The PBP 5 synthesis repressor (psr) gene of Enterococcus hirae ATCC 9790 is substantially longer than previously reported.

A reexamination of the nucleotide sequence of the psr gene of Enterococcus hirae revealed the presence of two additional nucleotides at residues 1190 and 1191. As a result, instead of a stop codon after 148 aa, the psr gene product would contain 293 aa residues. The revised size of the gene product was confirmed by subsequently cloning and expressing the psr gene in Escherichia coli. The derived amino acid sequence of the revised psr gene product was found to be similar to several other proteins in the combined GenBank/EMBL database. The protein products of some of these genes are thought to play regulatory role(s) in exo or capsular polysaccharide synthesis and/or in cell wall metabolism. All the putative homologs of the revised Psr appear to have a putative membrane-anchoring domain at their N-termini. Amino acid blocks with high degrees of similarity have been identified in the aligned sequences, and it is suggested that these common motifs could be of structural or functional importance.

Cellular heterogeneity in non-immune IgG-binding in a strain of Streptococcus mitis.

Electron microscopy revealed that Streptococcus mitis ATCC 903 bound gold probes conjugated with goat IgG by non-immune mechanisms. Only a few of the cells and the cell wall fragments could bind IgG, in contrast to Staphylococcus aureus and Streptococcus group G which showed a more homogeneous binding to nearly all cells or cell wall fragments.

Evidence that the PBP 5 synthesis repressor (psr) of Enterococcus hirae is also involved in the regulation of cell wall composition and other cell wall-related properties.

psr has been reported by M. Ligozzi, F. Pittaluga, and R. Fontana, (J. Bacteriol. 175:2046-2051, 1993) to be a genetic element located just upstream of the structural gene for the low-affinity penicillin-binding protein 5 (PBP 5) in the chromosome of Enterococcus hirae ATCC 9790 and to be involved in the repression of PBP 5 synthesis. By comparing properties of strains of E. hirae that contain a full-length, functional psr with those of strains that possess a truncated form of the gene, we have obtained data that indicate that psr is involved in the regulation of several additional surface-related properties. We observed that cells of strains that possessed a truncated psr were more sensitive to lysozyme-catalyzed protoplast formation, autolyzed more rapidly in 10 mM sodium phosphate (pH 6.8), and, in contrast to strains that possess a functional psr, retained these characteristics after the cultures entered the stationary growth phase. Cellular lytic properties did not correlate with differences in the cellular contents of muramidase-1 or muramidase-2, with the levels of PBP 5 produced, or with the penicillin susceptibilities of the strains. However, a strong correlation was observed with the amounts of rhamnose present in the cell walls of the various strains. All of the strains examined that possessed a truncated form of psr also possessed approximately one-half of the rhamnose content present in the walls of strains that possessed a functional psr. These data suggest that psr is also involved in the regulation of the synthesis of, or covalent linkage to the cell wall peptidoglycan of, a rhamnose-rich polysaccharide. These differences in cell wall composition could be responsible for the observed phenotypic differences. However, the multiple effects of psr suggest that it is part of a global regulatory system that, perhaps independently, affects several cell surface-related properties.

Bacterial walls, peptidoglycan hydrolases, autolysins, and autolysis.

