The N3 subdomain in a domain of fibronectin-binding protein B isotype I is an independent risk determinant predictive for biofilm formation of Staphylococcus aureus clinical isolates

Fibronectin-binding proteins (FnBP), FnBPA and FnBPB, are purported to be involved in biofilm formation of Staphylococcus aureus. This study was performed to find which of three consecutive N subdomains of the A domain in the FnBP is the key domain in FnBP. A total of 465 clinical isolates of S. aureus were examined for the biofilm forming capacity and the presence of N subdomains of FnBP. In the biofilm-positive strains, N2 and N3 subdomains of FnBPA, and N1 and N3 subdomains of FnBPB were significantly more prevalent. Multivariate logistic regression analysis of 246 biofilm-positive and 123 biofilm-negative strains identified only the FnBPB-N3 subdomain as an independent risk determinant predictive for biofilm-positive strains of S. aureus (Odds ratio [OR], 13.174; P<0.001). We also attempted to delete each of the fnbA-N2 and -N3 and fnbB-N1 and -N3 from S. aureus strain 8325-4 and examined the biofilm forming capacity in the derivative mutants. In agreement with the results of the multivariate regression analysis, deletion of either the fnbA-N2 or ?N3, or fnbB-N1 did not significantly diminish the capacity of strain 8325-4 to develop a biofilm, while deletion of the fnbB-N3 did. Therefore, it is suggested that the FnBPB-N3 subdomain of isotype I may be a key domain in FnBP which is responsible for the causing biofilm formation in S. aureus clinical isolates.     

Variation and association of fibronectin‐binding protein genes fnbA and fnbB in Staphylococcus aureus Japanese isolates

Fibronectin‐binding proteins A and B (FnBPA and FnBPB) mediate adhesion of Staphylococcus aureus to fibrinogen, elastin and fibronectin. FnBPA and FnBPB are encoded by two closely linked genes, fnbA and fnbB, respectively. With the exception of the N‐terminal regions, the amino acid sequences of FnBPA and FnBPB are highly conserved. To investigate the genetics and evolution of fnbA and fnbB, the most variable regions, which code for the 67th amino acids of the A through B regions (A67–B) of fnbA and fnbB, were focused upon. Eighty isolates of S. aureus in Japan were sequenced and 19 and 18 types in fnbA and fnbB, respectively, identified. Although the phylogeny of fnbA and fnbB were found to be quite different, each fnbA type connected with a specific fnbB type, indicating that fnbA and fnbB mutate independently, whereas the combination of both genes after recombination is stable. Hence those fnbA–fnbB combinations were defined as FnBP sequence types (FnSTs). Representative isolates of each FnST were assigned distinct STs by multilocus sequence typing, suggesting correspondence of FnST with genome lineage. Linkage disequilibrium (LD) analysis of the A67–B region revealed that subdomains N2, N3 and FnBR1 form a LD block in fnbA, whereas N2 and N3 form two independent LD blocks in fnbB. N2–N3 three‐dimensional structural models indicated that not only the variable amino acid residues, but also well‐conserved amino acid residues between FnBPA and FnBPB, are located on the surface of the protein. These results highlight a molecular process of the FnBP that has evolved by mingled mutation and recombination with retention of functions.     

Subdomains N2N3 of Fibronectin Binding Protein A Mediate Staphylococcus aureus Biofilm Formation and Adherence to Fibrinogen Using Distinct Mechanisms

Health care-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) forms biofilm in vitro that is dependent on the surface-located fibronectin binding proteins A and B (FnBPA, FnBPB). Here we provide new insights into the requirements for FnBP-dependent biofilm formation by MRSA. We show that expression of FnBPs is sustained at high levels throughout the growth cycle in the HA-MRSA strain BH1CC in contrast to laboratory strain SH1000, where expression could be detected only in exponential phase. We found that FnBP-mediated biofilm accumulation required Zn²⁺, while the removal of Zn²⁺ had no effect on the ability of FnBPA to mediate bacterial adherence to fibrinogen. We also investigated the role of FnBPA expressed on the surface of S. aureus in promoting biofilm formation and bacterial adhesion to fibrinogen. The minimum part of FnBPA required for ligand binding has so far been defined only with recombinant proteins. Here we found that the N1 subdomain was not required for biofilm formation or for FnBPA to promote bacterial adherence to fibrinogen. Residues at the C terminus of subdomain N3 required for FnBPA to bind to ligands using the “dock, lock, and latch” mechanism were necessary for FnBPA to promote bacterial adherence to fibrinogen. However, these residues were not necessary to form biofilm, allowing us to localize the region of FnBPA required for biofilm accumulation to residues 166 to 498. Thus, FnBPA mediates biofilm formation and bacterial adhesion to fibrinogen using two distinct mechanisms. Finally, we identified a hitherto-unrecognized thrombin cleavage site close to the boundary between subdomains N1 and N2 of FnBPA.     

