transformation of Gram- and Gram+ bacteria. An array of unique restriction sites was placed upstream from the lytA coding region. Pneumococcal promoters can be screened from random DNA fragments cloned in these sites for the ability to direct the expression of the autolysin in transformed autolysin-deficient pneumococcal cells. Transformants showing a Lyt+ phenotype were selected on agar plates using a simple filter technique. Relative promoter strength was determined by direct assay of the cell wall lytic activity in cell extracts.

The pneumococcal lytA gene coding for the major autolysin (amidase) can be expressed in Streptococcus pneumoniae and Escherichia coli using unchanged promoter and termination signals. A region containing several -10, -35 and -44 promoter elements, identical to other previously described prokaryotic promoter sequences, has been found upstream from the transcription start point. A transcription terminator consisting of a hairpin structure (-20.8 kcal/mol) typical of Rho-independent prokaryotic terminators was also localized. The lytA gene has a rather long (240-bp) leader sequence with a high A + T content (70%) that contrasts with the very short (2-bp) untranslated region of the polA gene [López et al., J. Biol. Chem. 264 (1989) 4255-4263], the unique pneumococcal transcription unit that had been characterized so far. Although two open reading frames have been found in the leader region it seems unlikely that these sequences can be translated due to the absence of appropriate ribosome-binding sites.

The cloning in Escherichia coli of the 3' moieties of the lytA and cpl-1 genes is described, coding for the C-terminal regions of the lytic amidase of Streptococcus pneumoniae and the phage Cp-1 lysozyme, respectively. The truncated genes were overexpressed in E. coli and the purified polypeptides showed a great affinity for choline, although they were devoid of cell wall-degrading activity. Biochemical and circular dichroism analyses indicated that these are the domains responsible for the specific recognition of the choline-containing pneumococcal cell walls by the lytic enzymes. The data presented here suggested that these choline-binding domains can function independently of their catalytic domains.

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.

The major pneumococcal autolysin (N-acetylmuramoyl-L-alanine amidase) has been localized in the cellular envelope of Streptococcus pneumoniae and Escherichia coli by using immunocytochemical labeling on ultrathin sections and whole-mounted cells. Cell fractionation experiments in E. coli confirmed the peripheral localization of the pneumococcal amidase and suggested that this enzyme is weakly bound to the outer face of the cytoplasmic membrane. This interaction does not depend on the presence of choline but represents an intrinsic property of the amidase. The autolysin, that is synthesized without any N-terminal signal sequence (García, P., García, J. L., García, E., and López, R. (1986) Gene (Amst.) 43, 265-272) was not processed during translocation. A new regulatory mechanism that might be specific for bacterial autolysins is discussed.

Autolysins are enzymes that have several important biological functions and also seem to be responsible for the irreversible effects induced by the beta-lactam antibiotics. The pneumococcal autolysin gene (lyt) has been subcloned from the plasmid pGL30 [García et al., Mol. Gen. Genet. 201 (1985) 225-230] and we have found that the E form of the autolysin is synthesized in Escherichia coli using its own promoter. The high amount of autolysin obtained in the heterologous system when the lyt gene is present in different orientations in the recombinant plasmids studied supports the idea that the autolysin promoter could be a strong one. The nucleotide sequence of the HindIII fragment of pGL80 (1213 bp) containing the autolysin structural gene has been determined. A unique open reading frame (ORF) has been found, a consensus ribosome-binding site and -10 and -35 promoter-like sequences as well as A + T-rich regions farther upstream were also identified. The lyt ORF encodes a protein of 318 amino acid residues having a calculated Mr of 36,532, which agrees with previous size estimates based on electrophoretic migration [Höltje and Tomasz, J. Biol. Chem. 251 (1976) 4199-4207; Briese and Hakenbeck, Eur. J. Biochem. 146 (1985) 417-427]. Our results also demonstrate that the lyt-4 marker represents the first example of a mutation in a structural gene of a bacterial autolysin. The polarity profile of the pneumococcal autolysin supports previous suggestions about the localization of this enzyme in the normal cell.

Plasmids containing modifications at the 3' end of the lytA gene encoding the pneumococcal amidase were constructed by DNA recombinant techniques. Several deleted and fused amidases were obtained. These modified amidases were capable of degrading cell walls containing choline residues in their teichoic acid components without need of conversion (i.e., change of the inactive E form of amidase to the active C form). The reintroduction of as few as the terminal 11 amino acid (aa) residues present in the wild-type (wt) amidase into the sequence of the most extensively deleted form of the autolysin obtained in this work (E-520) partially restored the need of conversion. Our results demonstrate the importance of the C terminus for the catalytic activation of the wt amidase.

A 7.5 kb BclI-fragment of Streptococcus 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.