Knowledge of the chemistry, ultrastructure, biosynthesis, assembly, and function of bacterial cell walls has expanded enormously since the opening of this field of research approximately 40 years ago, primarily by the early work of Milton Salton. It has become abundantly clear that, in most environments, walls are essential to the survival and growth of bacteria and in many ways are structurally and functionally unique. A common but not universal feature of bacterial walls is the presence of peptidoglycan (PG; murein, or in the case of certain Archae the analogous structure-pseudomurein). PGs are considered to be primarily responsible for the protective and shape-maintaining properties of walls. They are a biologically unique class of macro-molecules in that they are not linear or even branched macromolecules. Instead they are two- or three-dimensional net like polymers that are linked together by three different chemical bonds (glycosidic, amide, and peptide). In addition, they contain the D-isomers of some amino acids and therefore may possess DL, LD, and DD linkages. Furthermore, the exact chemical structure of a PG may vary depending on environmental factors, however, retaining the essential protective and shape maintaining properties of the wall. Thus, the overall architectural plan of the wall may be more important than the exact shape of the bricks used for the construct. Another somewhat unique feature of PGs (and walls) is their final assembly in situ on the outside of the cellular permeability barrier. A broad variety of bacteria have been shown to possess enzymes that can hydrolyze bonds in the wall PG. Hydrolysis of a sufficient number of bonds can result in the weakening of, or serious damage to, the protective properties of the PG. Frequently, a bacterial strain may possess more than one PG hydrolase activity. A commonly believed, but as yet unproven, hypothesis is that PG hydrolases play one or more roles in PG assembly and/or surface growth and cell division. At a minimum, such potentially suicidal activities must be exquisitely well regulated. Currently we know little concerning the regulation of these activities, or how they communicate with, and integrate with, chromosome replication, synthesis of cytoplasmic macromolecules, cell growth, and division, although such, probably two-way, communications must occur in growing and dividing cells. Recent data indicate that the psr element in Enterococcus hirae described by Fontana and collaborators as a genetic element that is involved in the regulation of the synthesis of PBP 5, also is involved in the regulation of several other surface properties. These properties include (1) autolysis rates of exponential phase. cells, (2) the retention of this property after cells enter the stationary phase, (3) lysozyme sensitivity, and (4) the ratio of rhamnose-containing wall polysaccharide to PG in the walls. Thus the psr element may be a part of a "global" regulation and communication system in E. hirae.

Penicillin resistance and autolysis in enterococci.

Comparison of several cell wall-related properties of the ATCC 9790 strain and the R40 strain, a penicillin-resistant, PBP5 overproducing strain, and Rev14, a penicillin-hypersensitive, PBP5-deficient strain, is consistent with a role of the genetic element, psr, in the global regulation of lysozyme sensitivity, autolytic capacity, and wall-rhamnose-containing polysaccharide content. These parameters appear to be independently regulated by a system that involves psr in a currently unknown manner.

Binding site-shaped repeated sequences of bacterial wall peptidoglycan hydrolases.

The non-catalytic C-terminal regions of the N-acetylmuramidase (lysozyme) of Clostridium acetobutylicum and N-acetylmuramoyl(D-lactyl)-L-alanine amidases CwlA of Bacillus subtilis, ORFL3 and CwlL of Bacillus licheniformis were previously reported to have similarities with the amino acid sequence of the non-catalytic N-terminal module of the Streptomyces albus G Zn DD-peptidase. This peptidase is a bipartite protein of known three-dimensional structure. Its non-catalytic N-terminal module possesses, exposed at the surface, an elongated crevice which is defined by a loop-helix-loop-helix motif that consists of two repeats, each 16 amino acid residues long, connected by a heptapeptide and whose design is compatible with its possible functioning as a substrate recognition and binding site. Amino acid alignments suggest that cavities nearly identical in shape to that present in the non-catalytic module of the S. albus peptidase, are borne by the C-terminal regions of the CwlA amidase (in one copy), the lysozyme and the ORFL3 and CwlL amidases (in two copies). Since a common feature of the five enzymes is their substrate, the bacterial cell wall peptidoglycan, we interpret the striking similarity of their non-catalytic N- or C-terminal modules to suggest that these modules are involved in the binding of these exocellular enzymes to their insoluble wall substrate.

The autolytic ('suicidase') system of Enterococcus hirae: from lysine depletion autolysis to biochemical and molecular studies of the two muramidases of Enterococcus hirae ATCC 9790.

Autolysis of Enterococcus hirae ATCC 9790 is the result of the action of endogenous enzymes that hydrolyze bonds in the protective and shape-maintaining cell wall peptidoglycan. It is thought that these potentially suicidal enzymes play a positive role(s) in wall growth and division and are expressed as autolysins when cell wall assembly and/or repair are inhibited. E. hirae possesses two potentially autolytic enzymes, both of which are muramidases. Although they hydrolyze the same bond as hen egg-white lysozyme, both are high-molecular-mass, complex enzymes. Muramidase-1 is synthesized as a zymogen, requiring protease activation. It is a glucoenzyme that is also multiply nucleotidylated with an unusual nucleotide, 5-mercaptouridine monophosphate. Muramidase-2 is almost certainly a product of a separate gene. The deduced amino acid sequence of a cloned gene for extracellular muramidase-2 showed several unusual features. It appears to be a two-, or perhaps three-domain protein with a putative glycosidase-active site near the N-terminal end and six 45-amino-acid-long repeats at the C-terminal end which are presumed to be involved with high-affinity binding to the insoluble peptidoglycan substrate. Muramidase-2 binds penicillin with low affinity. The presence of several amino acid groupings characteristic of serine-active site beta-lactam-interactive proteins is consistent with the possible presence of a penicillin-binding, third domain. Indirect evidence consistent with a role(s) for these enzymes in cell wall growth and division has been obtained. However, proof of such role(s) awaits modern genetic, molecular, and biochemical analyses.

