The Caulobacter crescentus paracrystalline S-layer protein is secreted by an ABC transporter (type I) secretion apparatus

Caulobacter crescentus is a gram-negative bacterium that produces a two-dimensional crystalline array on its surface composed of a single 98-kDa protein, RsaA. Secretion of RsaA to the cell surface relies on an uncleaved C-terminal secretion signal. In this report, we identify two genes encoding components of the RsaA secretion apparatus. These components are part of a type I secretion system involving an ABC transporter protein. These genes, lying immediately 3' of rsaA, were found by screening a Tn5 transposon library for the loss of RsaA transport and characterizing the transposon-interrupted genes. The two proteins presumably encoded by these genes were found to have significant sequence similarity to ABC transporter and membrane fusion proteins of other type I secretion systems. The greatest sequence similarity was found to the alkaline protease (AprA) transport system of Pseudomonas aeruginosa and the metalloprotease (PrtB) transport system of Erwinia chrysanthemi. The prtB and aprA genes were introduced into C. crescentus, and their products were secreted by the RsaA transport system. Further, defects in the S-layer protein transport system led to the loss of this heterologous secretion. This is the first report of an S-layer protein secreted by a type I secretion apparatus. Unlike other type I secretion systems, the RsaA transport system secretes large amounts of its substrate protein (it is estimated that RsaA accounts for 10 to 12% of the total cell protein). Such levels are expected for bacterial S-layer proteins but are higher than for any other known type I secretion system.     

Fine chemical modifications at N- and C-termini enhance peptide presentation to T cells by increasing the lifespan of both free and MHC-complexed peptides

We investigated the effect of modifying the N- and/or C-termini of the snake toxin peptide 24-36 on its presentation to T cells. Acetylation at the N-terminus as well as amidation at the C-terminus enhanced the capacity of the peptide to activate T cells. Simultaneous modifications further increased the stimulating activity, the peptide becoming approximately 100-fold more potent than the unmodified peptide. Clearly, the introduced modifications increased the lifetime of the peptide free in solution, by decreasing its proteolytic degradation, during the T cell stimulation assays. Paradoxically, however, at similar concentrations of free peptides, the modified ones, especially those having an acetylated N-terminus, were much more active than the unmodified peptide, irrespective of the experimental conditions. These observations suggested that components other than protection from proteolytic degradation should be associated with the higher stimulating activities of the modified peptides. Accordingly, chasing experiments with APC revealed that acetylation at N-terminus caused a higher persistence of the peptides at APC surface. Together, our data indicate that (i) the T cell stimulating capacity of a peptide is associated with its lifespans in the free and MHC II bound states; and (ii) these lifespans can be greatly enhanced by introducing fine chemical modifications at N- and C-termini. These data may have some implications in designing more potent peptidic immunomodulators.     

Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element

A novel protein purification system has been developed which enables purification of free recombinant proteins in a single chromatographic step. The system utilizes a modified protein splicing element (intein) from Saccharomyces cerevisiae (Sce VMA intein) in conjunction with a chitin-binding domain (CBD) from Bacillus circulans as an affinity tag. The concept is based on the observation that the modified Sce VMA intein can be induced to undergo a self-cleavage reaction at its N-terminal peptide linkage by 1,4-dithiothreitol (DTT), beta-mercaptoethanol (beta-ME) or cysteine at low temperatures and over a broad pH range. A target protein is cloned in-frame with the N-terminus of the intein-CBD fusion, and the stable fusion protein is purified by adsorption onto a chitin column. The immobilized fusion protein is then induced to undergo self-cleavage under mild conditions, resulting in the release of the target protein while the intein-CBD fusion remains bound to the column. No exogenous proteolytic cleavage is needed. Furthermore, using this procedure, the purified free target protein can be specifically labeled at its C-terminus.     

