Physical and genetic map of Streptococcus mutans GS-5 and localization of five rRNA operons.

The physical map of the 2.1 megabase chromosome of Streptococcus mutans GS-5 has been refined by including all ApaI and SmaI fragments of 5 kbp or greater, and by positioning the fragments generated by the endonuclease I-CeuI. Sixty-three new genetic loci have been added to the map, so that it now contains 90 loci. The new loci include those for 35 cloned streptococcal genes of established function and for 23 S. mutans genes of putative function. In addition, five rrn operons were identified and placed on the map of the chromosome. The presence of a SmaI site in each of the rrn operons allowed the direction of transcription of each operon to be deduced. The orientation of the rrn loci indicates that their transcription is directed away from a small region of the chromosome, identifying a possible region for the initiation of chromosome replication.

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.

Molecular epidemiology by ribotyping and PCR-ribotyping of Enterococcus faecium strains isolated from intercontinental areas.

In this study classical ribotyping based on hybridization of an enteroccocal ribosomal operon previously cloned from Enterococcus hirae (Sechi and Daneo-Moore, 1993) with XbaI cut chromosomal DNA and PCR-ribotyping were used to characterize the molecular epidemiology of 131 Enterococcus faecium, with high-level resistance to gentamicin, isolated from different hospitals in Italy and the United States. The ribotyping was able to differentiate all 131 clinical isolates into 96 family patterns. These family patterns appeared to be useful in establishing epidemiological spread. The results obtained were in agreement with those previously published, suggesting the presence of five to six operons in the Enterococcus genus (Sechi et al., 1994). We performed PCR-ribotyping, based on conserved sequences at the 3' end of the enterococcal 16S rrn and the 5' end of the 23S rrn, on 131 clinical isolates as well as on several enterococcal ATCC strains tested. The results were then compared with those obtained with the classical ribotyping method. The results suggest the presence of at least four classes of intergenic spacers among enterococci, but these classes are not helpful in differentiating between Enterococci or among Enterococcal isolates.

Characterization of new insertion-like sequences of Enterococcus hirae and their dissemination among clinical Enterococcus faecium isolates.

Sequence analysis of different fragments that hybridized with a 4.5-kb EcoRI fragment originally cloned from Enterococcus hirae ATCC 9790 showed 66% homology to IS-like sequences found in staphylococci and lactococci. We tested several enterococcal ATCC strains and found that only E. hirae ATCC 9790 and Enterococcus faecium ATCC 19434 hybridized with the IS-like sequence. Moreover, we wanted to investigate the dissemination of this new IS among E. faecium strains. We analyzed 131 clinical E. faecium isolated in Italy and the USA for the presence of the IS and we found its presence in more than 63% of the isolates. The hybridization patterns obtained vary considerably between unrelated strains and allow further classification among ribotype-grouped species.

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.

A novel tetracycline-resistant determinant, tet(U), is encoded on the plasmid pKq10 in Enterococcus faecium.

Nine tetracycline (Tc)-resistant clinical isolates of Enterococcus faecium were screened for plasmid content using agarose gel electrophoresis. pKQ10, a 1.9-kb plasmid carrying a novel Tc resistance determinant, was isolated from one of the isolates. The nucleotide sequence of this plasmid revealed an open reading frame corresponding to an 11.8-kDa protein and containing 105 amino acid residues. There was some limited similarity between this protein and tet(M), tet(O), tet(Q), tet(S), tetB(P), and otr(A), which overlapped, but did not include, the consensus GTP-binding sequences. The low-level, Tc-resistant determinant of pKQ10, named tet(U), does not appear to correspond to any other known Tc resistance determinant.

Ribosomal RNA gene (rrn) organization in enterococci.

A cloned 1.8-kb probe containing the 3' end of 16S ribosomal RNA and the 5' end of 23S ribosomal RNA from Enterococcus hirae was used to analyze various endonuclease digests of enterococci. In the ATCC strains tested we observed a remarkable conservation of the ApaI sites in the rrn operons, and a partial conservation of EcoRI sites. Using a number of other endonuclease digestions with the ApaI rrn probe, we estimate the number of rrn operons in enterococci to be between five and six.

Characterization of intergenic spacers in two rrn operons of Enterococcus hirae ATCC 9790.