Extracellular and cellular distribution of muramidase-2 and muramidase-1 of Enterococcus hirae ATCC 9790.

A substantial portion of the second peptidoglycan hydrolase (muramidase-2) activity of Enterococcus hirae ATCC 9790 (formerly Streptococcus faecium) is present in the supernatant culture medium. In contrast, nearly all muramidase-1 activity is associated with cells in the latent, proteinase-activatable form. Muramidase-2 activity is produced and secreted throughout growth, with maximal levels attained at or near the end of exponential growth in a rich organic medium. Muramidase-2 activity in the culture medium remained high even during overnight incubations in the absence of proteinase inhibitors. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of supernatant culture medium concentrated by 60% saturated ammonium sulfate precipitation showed the presence of several Coomassie blue-staining bands. One intensely staining protein band, at about 71 kDa, selectively adsorbed to the insoluble peptidoglycan fraction of cell walls of E. hirae, retained muramidase-2 activity, and reacted in Western immunoblots with monoclonal antibodies to muramidase-2. The mobility of extracellular muramidase-2 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis was indistinguishable from that of muramidase-2 extracted with 6 M guanidine hydrochloride from intact bacteria. Muramidase-2 appears to have only a limited number of binding sites on the peptidoglycan of E. hirae cell walls but binds with high affinity. Although high levels of muramidase-2 activity were present in supernatants of stationary-phase cultures, the bacteria were resistant to autolysis. Thus it appears that the peptidoglycan in walls of intact cells of E. hirae is somehow protected from the hydrolytic action of extracellular muramidase-2.

Cloning and sequence analysis of the muramidase-2 gene from Enterococcus hirae.

Extracellular muramidase-2 of Enterococcus hirae ATCC 9790 was purified to homogeneity by substrate binding, guanidine-HCl extraction, and reversed-phase chromatography. A monoclonal antibody, 2F8, which specifically recognizes muramidase-2, was used to screen a genomic library of E. hirae ATCC 9790 DNA in bacteriophage lambda gt11. A positive phage clone containing a 4.5-kb DNA insert was isolated and analyzed. The EcoRI-digested 4.5-kb fragment was cut into 2.3-, 1.0-, and 1.5-kb pieces by using restriction enzymes KpnI, Sau3AI, and PstI, and each fragment was subcloned into plasmid pJDC9 or pUC19. The nucleotide sequence of each subclone was determined. The sequence data indicated an open reading frame encoding a polypeptide of 666 amino acid residues, with a calculated molecular mass of 70,678 Da. The first 24 N-terminal amino acids of purified extracellular muramidase-2 were in very good agreement with the deduced amino acid sequence after a 49-amino-acid putative signal sequence. Analysis of the deduced amino acid sequence showed the presence at the C-terminal region of the protein of six highly homologous repeat units separated by nonhomologous intervening sequences that are highly enriched in serine and threonine. The overall sequence showed a high degree of homology with a recently cloned Streptococcus faecalis autolysin.

Modular design of the Enterococcus hirae muramidase-2 and Streptococcus faecalis autolysin.

The mature forms of the extracellular muramidase-2 of Enterococcus hirae and Streptococcus faecalis autolysin have very similar primary structures. Each consists of an active-site-containing N-terminal domain fused to a multiple-repeat C-terminal domain. Polypeptide segments occurring at equivalent places in these two bacterial wall lytic enzymes have homologues in two phage lysozymes and in three functionally unrelated proteins, illustrating the principle that protein molecules frequently are constructed from modules that are linked in a single polypeptide chain.

Determination of non-immune binding of immunoglobulin G to Staphylococcus aureus by enzyme-linked immunosorbent assays.

Non-immune binding of human IgG to Staphylococcus aureus was studied by enzyme-linked immunosorbent assays using whole bacteria or bacterial cell walls as the solid phase. Two types of anti-human IgG peroxidase conjugates each with a low affinity for protein A, were used: F(ab')2-fragments of goat IgG and chicken IgY.

Properties of cell wall-associated DD-carboxypeptidase of Enterococcus hirae (Streptococcus faecium) ATCC 9790 extracted with alkali.