Unidirectional reconstitution and characterization of purified Na+/proline transporter of Escherichia coli

A simple approach for large-scale purification and unidirectional reconstitution of the Na+/proline transporter of Escherichia coli (PutP) is described. The procedure is based on the insertion of a highly polar peptide composed of 17 amino acids including a 6His tag at the C-terminus of the transporter. Purification of the hybrid protein is achieved by Ni+-NTA affinity (purity >95%) and ion exchange chromatography (purity >99%). The purified transporter is reconstituted into preformed, detergent-destabilized liposomes. Detergent is removed slowly by adsorption to polystyrene beads. The highest activities [Vmax = 1.1 x 10(3) nmol min-1 (mg of protein)-1] are measured when Triton X100 is used for liposomes destabilization at a concentration corresponding to the onset of lipid solubilization. Site-directed labeling of PutP and site-specific proteolytic cleavage indicate that the transporter is inserted into proteoliposomes in an inside-out orientation. Reconstituted PutP is able to accumulate proline against a concentration gradient in the presence of an inwardly directed electrochemical Na+ or Li+ gradient, while a pH gradient does not affect transport. The apparent proline affinity of PutP in proteoliposomes is similar to the value determined with intact cells. Interestingly however, the apparent Na+ affinity of reconstituted PutP is reduced by a factor of about 25 compared to cells, suggesting a lower cation affinity on the cytosolic side of PutP relative to the outside.     

Serratia marcescens S-layer protein is secreted extracellularly via an ATP-binding cassette exporter, the Lip system

The Serratia marcescens Lip exporter belonging to the ATP-binding cassette (ABC) exporter is known to be involved in signal peptide-independent extracellular secretion of a lipase and a metalloprotease. Although the genes of secretory proteins and their ABC exporters are usually all reported to be linked in several gram-negative bacteria, neither the lipase nor the protease gene is located close to the Lip exporter genes, lipBCD. A gene (slaA) located upstream of the lipBCD genes was cloned, revealing that it encodes a polypeptide of 100 kDa and is partially similar to the Caulobacter crescentus paracrystalline cell surface layer (S-layer) protein. The Lip exporter-deficient mutants of S. marcescens failed to secrete the SlaA protein. Electron micrography demonstrated the cell surface layer of S. marcescens. The S-layer protein was secreted to the cultured media in Escherichia coli cells carrying the Lip exporter. Three ABC exporters, Prt, Has and Hly systems, could not allow the S-layer secretion, indicating that the S. marcescens S-layer protein is strictly recognized by the Lip system. This is the first report concerning secretion of an S-layer protein via its own secretion system.     

DNA binding and helicase domains of the Escherichia coli recombination protein RecG

The Escherichia coli RecG protein is a unique junction-specific helicase involved in DNA repair and recombination. The C-terminus of RecG contains motifs conserved throughout a wide range of DNA and RNA helicases and it is thought that this C-terminal half of RecG contains the helicase active site. However, the regions of RecG which confer junction DNA specificity are unknown. To begin to assign structure-function relationships within RecG, a series of N- and C-terminal deletions have been engineered into the protein, together with an N-terminal histidine tag fusion peptide for purification purposes. Junction DNA binding, unwinding and ATP hydrolysis were disrupted by mutagenesis of the N-terminus. In contrast, C-terminal deletions moderately reduced junction DNA binding but almost abolished unwinding. These data suggest that the C-terminus does contain the helicase active site whereas the N-terminus confers junction DNA specificity.     

Proteasomes generate in vitro a natural peptide of influenza-A nucleoprotein functional in HLA-B27 antigen assembly

We have studied the degradation of a set of long peptides (9-30 amino acids) from the nucleoprotein of influenza A. In common for all these peptides is the core sequence NH2-Ser-Arg-Tyr-Trp-Ala-Ile-Arg-Thr-Arg-COOH, NP383-391, known as an antigenic peptide specific for the HLA-B27 class I antigen. We show that this peptide is generated by enriched cytosolic proteasomes of two sizes, 20S and 12S. The 12S proteasome is the precursor, the preproteasome, to the 20S mature proteasome as shown by pulse-chase experiment and is most likely responsible for the proteolytic activity in the 12S region. Cleavage at the N-terminus is distinct and restricted to residue 383, independent of the N-terminal extension of the peptide. The C-terminus is generated via cleavage at three sites. Intermediate and final peptide products were identified by mass spectrometry. Finally, we show that the NP383-391 peptide generated by proteasomes in vitro is functional inasmuch as it possesses the ability to stimulate assembly of in vitro translated HLA-B27 antigens.     

