Influence of proteins Bsp and FemH on cell shape and peptidoglycan composition in group B streptococcusc

Dieter J. Reinscheid1,2, Claudia Stößer1, Kerstin Ehlert3, Ralph W. Jacka,4, Kerstin Möller2, Bernhard J. Eikmanns1 and Gursharan S. Chhatwal2

Department of Microbiology and Biotechnology, University of Ulm, D-89069 Ulm, Germany1
Department of Microbiology, GBF-National Research Centre for Biotechnology,D-38124 Braunschweig, Germany2
Bayer AG, PH Research Antiinfectives I, D-42096 Wuppertal, Germany3
Institute for Organic Chemistry, University of Tübingen, D-72070 Tübingen, Germany4

Author for correspondence: Dieter Reinscheid. Tel: +49 731 5024853. Fax: +49 731 5022719. e-mail: dieter.reinscheid{at}biologie.uni-ulm.de


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Group B streptococcus (GBS) is surrounded by a capsule. However, little is known about peptidoglycan metabolism in these bacteria. In the present study, a 65 kDa protein was isolated from the culture supernatant of GBS and N-terminally sequenced, permitting isolation of the corresponding gene, termed bsp. The bsp gene was located close to another gene, designated femH, and reverse transcription-PCR revealed a bicistronic transcriptional organization for both genes. The Bsp protein was detected in the culture supernatant from 31 tested clinical isolates of GBS, suggesting a wide distribution of Bsp in these bacteria. Overexpression of bsp resulted in lens-shaped GBS cells, indicating a role for bsp in controlling cell morphology. Insertional disruption of femH resulted in a reduction of the L-alanine content of the peptidoglycan, suggesting that femH is involved in the incorporation of L-alanine residues in the interpeptide chain of the peptidoglycan of GBS.

Keywords: Streptococcus agalactiae, murein hydrolase, fem-like genes

Abbreviations: GBS, group B streptococcus

c The GenBank accession number for the sequence reported in this paper is AJ305309.

a Present address: Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon Tong, Hong Kong SAR, China.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Group B streptococcus (GBS), also named Streptococcus agalactiae, is a frequent colonizer of the human respiratory, gastrointestinal and urogenital tracts of humans and several mammals. GBS is also the major cause of bacterial sepsis and meningitis in human newborn infants, and poses a significant threat to parturient women (Baker & Edwards, 1995 ). As the incidence of GBS infections has increased significantly during the last decade, particularly in immunocompromised persons (Waite et al., 1996 ), considerable research has been focused on the identification of putative virulence factors from GBS. A variety of studies have addressed the capsule of GBS (Rubens et al., 1991 ; Wessels et al., 1992 ; Kogan et al., 1996 ). In contrast, the function of most extracellular proteins and the nature of peptidoglycan metabolism in these bacteria are only poorly understood. In common with many other Gram-positive bacteria, the peptidoglycan stem peptide of GBS consists of the pentapeptide L-Ala-D-iGln-L-Lys-D-Ala-D-Ala (iGln, isoglutamine). However, in GBS, different peptidoglycan strands are cross-linked by short interpeptide bridges of L-Ala-L-Ala or L-Ala-L-Ser dipeptides, connecting the L-lysine of one stem peptide to the D-alanine in position 4 of a neighbouring subunit (Schleifer & Kandler, 1972 ).

Since the interpeptide bridge is a species-specific feature, it has been the focus of intense research in other pathogens. In Staphylococcus aureus, the interpeptide bridge consists of five glycine residues which are synthesized by the sequential addition of glycyl residues in the presence of the proteins FmhB, FemA and FemB, respectively. FmhB was shown to be required for the first step of interpeptide synthesis by attaching the first glycine to the {epsilon}-amino group of a lysine residue in the stem peptide (Rohrer et al., 1999 ; Tschierske et al., 1999 ), FemA directs the incorporation of the second and the third glycine, while FemB is required for the addition of the fourth and fifth glycines (Stranden et al., 1997 ).

In Streptococcus pneumoniae, the peptidoglycan stem peptides can be either directly linked to each other or cross-linked by an interpeptide bridge carrying L-Ala-L-Ala or L-Ser-L-Ala dipeptides (Garcia-Bustos et al., 1987 ). Recently, the fem-like genes murM and murN were identified in Strep. pneumoniae and shown to be required for the formation of the interpeptide bridge (Filipe & Tomasz, 2000 ; Weber et al., 2000 ). Insertional mutagenesis revealed that murM is involved in the incorporation of the first amino acid, while murN is required for the addition of the second amino acid of the interpeptide bridge (Filipe et al., 2000 ).

As the interpeptide bridge has a species-specific amino acid composition, it represents the target of specific bacteriolytic enzymes that cleave the interpeptide bridge and cause lysis of the target cell. Staphylococcus simulans biovar staphylolyticus and Staphylococcus capitis, respectively, secrete the glycyl-glycine endopeptidases lysostaphin and Ale-1, which recognize and cleave the pentaglycine interpeptide chain in the peptidoglycan of Staph. aureus, resulting in the lysis of this organism (Sugai et al., 1997a ; Thumm & Götz, 1997 ). The former strains protect their own cell walls from cleavage by the incorporation of serine molecules at positions 3 and 5 within the interpeptide chain (Ehlert et al., 2000 ). The immunity factors that mediate the incorporation of serine into the interpeptide chain reveal high similarity to Fem-like proteins (Sugai et al., 1997b ; Thumm & Götz, 1997 ). Similar bacteriolytic enzymes and immunity factors from different streptococcal species have also been described (Beatson et al., 1998 ; Simmonds et al., 1997 ; Beukes & Hastings, 2001 ).

The present study describes the identification and characterization of the genes bsp and femH, which appear to play a role in cell morphogenesis and peptidoglycan metabolism in GBS. By insertional mutagenesis, the bsp gene was deleted and the femH gene was disrupted in the genome of GBS. The mutant strains were characterized for their growth behaviour, ß-lactam susceptibility and cell wall composition. Furthermore, the bsp gene was overexpressed in GBS, and the shape of the resultant strain was compared with that of the parental strain. The results obtained reveal that overexpression of bsp causes altered cell morphology, while the disruption of femH changes the amino acid composition of the peptidoglycan of GBS.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains and culture conditions.
GBS strain 6313 is a serotype III clinical isolate obtained from an infected neonate (Valentin-Weigand et al., 1996 ). GBS strain SMB is a bsp deletion mutant of strain 6313, and strain FBH is a femH::pG+host6 derivate of GBS 6313, carrying an insertionally disrupted femH gene in the chromosome. The GBS strains belonging to different serotypes are clinical isolates and have been described elsewhere (Chhatwal et al., 1984 ). Escherichia coli DH5{alpha} (Hanahan, 1985 ) served as host for the pTEX5236 cosmid gene library and the recombinant pG+host6 plasmids. E. coli BL21(DE3) (Dubendorff & Studier, 1991 ) harboured the recombinant pET28 plasmid and was used for production of the hexahistidyl-tagged Bsp fusion protein. GBS was cultivated at 37 °C in Todd–Hewitt yeast (THY) broth containing 1% yeast extract. GBS strains carrying recombinant pG+host6 or pAT28 derivatives were selected in the presence of erythromycin (5 µg ml-1) and spectinomycin (200 µg ml-1), respectively. E. coli was grown at 37 °C in Luria broth (LB), and clones carrying cosmid pTEX5236, plasmid pET28a or plasmid pAT28 were selected in the presence of chloramphenicol (15 µg ml-1), kanamycin (50 µg ml-1) or spectinomycin (100 µg ml-1). For visual inspection of the mean chain length, the GBS strains were cultivated overnight in THY liquid medium and examined by light microscopy.