Two DNA restriction enzyme fragments coding for the 3' termini of 16S rRNA, the 5' termini of 23S rRNA, and the intergenic spaces between them in Enterococcus hirae ATCC 9790 were cloned and sequenced. The intergenic space of one of these genes contains a tRNA(Ala) sequence, whereas the other does not. Nevertheless, the intergenic spaces contain several regions that exhibit high levels of sequence homology and are capable of forming structures with similar base pairs. An analysis of Southern blots of chromosomal DNA cut with one and two restriction enzymes indicated that E. hirae has a total of six rrn operons.

Chromosome organization of Streptococcus mutans GS-5.

Twenty-eight genetic loci have been physically mapped to specific large restriction fragments of the Streptococcus mutans GS-5 chromosome by hybridization with probes of cloned genes or, for transposon-generated amino acid auxotrophs, with probes for Tn916. In addition, restriction fragments generated by one low-frequency-cleavage enzyme were used as probes to identify overlapping fragments generated by other restriction enzymes. The approach allowed construction of a low resolution physical map of the S. mutans GS-5 genome using restriction enzymes ApaI (5'-GGGCC/C), SmaI (5'-CCC/GGG), and NotI (5'-GC/GGCCGC).

A truncated Tn916-like element in a clinical isolate of Enterococcus faecium.

A 58.7-kb nonconjugative plasmid (pKQ1) previously reported in a clinical isolate of Enterococcus faecium was found to contain both a tetM and an erythromycin resistance (erm) determinant. The plasmid contained a region homologous to the A, F, H, and G HincII fragments of Tn916. However, the 4.8-kb B fragment of Tn916 which contained the tetM determinant was replaced by a 7.3-kb fragment, and the 3.6-kb HincII C fragment of Tn916 was missing. An element homologous to Tn917 was juxtaposed to the truncated Tn916-like element. The Tn917-like element was similar in size to the erm transposon Tn917 as determined by a ClaI restriction digest which spanned approximately 99% of the transposon. When Bacillus subtilis or Streptococcus sanguis were transformed with pKQ1, no zygotically induced transposition of the tetM element was detected. Similarly no transposition of the Tn917-like element was detected.

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.

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.

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.

Size of the Streptococcus mutans GS-5 chromosome as determined by pulsed-field gel electrophoresis.

Rare cutting restriction endonucleases were used to cut the Streptococcus mutans chromosome into large fragments. Restriction enzymes utilizing recognition sites containing 6-, 7-, or 8-base-pair sequences with only G and C nucleotides produced few fragments, most of which were greater than 100 kilobase pairs in size. Addition of the fragments from digests of SmaI, NotI, ApaI, RsrII, and EagI yielded a molecular size for the S. mutans GS-5 genome of 2,819 +/- 60 kilobase pairs.

Transposon-916-like elements in clinical isolates of Enterococcus faecium.

Tetracycline (Tc) resistance was found in nine out of ten clinical isolates of Enterococcus faecium. Conjugative transposons, designated Tn5031, Tn5032 and Tn5033, were present in the chromosome of three isolates. The transposons were similar both structurally and functionally to Tn916 containing the tetM determinant. A large non-conjugative plasmid found in a fourth isolate contained an element homologous to Tn916. The four isolates containing the element showing homology to Tn916 exhibited a substantially higher level of Tc resistance than the remaining five Tc-resistant isolates. Tc-resistance genes which have not been identified are apparently responsible for the low-level Tc resistance in five clinical isolates.

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.

Effect of cell cycle stages on the central density of Enterococcus faecium ATCC 9790.

Cultures of Enterococcus faecium growing at various rates were examined for timing of cell division cycle events by using the method of residual divisions and a morphological analysis. Both methods gave essentially the same timing for the onset of D1 (completion of chromosome replication) and of D2 (completion of septation). Frequencies of cells exhibiting a phase-reversed center in bovine serum albumin at various growth rates were determined. The data fit a model in which rapidly growing cells increase in refractive index (which is assumed to represent central density) at completion of the chromosome replication cycle involved in the ongoing division, whereas slowly growing cultures increase in central density at the time of completion of septation. There was no correlation between the timing of increase in central density and the timing of initiation of new sites of surface growth.