Influence of the secondary cell wall polymer on the reassembly, recrystallization, and stability properties of the S-layer protein from Bacillus stearothermophilus PV72/p2

The high-molecular-weight secondary cell wall polymer (SCWP) from Bacillus stearothermophilus PV72/p2 is mainly composed of N-acetylglucosamine (GlcNAc) and N-acetylmannosamine (ManNAc) and is involved in anchoring the S-layer protein via its N-terminal region to the rigid cell wall layer. In addition to this binding function, the SCWP was found to inhibit the formation of self-assembly products during dialysis of the guanidine hydrochloride (GHCl)-extracted S-layer protein. The degree of assembly (DA; percent assembled from total S-layer protein) that could be achieved strongly depended on the amount of SCWP added to the GHCl-extracted S-layer protein and decreased from 90 to 10% when the concentration of the SCWP was increased from 10 to 120 microg/mg of S-layer protein. The SCWP kept the S-layer protein in the water-soluble state and favored its recrystallization on solid supports such as poly-L-lysine-coated electron microscopy grids. Derived from the orientation of the base vectors of the oblique S-layer lattice, the subunits had bound with their charge-neutral outer face, leaving the N-terminal region with the polymer binding domain exposed to the ambient environment. From cell wall fragments about half of the S-layer protein could be extracted with 1 M GlcNAc, indicating that the linkage type between the S-layer protein and the SCWP could be related to that of the lectin-polysaccharide type. Interestingly, GlcNAc had an effect on the in vitro self-assembly and recrystallization properties of the S-layer protein that was similar to that of the isolated SCWP. The SCWP generally enhanced the stability of the S-layer protein against endoproteinase Glu-C attack and specifically protected a potential cleavage site in position 138 of the mature S-layer protein.     

Improved mimicry of a foot-and-mouth disease virus antigenic site by a viral peptide displayed on beta-galactosidase surface

A major antigenic site (site A) of foot-and-mouth disease virus includes multiple overlapping epitopes located within the flexible G-H loop of capsid protein VP1. We have studied the antigenicity of several recombinant E. coli beta-galactosidases displaying the site A from a serotype C virus in different surface regions of the bacterial enzyme. In each one of the explored insertion sites, the recombinant peptide shows different specificity with a set of anti-virus monoclonal antibodies directed to site A. In some of them, the inserted stretch mimics better than free or haemocyanin-coupled peptide the antigenicity of site A in the intact virus. In particular, an insertion within an exposed loop involved in the activating interface of beta-galactosidase (amino acids 272 to 287) led to a significant improvement of the overall reactivity. Since insertions at this site renders proteins enzymatically active, the activating interface could be an adequate place for the presentation of foreign antigens in correctly assembled beta-galactosidase tetramers. These results also suggest that anti-virus antibodies directed against the major antigenic site of FMDV recognize different conformations of the G-H loop, which are better reproduced in some of the recombinant proteins because of the dissimilar restrictions imposed by each particular insertion site.     

Presynaptic protein interactions in vivo: evidence from botulinum A, C, D and E action at frog neuromuscular junction

The present study examines the paralytic action of botulinum neurotoxins at their natural target, the neuromuscular junction. We asked whether syntaxin, synaptosome-associated protein of 25 kDa (SNAP-25) and vesicle-associated membrane protein (VAMP/synaptobrevin), the proteins proteolysed by botulinum, are susceptible to cleavage in frog nerve terminals, and whether they form complexes in vivo. In control terminals, the three SNAREs were distributed in broad bands at 1 micrometer intervals, at sites consistent with presynaptic Ca2+ channels. Within 3 h, botulinum A, C, D and E (BoNT/A/C/D/E) blocked nerve-evoked muscle contractions but their effects on substrate immunoreactivity varied. The effect of BoNT/A on either C-terminus or N-terminus immunoreactivity of SNAP-25 was undetectable after 3-h incubation, although C-terminus immunoreactivity was reduced after 24 h; N-terminus immunoreactivity was not affected even after 36 h. BoNT/E reduced C-terminus immunoreactivity of SNAP-25 1.5 h after toxin application when transmitter release was blocked, but required 24 h to reduce N-terminus immunoreactivity. BoNT/C reduced syntaxin immunoreactivity after 24-h incubation but did not affect SNAP-25. BoNT/D reduced VAMP immunoreactivity at 3 h while it increased SNAP-25 C-terminal staining fourfold. BoNT/A and BoNT/C applied together for 24 h reduced syntaxin immunoreactivity and that of both C- and N-terminus of SNAP-25, indicating that retention of SNAP-25 N-terminus after cleavage by BoNT/A depended on intact syntaxin. Therefore, we infer that SNAP-25 interacts with VAMP and with syntaxin in vivo. Neurotoxin action abolished only 40-60% of SNAP-25, VAMP or syntaxin immunoreactivity suggesting that distinct pools of these proteins, not immediately involved in triggered exocytosis, are resistant to proteolysis.     