Plasmids and cosmids used for cloning purposes.
A pTEX5236-based (Teng et al., 1998 ) cosmid gene library from GBS was used for the isolation of the bsp-encoding region (Reinscheid et al., 2001 ). Plasmid pUC18 (Vieira & Messing, 1982 ) was used for subcloning of the bsp gene after partial digestion of a bsp-carrying pTEX5236 cosmid with Sau3AI. Plasmid pET28a (Novagen) was used for the expression of the hexahistidyl-tagged Bsp fusion protein, which was constructed as follows. A truncated bsp gene, devoid of its signal-peptide-encoding sequence, was amplified by PCR using the primers 5'-CGCGGATCCGATCAAACTACATCGG-3' and 5'-TGGCACAAGCTTCAATATAGCGACGAA-3'. The BamHI and HindIII restriction sites used for cloning are underlined. After digestion of the bsp PCR product and of plasmid pET28a with BamHI and HindIII, the bsp gene was ligated into pET28 and transformed into E. coli BL21. Plasmid pAT28 (Trieu-Cuot et al., 1990 ) was used for the overexpression of bsp in GBS. For this purpose, the bsp gene was amplified from the genome of GBS by PCR using the primers 5'-GCTAGAATTCGGAACGATGAATTCAACCC-3' and 5'-CGTGACTCTAGAGACGTAAATCTCCACTG-3'. After digestion of the PCR product and of plasmid pAT28 with EcoRI and XbaI, the bsp gene was ligated into pAT28, resulting in plasmid pATbsp. Transformation of recombinant plasmids in GBS was performed as described by Ricci et al. (1994) .

Construction of GBS deletion and insertion mutants.
As the bsp and femH genes are transcriptionally linked, the bsp gene was deleted in the chromosome to rule out a polar effect on the expression of the downstream-located femH gene. For the deletion of the bsp gene, the thermosensitive plasmid pG+host6 (Appligene) was used. Two bsp flanking fragments were amplified by PCR using the primer pairs bsp_del1 (5'-CGCGGATCCAAGCAGAAGGTGTAGAGC-3') and bsp_del2 (5'-CCCATCCACTAAACTTAAACACGTAGAGAGTAAGA-TTGC-3') as well as bsp_del3 (5'-TGTTTAAGTTTAGTGGATGGGTAGTAGATGGTCATCAGTGG-3') and bsp_del4 (5'-TGGCACAAGCTTTTGGCATAGCCTTGCAATGC-3'). Complementary DNA sequences in primers bsp_del2 and bsp_del3 are shown in italics, and the BamHI and HindIII restriction sites in primers bsp_del1 and bsp_del4 are shown underlined. The bsp flanking PCR products were mixed in equal amounts with each other and subjected to crossover PCR by using primers bsp_del1 and bsp_del4. The resulting PCR product consisted of the bsp flanking regions on a single DNA fragment. The crossover PCR product and plasmid pG+host6 were digested with BamHI and HindIII, ligated, and transformed into E. coli DH5{alpha}. The resulting plasmid, pG+bsp, was transformed into GBS 6313, and transformants were selected by growth on erythromycin agar at 30 °C. Cells in which pG+bsp had integrated into the GBS chromosome were obtained by growth of the transformants at 37 °C with erythromycin selection, as described elsewhere (Maguin et al., 1996 ). Four such integrant strains were serially passaged for 3 days in liquid medium at 30 °C without erythromycin selection, to facilitate the excision of plasmid pG+bsp, producing the desired bsp deletion in the chromosome. Dilutions of the serially passaged cultures were plated onto agar plates, and single colonies were tested for erythromycin sensitivity to identify pG+bsp excisants. Chromosomal DNA of GBS 6313 and of 24 erythromycin-sensitive GBS excisants was tested by Southern blotting after HindIII digestion, using a digoxigenin-labelled bsp flanking fragment obtained with primers bsp_del1 and bsp_del2.

For targeted disruption of femH, an internal femH fragment, ranging from bp 214 to bp 684 of the structural femH gene, was amplified by PCR using primers 5'-CGCGGATCCAATTCAGCCTTGCCTTC-3' and 5'-TGGCACAAGCTTCAGGACCAGTTCATTC-3'. The BamHI and HindIII restriction sites used for cloning are underlined. The resulting PCR product and plasmid pG+host6 were digested with BamHI and HindIII, and the PCR product was subsequently ligated into pG+host6 and transformed into E. coli DH5{alpha}, resulting in plasmid pG+femH. After transformation of pG+femH into GBS 6313, integration of the plasmid into the chromosome of GBS 6313 was performed by means of a temperature shift to 37 °C as described elsewhere (Maguin et al., 1996 ). Successful disruption of femH was confirmed by Southern blot analysis with ClaI-digested chromosomal DNA from GBS and a digoxigenin-labelled femH probe obtained by PCR with primers 5'-TTATGCCAGTCACTGGTGG-3' and 5'-AGAGCGTTGCCTATGATAC-3'. The femH mutation was stably maintained by growing the femH-inactived GBS strain at 37 °C in the presence of 5 µg erythromcyin ml-1.

RNA preparation and RT-PCR analysis.
Total RNA from 250 ml mid-exponential-phase GBS 6313 culture was prepared by using the RNeasy Midi extraction kit (Qiagen) and was treated for 30 min with 150 U RNase-free DNase (Promega). For analysis of the transcriptional organization of bsp and femH in GBS, 1 µg RNA samples were used for RT-PCR with primers 3' bsp (5'-TAGTAGATGGTCATCAGTGG-3') and 5' femH (5'-GGTCCTGAATCAATTTTC-3'). For comparative expression analysis of bsp in GBS strains 6313(pAT28) and GBS 6313(pATbsp), RNA samples from the two strains containing 5, 1, 0·5, 0, 1, 0·05 or 0·01 ng RNA were tested by RT-PCR for the presence of a bsp-specific transcript, using primers 5'-GAGACAAGTGCGTCAAGTG-3' and 5'-TGAGTTGGACTCGCTACC-3'. RT-PCR was performed using the OneStep RT-PCR kit (Qiagen) according to the instructions of the manufacturer.