Fibronectin-binding protein B variation in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Background  

Fibronectin binding proteins A and B (FnBPA and FnBPB) mediate adhesion of S. aureus to fibrinogen, elastin and fibronectin. We previously identified seven different isotypes of FnBPA based on divergence in the fibrinogen- and elastin-binding A domains. The variation created differences in antigenicity while ligand binding functions were retained. Here, FnBPB variation was examined in both human and bovine isolates and compared to that of FnBPA.     

Two different genes encode fibronectin binding proteins in Staphylococcus aureus. The complete nucleotide sequence and characterization of the second gene.

A gene encoding a fibronectin binding protein (FnBP) has recently been isolated and sequenced from Staphylococcus aureus strain 8325-4. In the same bacterial strain, 682 bp downstream to the stop codon of this gene (fnbA), a second gene termed fnbB has not been discovered, encoding another FnBP (FnBPB). The two genes show in large parts striking sequence homologies. The complete amino acid sequence encoded by fnbB has been deduced and compared to that deduced from fnbA. In FnBPB a stretch of 66 amino acids downstream to the signal peptide has 75% identity with the corresponding region in FnBPA. At the C-terminal site another 394 amino acid stretch is almost identical in both gene products. This stretch contains the 38 amino acid long D repeats, the wall spanning Wr repeats and the hydrophobic membrane spanning domain. In FnBPA each of the three D repeats has been identified as a fibronectin binding structure. These structures are highly conserved in FnBPB and most likely represent the major Fn-binding domain of this protein. However, a subclone of gene fnbB lacking the coding region for the D repeats also clearly expresses fibronectin binding activity. This additional binding site is so far unique for FnBPB and interacts like the D domains with the N-terminal 24-31-kDa fragment of fibronectin. The purified recombinant FnBP fragment (not containing the D repeats) completely inhibits the binding of fibronectin to whole cells of S. aureus.     

Sequence diversity in the A domain of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>fibronectin-binding protein A

Background  

Fibronectin-binding protein A (FnBPA) mediates adhesion of Staphylococcus aureus to fibronectin, fibrinogen and elastin. We previously reported that S. aureus strain P1 encodes an FnBPA protein where the fibrinogen/elastin-binding domain (A domain) is substantially divergent in amino acid sequence from the archetypal FnBPA of S. aureus NCTC8325, and that these variations created differences in antigenicity. In this study strains from multilocus sequence types (MLST) that spanned the genetic diversity of S.aureus were examined to determine the extent of FnBPA A domain variation within the S. aureus population and its effect on ligand binding and immuno-crossreactivity.     

The N-terminal A domain of fibronectin-binding proteins A and B promotes adhesion of Staphylococcus aureus to elastin

The ability of Staphylococcus aureus to adhere to components of the extracellular matrix is an important mechanism for colonization of host tissues during infection. We have previously shown that S. aureus binds elastin, a major component of the extracellular matrix. The integral membrane protein, elastin-binding protein (EbpS), binds soluble elastin peptides and tropoelastin via its surface-exposed N-terminal domain. In this study, we demonstrate that some strains of S. aureus adhere strongly to immobilized human elastin and that this interaction is independent of EbpS but instead is mediated by the fibronectin-binding proteins, FnBPA and FnBPB. Our results show that EbpS mutant cells adhere to elastin-coated plates, whereas the cells negative for FnBPA and FnBPB do not adhere to the plates. Furthermore, only wild-type cells from the exponential phase of growth adhered when FnBPs were expressed maximally. We show that adherence to elastin promoted by FnBPA was not affected by soluble fibronectin, suggesting that the elastin binding domain is distinct from the fibronectin binding regions. Recombinant FnBPA(37-544) (rFnBPA(37-544)) protein corresponding to the A region of FnBPA and anti-FnBPA(37-544) antibodies inhibited FnBPA-mediated bacterial adherence to immobilized elastin. Finally, recombinant A domain proteins, rFnBPA(37-544) and rFnBPB(37-540), bound immobilized elastin dose-dependently and saturably. This interaction was inhibited by soluble elastin peptides, suggesting a specific receptor-ligand interaction.     