A selective medium for the rapid detection by an impedance technique of Pseudomonas spp. associated with poultry meat

IS511 is an endogenous insertion sequence (IS) of the bacterium Caulobacter crescentus strain CB15 and it is the first Caulobacter IS to be characterized at the molecular level. We determined the 1266-bp nucleotide sequence of IS511 and investigated its genetic organization, relationship to other ISs, and transposition properties. IS511 belongs to a distinct branch of the IS3 family that includes ISR1, IS476, and IS1222, based on nucleotide sequence similarity. The nucleotide sequence of IS511 encodes open reading frames (orfs) designated here as orfA and orfB, and their relative organization and amino acid sequences of the predicted protein products are very similar to those of orfAs and orfBs of other IS3 family members. Nuclease S1 protection assays identified an IS511 RNA, and its 5' end maps approximately 16 nucleotides upstream of orfA and about six nucleotides downstream of a sequence that is similar to the consensus sequence of C. crescentus housekeeping promoters. Evidence is presented that IS511 is capable of precise excision from the chromosome, and transposition from the chromosome to a plasmid. Transpositional insertions of IS511 occurred within sequences with a relatively high G + C content, and they were usually, but not always, flanked by a 4-bp direct repeat that matches a sequence at the site of insertion. We also determined the nucleotide sequence flanking the four endogenous IS511 elements that reside in the chromosome of C. crescentus. Our findings demonstrate that IS511 is a transposable IS that belongs to a branch of the IS3 family.     

Involvement of cell surface glycosyl-phosphatidylinositol-linked aspartyl proteases in alpha-secretase-type cleavage and ectodomain solubilization of human Alzheimer beta-amyloid precursor protein in yeast

Human beta-amyloid precursor protein (APP) introduced into yeast undergoes alpha-secretase-type cleavage, suggesting that yeast have alpha-secretase-like protease(s). Here we report that two structurally and functionally related glycosyl-phosphatidylinositol-linked yeast aspartyl proteases, Mkc7p and Yap3p (collectively termed yapsin), are responsible for alpha-secretase-type cleavage of APP expressed in yeast, resulting in release of soluble APP into the extracellular space. Disruption of MKC7 and YAP3 in a vacuolar protease-deficient strain abolished this APP cleavage/release, and APP cleavage/release could be restored by introduction of MKC7 or YAP3 on a single copy plasmid. Purified Mkc7p cleaved an internally quenched fluorogenic APP peptide substrate at the alpha-secretase cleavage site. Measurement of proteolytic activity either in yeast homogenates or on the yeast cell surface revealed that most Mkc7p and Yap3p activities were localized at the cell surface. These results establish a molecular basis for alpha-secretase-type cleavage in yeast and support the generally held concept that alpha-secretase cleavage of APP occurs at the cell surface.     

Proper spacing between heptad repeat B and the transmembrane domain boundary of the paramyxovirus SV5 F protein is critical for biological activity

The paramyxovirus, simian virus 5, fusion (F) protein contains seven amino acids between heptad repeat B (a domain required for a biologically active fusion protein) and the presumptive boundary of the transmembrane (TM) domain. The role of the seven membrane proximal residues in stability and fusion promotion was examined by construction of a series of insertion, substitution, and deletion mutants, as manipulation of this region to enable proteolytic cleavage would facilitate production of a soluble F protein. The majority of the mutant F proteins both oligomerized and had kinetics of intracellular transport similar to those of wild-type (wt) F protein. All mutant F proteins were expressed at the cell surface at or near the same level as the wt F protein. However, by using both a qualitative lipid mixing assay and a quantitative content mixing assay for membrane fusion, it was found that mutant F proteins containing insertions in the region between heptad repeat B and the TM domain were unable to induce fusion, whereas the mutant F proteins containing substitutions in this region, together with three of the four mutants with deletions in this region, could induce fusion. Four of the F protein mutants contained a Factor Xa cleavage site, IEGR; however, Factor Xa treatment of cell surfaces released either none or only very small amounts (< 1% of total protein) of the soluble heterodimer F1 + F2. As an alternative method of generating soluble F protein, a glycosyl phosphatidylinositol (GPI) anchor was added to the F protein at three membrane-proximal positions. The highest level of surface expression was observed when the final molecule did not contain a significant insertion of amino acids into the membrane proximal region. Two F-GPI mutants reached the surface at approximately 20% of the levels seen with the wt F protein, and approximately 25% of the cell surface population of these mutants could be cleaved with phosphatidylinositol phospholipase C (PI-PLC) to yield soluble F protein. However, all the F-GPI mutants oligomerized aberrantly and failed to promote fusion. Taken together, these data indicate that the spacing of the region immediately adjacent to the presumptive boundary of the TM domain is extremely important for the fusogenic activity of the SV5 F protein.     