General DNA techniques.
Chromosomal GBS DNA was isolated according to Pospiech & Neumann (1995)3. Conventional techniques for DNA manipulation, such as restriction enzyme digests, PCR, ligation, transformation by electroporation and Southern blotting, were performed as described by Sambrook et al. (1989) .

Electron microscopy and antibiotic testing.
Scanning electron microscopy of mid-exponential-phase cultures was performed as described previously (Reinscheid et al., 2001 ). Determination of the MICs for penicillin G, oxacillin, cefotaxim, imipenem and vancomycin was performed on sheep-blood agar plates by using antibiotic-containing E-test strips (AB Biodisk) according to the manufacturer’s instructions. Penicillin-induced lysis was measured as described by Fontana et al. (1990) . Briefly, a bacterial overnight culture was diluted in fresh THY medium to an OD600 of 0·2. Penicillin G was added to give concentrations of 0·032 µg ml-1, 0·064 µg ml-1 and 0·128 µg ml-1, respectively. Cultures were incubated with shaking at 37 °C, and 1 ml aliquots were removed every 30 min to determine the OD600.

N-terminal sequencing of proteins and peptides.
Proteins were separated by SDS-PAGE, transferred onto a PVDF membrane (Amersham/Pharmacia), and visualized with amido black. N-terminal amino acid sequencing was performed on excised bands, using an Applied Biosystems 447A pulsed-liquid protein sequencer. Generation and separation of internal Bsp peptides using endoproteinase Lys-C (Promega) was performed as described by Maiorino et al. (1996) . Briefly, a gel slice containing Bsp was washed twice with 100 µl 100 mM sodium bicarbonate and 50% acetonitrile. Cysteine residues were reduced for 30 min at 55 °C with 45 mM dithiothreitol in 50 µl 8 M urea and 0·4 M ammonium bicarbonate and alkylated for 30 min by the addition of 5 µl 100 mM iodoacetamide; this was followed by a washing step with H2O. Digestion of Bsp was performed at 37 °C for 20 h in a total volume of 50 µl containing 100 mM ammonium bicarbonate, 10% acetonitrile, 1% Triton X-100 and 0·5 µg endoproteinase Lys-C (sequencing grade; Promega). The solution was collected, vacuum-dried and subsequently dissolved in 20 µl 20% acetonitrile. Samples (10 µl each) were analysed by HPLC (Applied Biosystems) on an Aquapore OD-300 RP-18 column at 37 °C and at a flow rate of 40 µl min-1. Solvent A was 0·06% trifluoroacetic acid in water and solvent B 0·05% was trifluoroacetic acid in 80% acetonitrile. After sample injection, a linear gradient was started to reach 45% of solvent B in 75 min. Peaks were detected at 214 nm and collected manually in 0·5 ml micro-centrifuge vials.

Preparation of Bsp fusion protein and generation of anti-Bsp antibodies.
Bsp fusion protein was synthesized in recombinant E. coli BL21 by the addition of 1 mM IPTG after the culture had reached an OD600 of 1·0. The cells were disrupted using a French pressure cell, and purification of the fusion protein was performed according to the instructions of Clontech by using cobalt affinity chromatography. For the generation of anti-Bsp antibodies, affinity-purified Bsp fusion protein was size-separated by SDS-PAGE, blotted onto nitrocellulose and, after staining with Ponceau S, the Bsp-containing band was excised. After the nitrocellulose membrane had been dissolved in DMSO, the solution was used for the immunization of mice. Immunization consisted of two intramuscular applications of the purified protein within 2 weeks. Serum was collected 4 weeks after immunization.

Western blot analysis and quantification of cell-surface hydrophobicity.
Western blotting was performed essentially as described previously (Reinscheid et al., 2001 ), using a 1:500 dilution of the anti-Bsp antiserum, a 1:15000 dilution of goat anti-mouse-Fab fragments (Dianova), and subsequent detection by chemiluminescence using the ECL kit (Amersham/Pharmacia). Culture supernatant and cell wall proteins from GBS were isolated as described elsewhere (Kling et al., 1999 ). Cell-surface hydrophobicity was determined in an aqueous/hexadecane emulsion by quantification of the amount of bacteria in the aqueous phase as described by Rosenberg et al. (1981) .

Determination of murein hydrolase activity.
Peptidoglycan-lytic activity was analysed by zymography and by using a turbidity assay as described previously (Reinscheid et al., 2001 ). The bacteriolytic activity of Bsp fusion protein was tested according to Simmonds et al. (1997) . Cell autolysis was performed essentially as described by Qin et al. (1998) . Briefly, samples from exponential and stationary growth phase were washed three times with 100 mM phosphate buffer (pH 7·4) and the suspension was subsequently adjusted to an OD600 of 0·3. The suspension was then incubated at 37 °C, and the OD600 was measured at 30 min intervals for 6 h.

Preparation and separation of muropeptides.
After growth of GBS at 37 °C, peptidoglycan was prepared as described by Hakenbeck et al. (1998) . Digestion of lyophilized peptidoglycan with Streptomyces globisporus mutanolysin (25 µg ml-1) and reduction of the muropeptides with borohydride were performed as described by Weber et al. (2000) . Muropeptides were separated by reversed-phase HPLC as previously described (Hakenbeck et al., 1998 ). The analysis of the muropeptide profiles, including the preparation of peptidoglycan, was done in duplicate.

Amino acid analysis of peptidoglycan.
Purified peptidoglycan was hydrolysed in 6 M HCl at 166 °C for 1 h. Samples were subsequently subjected to amino acid analysis (Biotronic LC 5000) after drying. Amino acid enantiomers were quantified by the technique of enantiomer labelling (Frank et al., 1978 ) after separation by GC and detection by electron-impact MS as previously reported (Höltzel et al., 2001 ). The total amount of an amino acid was calculated by adding the amounts of the two enantiomers of the amino acid. Lyophilized peptidoglycan (1 mg) was hydrolysed in 6 M DCl/D2O (containing 20 mM thioglycolic acid as antioxidant) at 110 °C for 16 h. Amino acids, derivatized to their N-trifluoracetyl-O-ethyl esters, were analysed; the data collected were processed on a model 5973 gas chromatograph coupled on-line with a model 6890 electron-impact mass spectrometer using the manufacturer’s protocols and software (Hewlett Packard). Separation was achieved on a 250 µmx25 m column of fused silica modified with 30% 2,6-dipentyl-3-butyrylcyclodextrin in PS255 with a film thickness of 0·13 mm and a 250 µmx25 m Chirasil-L-Val capillary using appropriate temperature gradients.