A Staphylococcus capitis strain with unusual bacteriocin production

Staphylococcus capitis is a member of the human and mammal skin microbiomes and is considered less harmful than Staphylococcus aureus. S. capitis subsp. urealyticus BN2 was isolated from a cat and expressed strong antibacterial activity against a range of Gram-positive species, most notably including S. aureus strains with resistance to methicillin (MRSA) and strains with intermediate resistance to vancomycin (VISA). These latter strains are normally relatively resistant to bacteriocins, due to cell wall and cell membrane modifications. Genomic sequencing showed that the strain harboured at least two complete gene clusters for biosynthesis of antagonistic substances. The complete biosynthetic gene cluster of the well-known lantibiotic gallidermin was encoded on a large plasmid and the mature peptide was present in isopropanol cell extracts. In addition, a chromosomal island contained a novel non-ribosomal peptide synthetase (NRPS) gene cluster. Accidental deletion of two NRPS modules and partial purification of the anti-VISA activity showed that this novel bacteriocin represents a complex of differently decorated, non-ribosomal peptides. Additionally, a number of phenol-soluble modulins (PSMs) was detected by mass spectrometry of whole cells. Producing these compounds, the strain was able to outcompete several S. aureus strains, including MRSA and VISA, in tube cultures.     

Fibronectin-binding proteins of Staphylococcus aureus mediate activation of human platelets via fibrinogen and fibronectin bridges to integrin GPIIb/IIIa and IgG binding to the FcgammaRIIa receptor

Staphylococcus aureus is a leading cause of infective endocarditis (IE). Platelet activation promoted by S. aureus resulting in aggregation and thrombus formation is an important step in the pathogenesis of IE. Here, we report that the fibrinogen/fibronectin-binding proteins FnBPA and FnBPB are major platelet-activating factors on the surface of S. aureus from the exponential phase of growth. Truncated derivatives of FnBPA, presenting either the fibrinogen-binding A domain or the fibronectin-binding BCD region, each promoted platelet activation when expressed on the surface of S. aureus or Lactococcus lactis, indicating two distinct mechanisms of activation. FnBPA-promoted platelet activation is mediated by fibrinogen and fibronectin bridges between the A domain and the BCD domains, respectively, to the low affinity form of the integrin GPIIb/IIIa on resting platelets. Antibodies recognizing the FnBPA A domain or the complex between the FnBPA BCD domains and fibronectin were essential for activation promoted by bacteria expressing the A domain or the BCD domain respectively. Activation was inhibited by a monoclonal antibody (IV-3) specific for the FcgammaRIIa IgG receptor on platelets. We propose that the activation of quiescent platelets by bacteria expressing FnBPs involves the formation of a bridge between the bacterial cell and the platelet surface by (i) fibronectin and fibrinogen interacting with the low affinity form of GPIIb/IIIa and (ii) by antibodies specific to FnBPs that engage the platelet Fc receptor FcgammaRIIa. Platelet activation by S. aureus clinical IE isolates from both the exponential and stationary phases of growth was completely inhibited by monoclonal antibody IV-3 suggesting that the IgG-FcgammaRIIa interaction is of fundamental importance for platelet activation mediated by this organism. This suggests new avenues for development of therapeutics against vascular infections.     

Staphylococcus aureus keratinocyte invasion is dependent upon multiple high-affinity fibronectin-binding repeats within FnBPA

Staphylococcus aureus is a commensal organism and a frequent cause of skin and soft tissue infections, which can progress to serious invasive disease. This bacterium uses its fibronectin binding proteins (FnBPs) to invade host cells and it has been hypothesised that this provides a protected niche from host antimicrobial defences, allows access to deeper tissues and provides a reservoir for persistent or recurring infections. FnBPs contain multiple tandem fibronectin-binding repeats (FnBRs) which bind fibronectin with varying affinity but it is unclear what selects for this configuration. Since both colonisation and skin infection are dependent upon the interaction of S. aureus with keratinocytes we hypothesised that this might select for FnBP function and thus composition of the FnBR region. Initial experiments revealed that S. aureus attachment to keratinocytes is rapid but does not require FnBRs. By contrast, invasion of keratinocytes was dependent upon the FnBR region and occurred via similar cellular processes to those described for endothelial cells. Despite this, keratinocyte invasion was relatively inefficient and appeared to include a lag phase, most likely due to very weak expression of α(5)β(1) integrins. Molecular dissection of the role of the FnBR region revealed that efficient invasion of keratinocytes was dependent on the presence of at least three high-affinity (but not low-affinity) FnBRs. Over-expression of a single high-affinity or three low-affinity repeats promoted invasion but not to the same levels as S. aureus expressing an FnBPA variant containing three high-affinity repeats. In summary, invasion of keratinocytes by S. aureus requires multiple high-affinity FnBRs within FnBPA, and given the importance of the interaction between these cell types and S. aureus for both colonisation and infection, may have provided the selective pressure for the multiple binding repeats within FnBPA.     