Structure of the membrane-bound form of the pore-forming domain of colicin A: a partial proteolysis and mass spectrometry study

The ion-channel-forming thermolytic fragment (thA) of colicin A binds to negatively charged vesicles and provides an example of the insertion of a soluble protein into a lipid bilayer. The soluble structure is known and consists of a 10-helix bundle containing a hydrophobic helical hairpin. In this study, partial proteolysis and mass spectrometry were used to determine the accessible sites to proteolytic attack by trypsin and alpha-chymotrypsin in the thA fragment in its membrane-bound state. Electrospray mass spectrometry was quite an efficient method for the identification of the cleavage products, even with partially purified peptide mixtures and with only few controls by N-terminal sequencing. This work confirms that a major part of the peptide chain lies at the membrane surface and that even the hydrophobic hairpin is not protected by the lipid bilayer from proteolytic degradation. In the absence of a membrane potential, the hydrophobic hairpin in the colicin A membrane-bound form seems not fixed in a transmembrane orientation.     

Sec-independent insertion of thylakoid membrane proteins. Analysis of insertion forces and identification of a loop intermediate involving the signal peptide

A group of membrane proteins are synthesized with cleavable signal sequences but inserted into the thylakoid membrane by an unusual Sec/SRP-independent mechanism. In this report we describe a key intermediate in the insertion of one such protein, photosystem II subunit W (PSII-W). A single mutation in the terminal cleavage site partially blocks processing and leads to the formation of an intermediate-size protein in the thylakoid membrane during chloroplast import assays. This protein is in the form of a loop structure: the N and C termini are exposed on the stromal face, whereas the cleavage site has been translocated into the lumen. In this respect the insertion of this protein resembles that of M13 procoat, which also adopts a loop structure during insertion, and we present preliminary evidence that a similar mechanism is used by another thylakoid protein, PSII-X. However, whereas the negatively charged region of procoat is translocated by an apparently electrophoretic mechanism using the DeltamuH+, the corresponding region of PSII-W is equally acidic but insertion is DeltamuH+ independent. We furthermore show that neutralization of this region has no apparent effect on the insertion process. We propose that a central element in this insertion mechanism is a loop structure whose formation is driven by hydrophobic interactions.     

Ocular manifestations and sequelae of Lyell syndrome caused by sulfadoxine-pyrimethamine in Cameroon]

Missense point mutations, leading to inactivation of the p53 tumor suppressor gene product, are currently the most frequent alterations in human cancer. Little, however, is known about small intragenic deletions or insertions occurring in this locus of chromosome 17. We have analyzed 56 primary ovarian tumors for the presence of such abnormalities. The analysis was based on multiplex PCR amplification of exons 1 through 11 of the p53 gene and fragment analysis of the generated PCR products. Mutations were detected in 14% (8 of 56) of the tumors. Deletions were much more prevalent than insertions (seven vs one). Six of the deletions and the insertion affected exon 5, and the other deletion was in exon 7. Two deletions and the insertion did not disrupt the reading frame; the protein product was expressed in the tumor at high concentrations in all three cases. The other five deletions generated a frameshift, which is predicted to result in the production of a truncated protein product. In the case of the deletions, a 2-5-bp repeat was present close to the detected deletion, whereas the insertion duplicated the sequence immediately upstream of the insertion site. Overall our findings indicate that small intragenic p53 deletions/insertions are not rare events in ovarian cancer, and that p53 exon 5 is the target in the vast majority (88%) of the cases.     