Both procedures for amino acid analysis, including the preparation of peptidoglycan, were done in duplicate.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
N-terminal sequencing of proteins in the culture supernatant of GBS
SDS-PAGE of the concentrated culture supernatant of GBS strain 6313 previously identified three major secreted proteins (Reinscheid et al., 2001 ). However, by loading higher quantities of concentrated culture supernatant onto the gel, three further protein bands with sizes of 90, 65 and 60 kDa could be visualized (data not shown); these bands were isolated and subjected to N-terminal sequencing. The N-terminus (SKIIGIDLGTTNSAVAVLEG) of the 90 kDa polypeptide revealed significant similarity to the chaperone Hsp70 from Lactococcus lactis, while the N-terminus (EPDSVWAAR) of the 60 kDa protein did not show any similarity to the N-terminal regions of polypeptides available in public-domain databases. Interestingly, the N-terminus (DQTTSVQVNN) of the 65 kDa protein (P65) revealed 60% identity to the N-terminal region (DSNNSVSVNN) of an M-like protein from Streptococcus pyogenes M65. Since M-like proteins are important virulence factors in Strep. pyogenes, we initiated studies to isolate the P65 gene and to identify and characterize the function of the P65 protein in GBS. As P65 represents a group B streptococcal secreted protein, it was designated Bsp.

Isolation and characterization of the bsp gene
Endoproteolytic digestion of Bsp with endoproteinase Lys-C and N-terminal sequencing of five peptides yielded the sequences Bsp1 (TGVYNIIGSTEVK), Bsp2 (DQTTSVQVNN), Bsp3 (VASPTQFTLDK), Bsp4 (TLPEQGNYVYS) and Bsp5 (VSSPVEFNFQK), respectively. Two degenerate primers (5'-GTWCARGTNAAYAAYCARAC-3' and 5'-CCDATDATRTTRTANACNCC-3') were synthesized, according to the N-terminus and the internal sequence Bsp1, and used to amplify a bsp internal fragment of about 1·0 kb from the chromosome of GBS. The bsp-specific PCR product was used as a digoxigenin-labelled probe to isolate the entire bsp gene from a GBS cosmid library in E. coli. Subcloning of the bsp-encoding region in pUC18 resulted in the identification of a 3·7 kb insert which was finally sequenced. As shown in Fig. 1, analysis of the bsp-encoding region identified two ORFs extending from bp 590 to bp 2230 (ORF1) and from bp 2374 to bp 3609 (ORF2). ORF1 is preceded by a typical ribosome-binding site (AAGGAAG) and encodes a polypeptide of 544 aa with a predicted molecular mass of 60·417 Da. As the six peptide sequences obtained by N-terminal sequencing of Bsp and Bsp-derived peptides exactly matched the deduced polypeptide of ORF1, it was concluded that ORF1 represents the bsp gene. The deduced Bsp protein carries, at its N-terminus, a typical signal peptide sequence (Nielsen et al., 1997 ) of 42 aa, and possesses, at its C-terminus, a cell wall anchor motif (Schneewind et al., 1993 ) (LPKTG), suggesting that Bsp is transported across the cytoplasmic membrane and is covalently attached to the cell wall of GBS. Interestingly, analysis of Bsp with the SMART program (http://smart.embl-heidelberg.de/smart) identified four SH3 domains, which are suggested to be involved in the binding of proteins to the bacterial cell wall (Ponting et al., 1999 ).



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Fig. 1. Restriction map of the bsp- and femH-encoding region in GBS, and a diagrammatic representation of structural features and domains of the deduced Bsp protein. Open arrows indicate the positions of the bsp and femH genes, and ’T’ represents the proposed transcriptional terminator downstream of the femH gene. SP, putative signal peptide; SH3, putative cell-wall-binding SH3 domain.

 
ORF2 starts 144 bp downstream of the bsp gene, is preceded by a ribosome-binding site (AAGAAAGG) and is transcribed in the same direction as the bsp gene. Since the deduced polypeptide revealed significant homology to Fem-like proteins (see below), ORF2 was designated femH. The femH gene is followed by a putative rho-independent terminator structure, suggesting transcriptional termination downstream of the femH gene. To test the possibility of bsp and femH co-transcription, RT-PCR was used to amplify from total RNA of GBS the junction region between 3' bsp and 5' femH. By using RT-PCR, a DNA fragment of 432 bp was amplified, of which the DNA sequence was identical to the targeted bspfemH region (data not shown). No PCR product was obtained without the addition of reverse transcriptase. These results indicate that bsp and femH have a bicistronic organization in GBS.

Homology search of Bsp and FemH
Database analysis of the deduced Bsp protein revealed 42·6% similarity to an unknown ORF in the incomplete genome sequence of Streptococcus mutans (http://www.genome.ou.edu), 25·2% similarity to the glycyl-glycine endopeptidase lysostaphin from Staph. simulans biovar staphylolyticus, and 22·2% similarity to the glycyl-glycine endopeptidase Ale-1 from Staph. capitis. The deduced FemH protein shows 62·3% similarity to the MurN protein from Strep. pneumoniae, 58·6% similarity to the zoocin immunity factor (Zif) from Streptococcus equi subsp. zooepidermicus, and 36·4% similarity to the FemA protein from Staph. aureus. Since MurN, Zif and FemA are known to be involved in the formation of the interpeptide bridge during peptidoglycan biosynthesis, the database analysis indicates that FemH is involved in the synthesis of the interpeptide bridge of the peptidoglycan in GBS, whereas Bsp appears to be a putative peptidoglycan-cleaving endopeptidase.

Functional analysis and serological detection of the Bsp protein
Because of the sequence similarity of Bsp to the bacteriolytic endopeptidases lysostaphin and Ale-1, a hexahistidyl-tagged recombinant Bsp protein and Bsp-containing culture supernatant of GBS were tested in agar diffusion tests for bacteriocidal activity against different bacterial species. However, no such activity could be observed against Lactococcus lactis, Enterococcus faecalis, Micrococcus luteus, Staph. aureus and streptococci of the serological groups A, B, C, G and L, indicating that Bsp does not represent a bacteriolytic enzyme from GBS. In addition, neither the Bsp fusion protein nor Bsp in the culture supernatant of GBS revealed autolytic activity against cell walls of GBS (data not shown).

To investigate the distribution of Bsp in different GBS serotypes, purified Bsp fusion protein was used for the production of antibodies against Bsp. Culture supernatants of 31 clinical isolates of GBS, belonging to six serological groups, were subsequently tested for the presence of the Bsp protein (Fig. 2). In the culture supernatant of every GBS strain, the anti-Bsp antibodies detected a single protein of 65 kDa, indicating a wide distribution of Bsp in GBS and a high degree of conservation of the size of the Bsp protein.