DD-Carboxypeptidase (DD-CPase) activity of Enterococcus hirae (Streptococcus faecium) ATCC 9790 was extracted from intact bacteria and from the insoluble residue (crude cell wall fraction) of mechanically disrupted bacteria by a brief treatment at pH 10.0 (10 mM glycine-NaOH) at 0 degrees C or by extraction with any of several detergents. Extractions with high salt concentrations failed to remove DD-CPase activity from the crude wall fraction. In contrast to N-acetylmuramoylhydrolase (both muramidase 2 and muramidase 1) activities, DD-CPase activity failed to bind to insoluble cell walls or peptidoglycan matrices. Thus, whereas muramidase 1 and muramidase 2 activities can be considered to be cell wall proteins, the bulk of the data are consistent with the interpretation that the DD-CPase of this species is a membrane protein that is sometimes found in the cell wall fraction, presumably because of hydrophobic interactions with other proteins and cell wall polymers. The binding of [14C]penicillin to penicillin-binding protein 6 (43 kilodaltons) was proportional to DD-CPase activity. Kinetic parameters were also consistent with the presence of only one DD-CPase (penicillin-binding protein 6) in E. hirae.

The second peptidoglycan hydrolase of Streptococcus faecium ATCC 9790 covalently binds penicillin.

A second peptidoglycan hydrolase (muramidase-2) of Streptococcus faecium ATCC 9790 (Enterococcus hirae) has been purified to apparent homogeneity. The enzyme has been shown to be a beta-1,4-N-acetylmuramoylhydrolase (muramidase; EC 3.2.1.17) and to differ in substrate specificity from a previously isolated muramidase. Purified enzyme appears as two protein staining bands with molecular masses of 125 and 75 kilodaltons (kDa) on polyacrylamide gels after sodium dodecyl sulfate electrophoresis. Elution and renaturation of protein bands from sodium dodecyl sulfate-polyacrylamide gels showed that both proteins have muramidase-2 activity. Both proteins have been shown to bind radioactive benzylpenicillin and have the same electrophoretic mobilities as penicillin-binding proteins 1 and 5 present in membrane preparations of this organism, respectively. Incubation of a [14C]penicillin G-labeled 125-kDa form of the enzyme with crude alkaline extracts from S. faecium (which did not contain added proteinase inhibitors) showed the endogenous conversion of the radiolabeled 125-kDa form to the radiolabeled 75-kDa form of the enzyme.

Tn916 insertional inactivation of multiple genes on the chromosome of Streptococcus mutans GS-5.

Streptococcus mutans GS-5 was transformed with the Escherichia coli plasmid pAM150 containing the cloned streptococcal transposon Tn916. Southern blot analyses with the tetracycline-resistant determinant of Tn916 showed that Tn916 was inserted into the chromosome of S. mutans at a variety of different sites. Tn916 insertions resulted in the inactivation of genes that code for various steps in the biosynthesis of several different amino acids. Two auxotrophs which contained a single copy of Tn916 were shown to revert to prototrophy at frequencies of about 10(-8). All of the revertant prototrophs were susceptible to tetracycline, indicating regeneration of the functional gene by excision of Tn916.

Covalent modification of the beta-1,4-N-acetylmuramoylhydrolase of Streptococcus faecium with 5-mercaptouridine monophosphate.

Purified beta-1,4-N-acetylmuramoylhydrolase (muramidase-1; EC 3.2.1.17) of Streptococcus faecium ATCC 9790 has been shown to be covalently substituted with approximately 12 mol equivalents of monomeric 5-mercaptouridine monophosphate. All 12 residues are present on the proteolytically processed 87-kDa active form of the enzyme. A peptide fragment containing 5-mercaptouridine, tyrosine, alanine, glycine, and leucine was isolated consistent with an O-phosphate linkage of the nucleotide to tyrosine.

The ability to sensitize host cells for destruction by autologous complement is a general property of lipoteichoic acid.