Adhesion properties of mutants of Staphylococcus aureus defective in fibronectin-binding proteins and studies on the expression of fnb genes

Staphylococcus aureus 8325-4 has the potential to express two distinct cell wall-associated fibronectin-binding proteins called FnBPA and FnBPB. In order to test if both proteins are expressed in S. aureus and if both are required for promoting bacterial adhesion to fibronectin-coated surfaces, insertion mutations were isolated in each gene. A DNA fragment encoding tetracycline resistance was inserted into fnbA and a fragment encoding erythromycin resistance was inserted into fnbB . A double fnbA fnbB mutant was also constructed. The fnbA and fnbB single mutants showed no significant reduction in their adhesion to polymethylmethacrylate coverslips that had been coated in vitro with fibronectin. However, the double mutant was completely defective in adhesion. Monospecific antibodies directed against the non-conserved N-terminal regions of both proteins confirmed the lack of expression of FnBPs in the mutant strains. Wild-type fnbA and fnbB genes cloned seperately on a multicopy plasmid were each able to restore fully the adhesion-defective phenotype of the 8325-4 fnbA fnbB mutant. This demonstrates that both fnb genes are expressed in S. aureus and that both contribute to the ability of strain 8325-4 to adhere to fibronectin-coated surfaces. The double mutant was also defective in adhesion to coverslips that had been removed from tissue cages implanted subcutaneously in guinea-pigs, which suggests that fibronectin is important in promoting attachment of S. aureus to biomaterial in vivo .     

Identification of the Immunodominant Regions of Staphylococcus aureus Fibronectin-Binding Protein A

Staphylococcus aureus is an opportunistic bacterial pathogen responsible for a diverse spectrum of human diseases and a leading cause of nosocomial and community-acquired infections. Development of a vaccine against this pathogen is an important goal. The fibronectin binding protein A (FnBPA) of S. aureus is one of multifunctional ‘microbial surface components recognizing adhesive matrix molecules'' (MSCRAMMs). It is one of the most important adhesin molecules involved in the initial adhesion steps of S. aureus infection. It has been studied as potential vaccine candidates. However, FnBPA is a high-molecular-weight protein of 106 kDa and difficulties in achieving its high-level expression in vitro limit its vaccine application in S. aureus infection diseases control. Therefore, mapping the immunodominant regions of FnBPA is important for developing polyvalent subunit fusion vaccines against S. aureus infections. In the present study, we cloned and expressed the N-terminal and C-terminal of FnBPA. We evaluated the immunogenicity of the two sections of FnBPA and the protective efficacy of the two truncated fragments vaccines in a murine model of systemic S. aureus infection. The results showed recombinant truncated fragment F1_30-500 had a strong immunogenicity property and survival rates significantly increased in the group of mice immunized with F1_30-500 than the control group. We futher identified the immunodominant regions of FnBPA. The mouse antisera reactions suggest that the region covering residues 110 to 263 (F1B_110-263) is highly immunogenic and is the immunodominant regions of FnBPA. Moreover, vaccination with F1B_110-263 can generate partial protection against lethal challenge with two different S. aureus strains and reduced bacterial burdens against non-lethal challenge as well as that immunization with F1_30-500. This information will be important for further developing anti- S. aureus polyvalent subunit fusion vaccines.     