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Fig. 2. Western blot analysis of culture supernatants from different GBS strains belonging to serotypes Ia, Ib, II, III, IV and V, respectively, for the presence of the Bsp protein. The strains represent different clinical isolates that had been isolated from infected neonates. Total protein (15 µg) isolated from culture supernatant of the respective strains was tested by using mouse polyclonal anti-Bsp antibodies.

 
Construction of a bsp deletion mutant and a femH insertion mutant of GBS
To analyse the importance of bsp and femH for GBS, bsp was deleted and femH insertionally inactivated in the genome of GBS 6313, resulting in the bsp mutant SMB and in the femH mutant FBH. The successful deletion of bsp and the disruption of femH in the two mutant strains was confirmed in both by Southern blot analysis (data not shown). In Western blotting experiments, culture supernatant of GBS SMB lacked a Bsp-specific band, while culture supernatants from the GBS strains 6313 and FBH revealed identical amounts of Bsp (data not shown). GBS mutants SMB and FBH exhibited growth rates and final optical densities similar to those of their parental strain, 6313, indicating that bsp and femH are not essential for GBS under these growth conditions. Since Bsp and FemH are suggested to be involved in the cell wall metabolism of GBS, the autolysis rate of the two mutant strains was compared with GBS 6313. However, no difference in autolysis could be observed between the strains (data not shown). Subsequently, the sensitivity of the GBS mutants SMB and FBH to different ß-lactam antibiotics and vancomycin was determined. The MICs of penicillin G, oxacillin, imipenem, cefotaxim and vancomycin for the GBS mutants SMB and FBH and the GBS parental strain were identical at 0·064 µg ml-1, 0·38 µg ml-1, 0·064 µg ml-1, 0·032 µg ml-1 and 0·5 µg ml-1, respectively. In addition, the lysis rates of the three strains in the presence of 0·032 µg ml-1, 0·064 µg ml-1 and 0·128 µg ml-1 penicillin G were identical (data not shown). These data indicate that bsp and femH do not influence the sensitivity of GBS to antibiotics that act on the cell walls of these bacteria.

The surface hydrophobicity of GBS mutants SMB and FBH was compared to that of GBS 6313. The assay was performed in an aqueous/hydrocarbon emulsion by photometric quantification of the bacteria in the aqueous phase. The amount of bacteria in the aqueous phase was comparable for the femH mutant FBH and the parental GBS strain (OD600 0·34). However, the amount of the bsp mutant SMB in the aqueous phase was reduced (OD600 0·225), indicating that the presence of Bsp decreases the cell-surface hydrophobicity of GBS.

Bsp affects the cell shape of GBS
To analyse the effect of bsp overexpression on the chain length and morphology of GBS, strains 6313 and SMB were transformed with the E. coli/Streptococcus shuttle vector pAT28 or the bsp-carrying plasmid pATbsp. RT-PCR analysis revealed that plasmid-mediated expression of bsp increased the amount of bsp-specific transcript in GBS 6313(pATbsp) about fivefold compared to GBS 6313(pAT28) (data not shown). In Western blotting experiments, overexpression of bsp resulted in threefold higher amounts of Bsp in the culture supernatant of GBS 6313(pATbsp), while Bsp was not detected in cell wall preparations of either of the GBS strains (data not shown). The 6313- and SMB-derived strains were subsequently subjected to a light-microscopic inspection, and the mean number of cells within 50 arbitrarily chosen chains was determined. No difference in the chain length could be observed between the different strains. Scanning electron microscopy revealed no differences in the cell morphology of GBS strain 6313(pAT28) and its bsp mutant, SMB(pAT28). However, as shown in Fig. 3, cells of GBS 6313(pAT28) exhibit a spherical shape, while those of GBS 6313(pATbsp) are lens-shaped. Similar differences were also observed when comparing cells from strain SMB(pAT28) with those from SMB pATbsp (data not shown).



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Fig. 3. Scanning electron micrographs of GBS 6313(pAT28) (a) and its bsp-overexpressing derivate, GBS 6313(pATbsp) (b). Cells of GBS 6313(pAT28) are spherical while those of GBS 6313(pATbsp) are lens-shaped. Bars, 2 µm.

 
FemH affects the muropeptide profile and amino acid composition of peptidoglycan
To examine the role of femH in cell wall biosynthesis in GBS, peptidoglycan of the GBS strains 6313 and FBH was isolated, digested with muramidase, and the muropeptides analysed by reversed-phase HPLC (Fig. 4). The disruption of femH in mutant FBH resulted in the virtual absence of four muropeptides (Fig. 4, peaks 11, 12, 13 and 16) and an increased amount of two muropeptides (Fig. 4, peaks 17 and 18), indicating significant differences in the peptidoglycan of GBS mutant FBH compared to GBS 6313. To analyse these changes in more detail, the peptidoglycan of GBS strains 6313 and FBH was subjected to total amino acid analysis by two experimental approaches: amino acid racemates were quantified both in an amino acid analyser and by GC followed by electron-impact MS. The latter method was also used to quantify the enantiomers of each amino acid. The results of the two experimental approaches, each performed in duplicate, are summarized in Fig. 5. Both analytical procedures exclusively identified the amino acids alanine, serine, isoglutamine and lysine within the peptidoglycan of GBS, indicating a high purity of the murein preparations. A comparison of the amino acid ratios within the peptidoglycan from GBS 6313 and mutant FBH revealed no differences in their molar ratios of serine, lysine and isoglutamine. However, for the peptidoglycan of GBS mutant FBH, both analytical methods showed a 14% lower amount of DL-alanine compared to that in GBS 6313. Quantification of amino acids by electron-impact MS allowed us to distinguish the relative proportions of the stereoisomers of each amino acid. Serine and lysine were present in the peptidoglycan of GBS exclusively as the L-enantiomer, isoglutamine as the D-isomer, and alanine as both D- and L-enantiomers. A detailed analysis of the data obtained by electron-impact MS revealed no difference in the molar ratio of D-alanine within the peptidoglycan of GBS 6313 and mutant FBH (Fig. 5). However, the amount of L-alanine within the peptidoglycan of GBS mutant FBH was reduced by 16%. Taking into account the presence of one L-alanine in the peptidoglycan stem peptide of the two strains, the L-alanine content of the interpeptide chain of GBS mutant FBH is reduced by 25% compared to that of GBS parental strain 6313. These data indicate that GBS mutant FBH carries shorter L-alanine-containing interpeptide chains than GBS 6313.



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Fig. 4. HPLC analysis of mutanolysin-digested cell walls of the GBS strains 6313 and FBH. Isolated muropeptides were subjected to reversed-phase HPLC as described in Methods. (a) GBS 6313; (b) GBS FBH. Peaks are numbered sequentially.