Previous studies have demonstrated that lipoteichoic acid (LTA) from Streptococcus pneumoniae binds to erythrocytes and renders them susceptible to lysis by autologous complement. The present study was performed to determine whether LTA from two other gram-positive bacterial species had the ability to render mammalian cells susceptible to lysis by autologous complement. Human erythrocytes were sensitized with LTA from S. pneumoniae, Streptococcus pyogenes, or Lactobacillus fermentum. Under incubation in normal autologous serum, lysis was observed with each of the LTA-sensitized erythrocyte preparations. When erythrocytes from a C2-deficient patient were sensitized with the LTA preparations and then incubated in autologous, C2-deficient serum, the erythrocytes sensitized with S. pyogenes or L. fermentum LTA demonstrated relatively little lysis, whereas the erythrocytes sensitized with S. pneumoniae LTA yielded near-total lysis. After reconstitution of the C2-deficient serum with purified human C2, lysis was observed with all three LTA preparations. When erythrocytes from an agammaglobulinemic patient were sensitized with either the S. pyogenes or the L. fermentum LTA, they were not lysed in the presence of autologous agammaglobulinemic serum, whereas the erythrocytes sensitized with S. pneumoniae LTA were completely lysed. Serum obtained from the agammaglobulinemic patient after reconstitution with intravenous pooled gamma globulin was able to lyse autologous erythrocytes sensitized with each of the three LTA preparations. These results demonstrate that the ability to render host cells susceptible to lysis by autologous complement is a general property of LTA. Whether activation of the autologous complement occurs by the classical or alternative pathways and whether it is antibody dependent depends on the nature of the bacterial LTA.

The autolytic peptidoglycan hydrolases of Streptococcus faecium.

Streptococcus faecium ATCC 9790 possesses two peptidoglycan hydrolase activities. The first enzyme, an N-acetylmuramoylhydrolase, has been purified and has been shown to be a glucoenzyme. Studies of hydrolysis of soluble, linear uncross-linked peptidoglycan chains showed that the enzyme bound strongly to the non-reducing ends of the chains and then sequentially (processively) hydrolysed susceptible bonds in that chain. The second peptidoglycan hydrolase does not appear to be a glycoprotein and differs from the first enzyme in substrate specificity and mechanism of hydrolysis. The presence of two partially redundant activities which may play different roles in surface growth and division could, at least in part, explain previous difficulties in obtaining mutants which completely lack autolytic activity.

Division of temperature-sensitive Streptococcus faecium mutants after return to the permissive temperature.

The regrowth of 27 temperature-sensitive division mutants of Streptococcus faecium ATCC 9790 was examined after various periods of incubation at the nonpermissive temperature. Several of the mutants blocked at various stages of septum formation or of daughter-cell separation divided in a partially or completely synchronous way after a short incubation at the nonpermissive temperature. All four lytic mutants blocked early in the cell division cycle divided at a normal rate after a brief lag.

The mechanism of soluble peptidoglycan hydrolysis by an autolytic muramidase. A processive exodisaccharidase.

The action of purified N-acetylmuramoylhydrolase (muramidase, EC 3.2.1.17) of Streptococcus faecium ATCC 9790 on linear, uncross-linked, soluble, peptidoglycan chains produced by the same organism in the presence of benzylpenicillin was characterized as a processive exodisaccharidase. Specific labels, one [( 14C]Gal) added to the nonreducing ends of chains, and the other (3H from [3H]NaBH4) incorporated into the reducing ends of the chains, were used to establish that an enzyme molecule binds at the nonreducing terminus and sequentially hydrolyzes the glycosidic bonds, releasing disaccharide-peptide units. An enzyme molecule remains bond to a chain, and is not released at a detectable rate, until hydrolysis of that chain is complete. Reaction rates increased with the length of the polymer chain to give a maximum of 91 bonds cleaved/min/enzyme molecule for hydrolysis of a continuous polymeric substrate. The relationship between hydrolytic rate and glycan chain length is consistent with hydrolysis of bonds within the chain followed by slow release of enzyme from the distal, reducing terminus. This mechanism was experimentally confirmed by analysis of product formation during hydrolysis with stoichiometric mixtures of enzyme and soluble peptidoglycan chains. Kinetic analyses showed an apparent Km of 0.17 microM for the enzyme, independent of substrate polymer length. The dissociation constant for the initial enzyme-substrate complex was calculated to be 1.5 nM. Kinetic analyses are consistent with one catalytic site per enzyme molecule. The Kcat/Km value of 9 X 10(6) M-1 S-1 is near the limit imposed by diffusion for the initial hydrolytic events when long chains are hydrolyzed. The kinetic and physical properties of this muramidase are highly consistent with its location outside of the cellular permeability barrier and its ability to remain with and hydrolyze appropriate bonds in the cell wall in such an environment.