Fibronectin-binding protein A of Staphylococcus aureus has multiple, substituting, binding regions that mediate adherence to fibronectin and invasion of endothelial cells

Invasive Staphylococcus aureus infection frequently involves bacterial seeding from the bloodstream to other body tissues, a process necessarily involving interactions between circulating bacteria and vascular endothelial cells. Staphylococcus aureus fibronectin‐binding protein is central to the invasion of endothelium, fibronectin forming a bridge between bacterial fibronectin‐binding proteins and host cell receptors. To dissect further the mechanisms of invasion of endothelial cells by S. aureus, a series of truncated FnBPA proteins that lacked one or more of the A, B, C or D regions were expressed on the surface of S. aureus and tested in fibronectin adhesion, endothelial cell adhesion and invasion assays. We found that this protein has multiple, substituting, fibronectin‐binding regions, each capable of conferring both adherence to fibronectin and endothelial cells, and endothelial cell invasion. By expressing S. aureus FnBPA on the surface of the non‐invasive Gram‐positive organism Lactococcus lactis, we have found that no other bacterial factor is required for invasion. Furthermore, we have demonstrated that, as with other cell types, invasion of endothelial cells is mediated by integrin α₅β₁. These findings may be of relevance to the development of preventive measures against systemic infection, and bacterial spread in the bacteraemic patient.     

Dissociation rate constants of human fibronectin binding to fibronectin-binding proteins on living Staphylococcus aureus isolated from clinical patients

Staphylococcus aureus is part of the indigenous microbiota of humans. Sometimes, S. aureus bacteria enter the bloodstream, where they form infections on implanted cardiovascular devices. A critical, first step in such infections is a bond that forms between fibronectin-binding protein (FnBP) on S. aureus and host proteins, such as fibronectin (Fn), that coat the surface of implants in vivo. In this study, native FnBPs on living S. aureus were shown to form a mechanically strong conformational structure with Fn by atomic force microscopy. The tensile acuity of this bond was probed for 46 bloodstream isolates, each from a patient with a cardiovascular implant. By analyzing the force spectra with the worm-like chain model, we determined that the binding events were consistent with a multivalent, cluster bond consisting of ~10 or ~80 proteins in parallel. The dissociation rate constant (k(off), s(-1)) of each multibond complex was determined by measuring strength as a function of the loading rate, normalized by the number of bonds. The bond lifetime (1/k(off)) was two times longer for bloodstream isolates from patients with an infected device (1.79 or 69.47 s for the 10- or 80-bond clusters, respectively; n = 26 isolates) relative to those from patients with an uninfected device (0.96 or 34.02 s; n = 20 isolates). This distinction could not be explained by different amounts of FnBP, as confirmed by Western blots. Rather, amino acid polymorphisms within the Fn-binding repeats of FnBPA explain, at least partially, the statistically (p < 0.05) longer bond lifetime for isolates associated with an infected cardiovascular device.     

Staphylococcus aureus SigB activity promotes a strong fibronectin–bacterium interaction which may sustain host tissue colonization by small‐colony variants isolated from cystic fibrosis patients

Genes encoding cell‐surface proteins regulated by SigB are stably expressed in Staphylococcus aureus small‐colony variants (SCVs) isolated from cystic fibrosis (CF) patients. Our hypothesis is that CF‐isolated SCVs are locked into a colonization state by sustaining the expression of adhesins such as fibronectin‐binding proteins (FnBPs) throughout growth. Force spectroscopy was used to study the fibronectin–FnBPs interaction among strains varying for their SigB activity. The fibronectin–FnBPs interaction was described by a strength of 1000 ± 400 pN (pulling rate of 2 μm s^?1), an energetic barrier width of 0.6 ± 0.1 Å and an off‐rate below 2 × 10^?4 s^?1. A CF‐isolated SCV highly expressed fnbA throughout growth and showed a sustained capacity to bind fibronectin, whereas a prototypic strain showed a reduced frequency of fibronectin‐binding during the stationary growth phase when its fnbA gene was down‐regulated. Reduced expression of fnbA was observed in sigB mutants, which was associated with an overall decrease adhesion to fibronectin. These results suggest that the fibronectin–FnBPs interaction plays a role in the formation of a mechanically resistant adhesion of S. aureus to host tissues and supports the hypothesis that CF‐isolated SCVs are locked into a colonization state as a result of a sustained SigB activity.     