 


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Fig. 5. Total amino acid analysis of purified peptidoglycan from GBS strains 6313 ({blacksquare}) and FBH ({square}). The amount of each amino acid is expressed as a molar ratio relative to iGln, for which the amount was set as 1·0. The amount of D,L-Ala, Ser, Lys and iGln was determined both in an amino acid analyser and by GC, followed by electron-impact MS. Each experimental approach was performed in duplicate. The amount of the D- and L- stereoisomers of alanine was determined in duplicate by GC, followed by electron-impact MS. Data represent means±SD.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Although the bacterial murein sacculus appears as a static structure that protects the cell against its intracellular pressure, it is a highly flexible meshwork that allows bacterial growth and separation during cell division (Höltje, 1998 ). This requires a permanent turnover of peptidoglycan, i.e. concomitant biosynthesis, cleavage and recycling. Despite the functional coupling of cell wall biosynthesis and degradation, a genetic linkage of enzymes involved in peptidoglycan biosynthesis and degradation is found only in a few bacteria, i.e. Staph. simulans biovar staphylolyticus, Staph. capitis, Streptococcus milleri and Streptococcus equi subsp. zooepidermicus. In these organisms, the genes for the bacteriolytic endopeptidases lysostaphin, Ale-1, millericin B and zoocin A, respectively, are clustered with genes encoding Fem-like resistance proteins which modify the interpeptide bridge of the peptidoglycan, thereby protecting these strains against their own bacteriolytic enzymes (Beatson et al., 1998 ; Sugai et al., 1997b ; Thumm & Götz, 1997 ; Beukes & Hastings 2001 ). In the present study, the bsp gene, whose product reveals similarity to the bacteriolytic enzyme lysostaphin, was found to be clustered and co-transcribed with the femH gene, encoding a Fem-like protein. This genetic organization and the apparent similarity of Bsp and FemH to bacteriolytic enzymes and immunity factors, respectively, might indicate that Bsp is a bacteriolytic enzyme and FemH a protein conferring protection against Bsp. However, functional analysis of a Bsp fusion protein and Bsp-containing culture supernatant revealed no bacteriolytic activity against a variety of different Gram-positive bacterial species. In addition, insertional mutagenesis of femH revealed no effect on the viability of GBS, which is in contrast to the Fem-like immunity factors that are essential for the viability of those strains that produce a bacteriolytic enzyme. Taken together, these data suggest that bsp and femH do not encode a bacteriolytic enzyme and an immunity factor, respectively, from GBS.

Bsp from GBS has limited similarity to the murein hydrolases lysostaphin and Ale-1, respectively, and possesses four SH3 domains which are suggested to mediate binding to the bacterial cell wall (Ponting et al., 1999 ). As both lysostaphin and Ale-1 contain SH3 domains (Ponting et al., 1999 ), it can be speculated that Bsp, like these murein hydrolases, is capable of recognizing, and binding to, the bacterial cell wall. However, in our studies, the Bsp protein could not be detected in cell wall preparations of GBS, indicating that Bsp is not tightly linked to the cell wall. Interestingly, overexpression of bsp resulted in a lens-shaped morphology for GBS. Although GBS cells are typically spherical, a shape which is generally believed to be the most simple cellular morphology, recent findings with different streptococci, including GBS, have clearly shown the importance of extracellular proteins for the cell morphology of these bacteria (Reinscheid et al., 2001 ; Chia et al., 2001 ; Mattos-Graner et al., 2001 ). It is therefore tempting to speculate that Bsp plays a role in controlling the cell shape of GBS. Although the bsp deletion mutant SMB did not reveal an altered cell morphology, its bsp deficiency might be compensated for by alternative mechanisms. Similarly, different components involved in the peptidoglycan metabolism of E. coli have been shown to be highly redundant (Höltje & Heidrich, 2001 ).

Disruption of femH in GBS resulted in a significant decrease in the amount of L-alanine in the cell wall of the GBS mutant FBH. As the interpeptide chain of the peptidoglycan of GBS is composed of L-alanyl-L-alanine and L-alanyl-L-serine dipeptides, FemH of GBS is conceivably involved in the incorporation of L-alanine residues into the interpeptide bridge of the peptidoglycan. Similarly, the disruption of femAB and murMN in Staph. aureus and Strep. pneumoniae, respectively, result in a significant reduction of the amount of glycine and alanine, respectively, in the interpeptide chains of the resultant mutants (Filipe et al., 2000 ; Stranden et al., 1997 ). The deduced protein sequence of femH reveals striking similarity to MurN from Strep. pneumoniae, which is required for the addition of the second L-alanine into the interpeptide chain of the bacteria (Filipe et al., 2000 ). Because of this sequence similarity, it is tempting to speculate that FemH from GBS incorporates L-alanine into position 2 of the interpeptide chain. However, the femH mutant FBH revealed no L-Ser reduction and a reduction of only 0·5 L-Ala per interpeptide chain, while, in the case of the murN mutant of Strep. pneumoniae, one L-Ala per interpeptide chain is lost (Filipe et al., 2000 ). This discrepancy might be explained by incomplete inactivation of femH in GBS mutant FBH. Although the femH gene was insertionally inactivated by a fragment, corresponding in size and location to the one used for disrupting murN in Strep. pneumoniae, the possibility that the truncated FemH protein in GBS retained some catalytic activity cannot be ruled out. Alternatively, the disruption of femH might be partially compensated for by another fem-like gene from GBS. Similarly, the Fem-like lysostaphin immunity factor Lif was shown to complement a FemB deficiency in Staph. aureus (Tschierske et al., 1997 ).

Although GBS remains uniformly susceptible to ß-lactam antibiotics, the treatment of GBS infections requires 4–10-fold higher doses of ß-lactam antibiotics compared to Strep. pyogenes infections (Fernandez et al., 1998 ). Penicillin resistance can result from the acquisition of specific penicillin-binding proteins that, in the presence of ß-lactam antibiotics, take over the function of the cells’ own suceptible penicillin-binding proteins in cell wall biosynthesis (de Jonge & Tomasz, 1993 ). Interestingly, the peptidoglycan interpeptide bridges of ß-lactam-resistant strains of Staph. aureus and Strep. pneumoniae are essential for the resistance of these strains against methicillin and penicillin, respectively. Thus, the disruption of the femAB genes in highly methicillin-resistant Staph. aureus strains results in virtually a complete loss of methicillin resistance (Stranden et al., 1997 ). Similarly, the inactivation of the murMN genes in pencillin-resistant Strep. pneumoniae strains causes a complete loss of the penicillin resistance of these strains (Filipe & Tomasz, 2000 ). Since FemH from GBS exhibits significant similarity to MurN and to FemA from Strep. pneumoniae and Staph. aureus, respectively, FemH might be required for the intrinsic reduced ß-lactam susceptibility of GBS. However, the disruption of femH in GBS did not increase the susceptibility of GBS to different ß-lactam antibiotics, revealing that femH is not involved in the lower ß-lactam sensitivity of GBS. It is interesting to note that in Strep. pneumoniae strains, which are intrinsically sensitive to ß-lactam antibiotics, the disruption of murMN does not further increase their susceptibility to ß-lactams (Filipe & Tomasz, 2000 ). Therefore, it would be interesting to analyse the effect of disrupting femH in ß-lactam-resistant GBS strains (Kim, 1985 ). Finally, it is tempting to speculate that FemH might be used as a target in future studies of ß-lactam-resistant strains of GBS.