Cloning and characterization of archaeal type I signal peptidase from Methanococcus voltae

Archaeal protein trafficking is a poorly characterized process. While putative type I signal peptidase genes have been identified in sequenced genomes for many archaea, no biochemical data have been presented to confirm that the gene product possesses signal peptidase activity. In this study, the putative type I signal peptidase gene in Methanococcus voltae was cloned and overexpressed in Escherichia coli, the membranes of which were used as the enzyme source in an in vitro peptidase assay. A truncated, His-tagged form of the M. voltae S-layer protein was generated for use as the substrate to monitor the signal peptidase activity. With M. voltae membranes as the enzyme source, signal peptidase activity in vitro was optimal between 30 and 40 degrees C; it was dependent on a low concentration of KCl or NaCl but was effective over a broad concentration range up to 1 M. Processing of the M. voltae S-layer protein at the predicted cleavage site (confirmed by N-terminal sequencing) was demonstrated with the overexpressed archaeal gene product. Although E. coli signal peptidase was able to correctly process the signal peptide during overexpression of the M. voltae S-layer protein in vivo, the contribution of the E. coli signal peptidase to cleavage of the substrate in the in vitro assay was minimal since E. coli membranes alone did not show significant activity towards the S-layer substrate in in vitro assays. In addition, when the peptidase assays were performed in 1 M NaCl (a previously reported inhibitory condition for E. coli signal peptidase I), efficient processing of the substrate was observed only when the E. coli membranes contained overexpressed M. voltae signal peptidase. This is the first proof of expressed type I signal peptidase activity from a specific archaeal gene product.     

Evidence that the N-terminal part of the S-layer protein from Bacillus stearothermophilus PV72/p2 recognizes a secondary cell wall polymer

The S-layer of Bacillus stearothermophilus PV72/p2 shows oblique lattice symmetry and is composed of identical protein subunits with a molecular weight of 97,000. The isolated S-layer subunits could bind and recrystallize into the oblique lattice on native peptidoglycan-containing sacculi which consist of peptidoglycan of the A1gamma chemotype and a secondary cell wall polymer with an estimated molecular weight of 24,000. The secondary cell wall polymer could be completely extracted from peptidoglycan-containing sacculi with 48% HF, indicating the presence of phosphodiester linkages between the polymer chains and the peptidoglycan backbone. The cell wall polymer was composed mainly of GlcNAc and ManNAc in a molar ratio of 4:1, constituted about 20% of the peptidoglycan-containing sacculus dry weight, and was also detected in the fraction of the S-layer self-assembly products. Extraction experiments and recrystallization of the whole S-layer protein and proteolytic cleavage fragments confirmed that the secondary cell wall polymer is responsible for anchoring the S-layer subunits by the N-terminal part to the peptidoglycan-containing sacculi. In addition to this binding function, the cell wall polymer was found to influence the in vitro self-assembly of the guanidinium hydrochloride-extracted S-layer protein. Chemical modification studies further showed that the secondary cell wall polymer does not contribute significant free amino or carboxylate groups to the peptidoglycan-containing sacculi.     

Targeting of muralytic enzymes to the cell division site of Gram-positive bacteria: repeat domains direct autolysin to the equatorial surface ring of Staphylococcus aureus

Staphylococcus aureus secretes autolysin (Atl) to complete cell division by hydrolyzing its thick cell wall layer at a designated site, known as the equatorial surface ring. Secreted pro-Atl (1256 amino acids) is cleaved at residues 198 and 775 to generate a pro-peptide, amidase and glucosaminidase, respectively. Here we examined the mechanism that directs amidase and glucosaminidase to the cell division site on the staphylococcal surface. Targeting of pro-Atl to the cell surface occurred prior to its proteolytic processing. Three repeat domains (R1, R2 and R3) located at the center of pro-Atl are necessary and sufficient for the targeting of reporter proteins to the equatorial surface ring. Pro-Atl cleavage at residue 775 separates the polypeptide such that R1 and R2 are linked to the C-terminus of amidase, whereas R3 is located at the N-terminus of glucosaminidase. Thus, it appears that the repeat domains direct pro-Atl, amidase and glucosaminidase to a specific receptor at the equatorial surface ring of staphylococci, thereby allowing localized peptidoglycan hydrolysis and separation of the dividing cells.