Purification and biochemical characterization of an organic solvent-tolerant and detergent-stable lipase from Staphylococcus capitis

A mesophilic bacterial culture, producing an extracellular alkaline lipase, was isolated from the gas-washing wastewaters generated from the Sfax phosphate plant of the Tunisian Chemical Group and identified as Staphylococcus capitis strain. The lipase, named S. capitis lipase (SCL), has been purified to homogeneity from the culture medium. The purified enzyme molecular weight was around 45 kDa. Specific activities about 3,900 and 500 U/mg were measured using tributyrin and olive oil emulsion as substrates, respectively at 37°C and pH 8.5. Interestingly, the SCL maintained more than 60% of its initial activity over a wide pH values ranging from 5 to 11 with a high stability between pH 9 and 11 after 1 hr of incubation at room temperature. The lipase activity was enhanced in the presence of 2 mM of Mg²⁺, Ca²⁺, and K⁺. SCL showed significant stability in the presence of detergents and organic solvents. Altogether, these features make the SCL useful for industrial applications. Besides, SCL was compatible with commercially available detergents, and its incorporation increases lipid degradation performances making it a potential candidate in detergent formulation.     

Fibronectin-binding protein acts as Staphylococcus aureus invasin via fibronectin bridging to integrin alpha5beta1

The ability of Staphylococcus aureus to invade mammalian cells may explain its capacity to colonize mucosa and to persist in tissues after bacteraemia. To date, the underlying molecular mechanisms of cellular invasion by S. aureus are unknown, despite its high prevalence and difficulties in treatment. Here, we show cellular invasion as a novel function for an S. aureus adhesin, previously implicated solely in attachment. S. aureus , but not S. epidermidis , invaded epithelial 293 cells in a temperature- and F-actin-dependent manner. Formaldehyde-fixed and live bacteria were equally invasive, suggesting that no active bacterial process was involved. All clinical S. aureus isolates analysed, but only a subset of laboratory strains, were invasive. Fibronectin-binding proteins (FnBPs) acted as S. aureus invasins, because: (i) FnBP deletion mutants of invasive laboratory strains lost invasiveness; (ii) expression of FnBPs in non-invasive strains conferred invasiveness; and (iii) the soluble isolated fibronectin-binding domain of FnBP (D1–D4) completely blocked invasion. Integrin α₅β₁ served as host cell receptor, which interacted with staphylococcal FnBPs through cellular or soluble fibronectin. FnBP-deficient mutants lost invasiveness for epithelial cells, endothelial cells and fibroblasts. Thus, fibronectin-dependent bridging between S. aureus FnBPs and host cell integrin α₅β₁ is a conserved mechanism for S. aureus invasion of human cells. This may prove useful in developing new therapeutic and vaccine strategies for S. aureus infections.     

In vivo immobilization of enzymatically active polypeptides on the cell surface of Staphylococcus carnosus

Many surface proteins of Gram-positive bacteria are covalently anchored to the cell wall by a ubiquitous mechanism, involving a specific, C-terminal sorting signal. To achieve cell-wall immobilization of a normally secreted enzyme in vivo, we constructed a hybrid protein consisting of Staphylococcus hyicus lipase and the C-terminal region of Staphylococcus aureus fibronectin binding protein B (FnBPB). This region comprised the authentic cell-wall-spanning region and cell-wall sorting signal of FnBPB. Expression of the hybrid protein in Staphylococcus carnosus resulted in efficient cell-wall anchoring of enzymatically active lipase. The cell-wall-immobilized lipase (approximately 10000 molecules per cell) retained more than 80% of the specific activity, compared to the C-terminally unmodified S. hyicus lipase secreted by S. carnosus cells. After releasing the hybrid protein from the cell wall by lysostaphin treatment, its specific activity was indistinguishable from that of the unmodified lipase. Thus, the C-terminal region of FnBPB per se was fully compatible with folding of the lipase to an active conformation. To study the influence of the distance between the cell-wall sorting signal and the C-terminus of the lipase on the activity of the immobilized lipase, the length of this spacer region was varied. Reduction of the spacer length gradually reduced the activity of the surface-immobilized lipase. On the other hand, elongation of this spacer did not stimulate the activity of the immobilized lipase, indicating that the spacer must exceed a critical length of approx. 90 amino acids to allow efficient folding of the enzyme, which probably can only be achieved outside the pep-tidoglycan web of the cell wall. When the lipase was replaced by another enzyme, the Escherichia coliβ-lactamase, the resulting hybrid was also efficiently anchored in an active conformation to the cell wall of S, carnosus. These results demonstrate that it is possible to immobilize normally soluble enzymes on the cell wall of S. carnosus - without radically altering their catalytic activity - by fusing them to a cell-wall-immobilization unit, consisting of a suitable cellwall-spanning region and a standard cell-wall sorting signal.