   ACKNOWLEDGEMENTS
 
We thank S. Sauter and K. Servan for excellent technical assistance.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Baker, C. J. & Edwards, M. S. (1995). Group B streptococcal infections. In Infectious Diseases of The Fetus and Newborn Infant , pp. 980-1054. Edited by J. S. Remington & J. O. Klein. Philadelphia:W. B. Saunders.

Beatson, S. A., Sloan, G. L. & Simmonds, R. S. (1998). Zoocin A immunity factor: a femA-like gene found in a group C streptococcus. FEMS Microbiol Lett 163, 73-77.[Medline]

Beukes, M. & Hastings, J. W. (2001). Self-protection against cell wall hydrolysis in Streptococcus milleri NMSCC 061 and analysis of the millericin B operon. Appl Environment Microbiol 67, 3888-3896.[Abstract/Free Full Text]

Chhatwal, G. S., Lämmler, C. & Blöbel, H. (1984). Guanidine extraction enhances the binding of human fibrinogen to group-B streptococci. Med Microbiol Immunol 173, 19-27.[Medline]

Chia, J. S., Chang, L. Y., Shun, C. T., Chang, Y. Y. & Chen, J. Y. (2001). A 60-kilodalton immunodominant glycoprotein is essential for cell wall integrity and the maintenance of cell shape in Streptococcus mutants. Infect Immun 69, 6987-6998.[Abstract/Free Full Text]

de Jonge, B. L. & Tomasz, A. (1993). Abnormal peptidoglycan produced in a methicillin-resistant strain of Staphylococcus aureus grown in the presence of methicillin: functional role for penicillin-binding protein 2A in cell wall synthesis. Antimicrob Agents Chemother 37, 342-346.[Medline]

Dubendorff, J. W. & Studier, F. W. (1991). Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor. J Mol Biol 219, 45-59.[Medline]

Ehlert, K., Tschierske, M., Mori, C., Schroder, W. & Berger-Bächi, B. (2000). Site-specific serine incorporation by Lif and Epr into positions 3 and 5 of the Staphylococcal peptidoglycan interpeptide bridge. J Bacteriol 182, 2635-2638.[Abstract/Free Full Text]

Fernandez, M., Hickman, M. E. & Baker, C. J. (1998). Antimicrobial susceptibilities of group B streptococci isolated between 1992 and 1996 from patients with bacteremia or meningitis. Antimicrob Agents Chemother 42, 1517-1519.[Abstract/Free Full Text]

Filipe, S. R. & Tomasz, A. (2000). Inhibition of the expression of penicillin resistance in Streptococcus pneumoniae by inactivation of cell wall muropeptide branching genes. Proc Natl Acad Sci USA 97, 4891-4896.[Abstract/Free Full Text]

Filipe, S. R., Pinho, M. G. & Tomasz, A. (2000). Characterization of the murMN operon involved in the synthesis of branched peptidoglycan peptides in Streptococcus pneumoniae. J Biol Chem 275, 27768-27774.[Abstract/Free Full Text]

Fontana, R., Boaretti, M., Grossato, A., Tonin, E. A., Lleo, M. M. & Satta, G. (1990). Paradoxical response of Enterococcus faecalis to the bactericidal activity of penicillin is associated with reduced activity of one autolysin. Antimicrob Agents Chemother 34, 314-320.[Medline]

Frank, H., Nicholson, G. J. & Bayer, E. (1978). Enantiomer labelling, a method for the quantitative analysis of amino acids. J Chromatogr 167, 187-196.[Medline]

Garcia-Bustos, J. F., Chait, B. T. & Tomasz, A. (1987). Structure of the peptide network of pneumococcal peptidoglycan. J Biol Chem 262, 15400-15405.[Abstract/Free Full Text]

Hakenbeck, R., Konig, A., Kern, I., van der Linden, M., Keck, W., Billot-Klein, D., Legrand, R., Schoot, B. & Gutmann, L. (1998). Acquisition of five high-Mr penicillin-binding protein variants during transfer of high-level ß-lactam resistance from Streptococcus mitis to Streptococcus pneumoniae. J Bacteriol 180, 1831-1840.[Abstract/Free Full Text]

Hanahan, D. (1985). Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166, 557-580.

Höltje, J. V. (1998). Growth of the stress-bearing and shape-maintaining murein sacculus of Escherichia coli. Microbiol Mol Biol Rev 62, 181-203.[Abstract/Free Full Text]

Höltje, J. V. & Heidrich, G. (2001). Enzymology of elongation and constriction of the murein sacculus of Escherichia coli. Biochimie 83, 103-108.[Medline]

Höltzel, A., Jack, R. W., Nicholson, G., Jung, G., Gebhardt, K., Fiedler, H.-P. & Süßmuth, R. (2001). Streptocidins A–D, novel cyclic decapeptide antibiotics from Streptomyces spp. TÜ 6071. II. Structure elucidation. J Antibiot 54, 434-440.[Medline]

Kim, K. S. (1985). Antimicrobial susceptibility of GBS. Antibiot Chemother 35, 83-89.[Medline]

Kling, D. E., Madoff, L. C. & Michel, J. L. (1999). Subcellular fractionation of group B streptococcus. Biotechniques 27, 24–6, 28.[Medline]

Kogan, G., Uhrin, D., Brisson, J. R., Paoletti, L. C., Blodgett, A. E., Kasper, D. L. & Jennings, H. J. (1996). Structural and immunochemical characterization of the type VIII group B streptococcus capsular polysaccharide. J Biol Chem 271, 8786-8790.[Abstract/Free Full Text]

Maguin, E., Prevost, H., Ehrlich, S. & Gruss, A. (1996). Efficient insertional mutagenesis in lactococci and other gram-positive bacteria. J Bacteriol 178, 931-935.[Abstract]

Maiorino, M., Roche, C., Kiess, M., Koenig, K., Gawlik, D., Matthes, M., Naldini, E., Pierce, R. & Flohe, L. (1996). A selenium-containing phospholipid-hydroperoxide glutathione peroxidase in Schistosoma mansoni. Eur J Biochem 238, 838-844.[Abstract]

Mattos-Graner, R. O., Jin, S., King, W. F., Chen, T., Smith, D. J. & Duncan, M. J. (2001). Cloning of the Streptococcus mutans gene encoding glucan binding protein B and analysis of genetic diversity and protein production in clinical isolates. Infect Immun 69, 6931-6941.[Abstract/Free Full Text]

Nielsen, H., Engelbrecht, J., Brunak, S. & von Heijne, G. (1997). Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10, 1-6.[Abstract]

Ponting, C. P., Aravind, L., Schultz, J., Bork, P. & Koonin, E. V. (1999). Eukaryotic signalling domain homologous in archaea and bacteria. Ancient ancestry and horizontal gene transfer. J Mol Biol 289, 729-745.[Medline]

Pospiech, A. & Neumann, B. (1995). A versatile quick-prep of genomic DNA from Gram-positive bacteria. Trends Genet 11, 217-218.[Medline]

Qin, X., Singh, K. V., Xu, Y., Weinstock, G. M. & Murray, B. E. (1998). Effect of disruption of a gene encoding an autolysin of Enterococcus faecalis OG1RF. Antimicrob Agents Chemother 42, 2883-2888.[Abstract/Free Full Text]

Reinscheid, D. J., Gottschalk, B., Schubert, A., Eikmanns, B. J. & Chhatwal, G. S. (2001). Identification and molecular analysis of PcsB, a protein required for cell wall separation of group B streptococcus. J Bacteriol 183, 1175-1183.[Abstract/Free Full Text]

Ricci, M. L., Manganelli, R., Berneri, C., Orefici, G. & Pozzi, G. (1994). Electrotransformation of Streptococcus agalactiae with plasmid DNA. FEMS Microbiol Lett 119, 47-52.[Medline]

Rohrer, S., Ehlert, K., Tschierske, M., Labischinski, H. & Berger-Bächi, B. (1999). The essential Staphylococcus aureus gene fmhB is involved in the first step of peptidoglycan pentaglycine interpeptide formation. Proc Natl Acad Sci USA 96, 9351-9356.[Abstract/Free Full Text]

Rosenberg, M., Perry, A., Bayer, E. A., Gutnick, D. L., Rosenberg, E. & Ofek, I. (1981). Adherence of Acinetobacter calcoaceticus RAG-1 to human epithelial cells and to hexadecane. Infect Immun 33, 29-33.[Medline]

Rubens, C. E., Kuypers, J. M., Heggen, L. M., Kasper, D. L. & Wessels, M. R. (1991). Molecular analysis of the group B streptococcal capsule genes. In Lactococci, Enterococci and Streptococci , pp. 179-183. Edited by G. M. Dunny, P. P. Cleary & L. L. McKay. Washington, DC:American Society for Microbiology.

Sambrook, J., Fritsch, E. F. & Maniatis, J. (1989). Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Schleifer, K. H. & Kandler, O. (1972). Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol Rev 36, 407-477.[Medline]

Schneewind, O., Mihaylova-Petkov, D. & Model, P. (1993). Cell wall sorting signals in surface proteins of gram-positive bacteria. EMBO J 12, 4803-4811.[Abstract]

Simmonds, R. S., Simpson, W. J. & Tagg, J. R. (1997). Cloning and sequence analysis of zooA, a Streptococcus zooepidermicus gene encoding a bacteriocin-like inhibitory substance having a domain structure similar to that of lysostaphin. Gene 189, 255-261.[Medline]

Stranden, A. M., Ehlert, K., Labischinski, H. & Berger-Bächi, B. (1997). Cell wall monoglycine cross-bridge and methicillin hypersusceptibility in a femAB null mutant of methicillin-resistant Staphylococcus aureus. J Bacteriol 179, 9-16.[Abstract]

Sugai, M., Fujiwara, T., Akiyama, T., Ohara, M., Komatsuzawa, H., Inoue, S. & Suninaka, H. (1997a). Purification and molecular characterization of glycylglycine endopeptidase produced by Staphylococcus capitis EPK1. J Bacteriol 179, 1193-1202.[Abstract]

Sugai, M., Fujiwara, T., Ohta, K., Komatsuzawa, H., Ohara, M. & Suninaka, H. (1997b). epr, which encodes glycylglycine endopeptidase resistance, is homologous to femAB and affects serine content of peptidoglycan cross bridges in Staphylococcus capitís and Staphylococcus aureus. J Bacteriol 179, 4311-4318.[Abstract]

Teng, F., Murray, B. E. & Weinstock, G. M. (1998). Conjugal transfer of plasmid DNA from Escherichia coli to enterococci: a method to make insertion mutations. Plasmid 39, 182-186.[Medline]

Thumm, G. & Götz, F. (1997). Studies on prolysostaphin processing and characterisation of the lysostaphin immunity factor (Lif) of Staphylococcus simulans biovar staphylolyticus. Mol Microbiol 23, 1251-1265.[Medline]

Trieu-Cuot, P., Carlier, C., Poyard-Salmeron, C. & Courvalin, P. (1990). A pair of mobilizable shuttle vectors conferring resistance to spectinomycin for molecular cloning in Escherichia coli and in gram-positive bacteria. Nucleic Acids Res 18, 4296.[Medline]

Tschierske, M., Ehlert, K., Stranden, A. M. & Berger-Bächi, B. (1997). Lif, the lysostaphin immunity factor, complements FemB in staphylococcal peptidoglycan interpeptide bridge formation. FEMS Microbiol Lett 153, 261-264.[Medline]

Tschierske, M., Mori, C., Rohrer, S., Ehlert, K., Shaw, K. & Berger-Bächi, B. (1999). Identification of three additional femAB-like open reading frames in Staphylococcus aureus. FEMS Microbiol Lett 171, 97-102.[Medline]

Valentin-Weigand, P., Benkel, P., Rohde, M. & Chhatwal, G. S. (1996). Entry and intracellular survival of group B streptococci in J774 macrophages. Infect Immun 64, 2467-2473.[Abstract]

Vieira, J. & Messing, J. (1982). The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19, 259-268.[Medline]

Waite, D. C., Alper, E. J. & Mady, B. J. (1996). Adult group B streptococcal disease. Ann Intern Med 125, 152-153.[Free Full Text]

Weber, B., Ehlert, K., Diehl, A., Reichmann, P., Labischinski, H. & Hakenbeck, R. (2000). The fib locus in Streptococcus pneumoniae is required for peptidoglycan crosslinking and PBP-mediated ß-lactam resistance. FEMS Microbiol Lett 188, 81-85.[Medline]

Wessels, M. R., Haft, R. F., Heggen, L. M. & Rubens, C. E. (1992). Identification of a genetic locus essential for capsule sialylation in type III group B streptococci. Infect Immun 60, 392-400.[Abstract]

Received 2 April 2002; revised 3 July 2002; accepted 5 July 2002.