Biomedical subjects

F Götz

Publications and source records attributed to F Götz.

At least 73 records · Page 4Linked to original sources

PubMed1997

Studies on prolysostaphin processing and characterization of the lysostaphin immunity factor (Lif) of Staphylococcus simulans biovar staphylolyticus.

Lysostaphin is an extracellular glycylglycine endopeptidase produced by Staphylococcus simulans biovar staphylolyticus ATCC1362 that lyses staphylococcal cells by hydrolysing the polyglycine interpeptide bridges of the peptidoglycan. Renewed analysis of the sequence of the lysostaphin gene (lss), and the sequencing of the amino-terminus of purified prolysostaphin and of mature lysostaphin revealed that lysostaphin is organized as a preproprotein of 493 amino acids (aa), with a signal peptide consisting of 36 aa, a propeptide of 211 aa from which 195 aa are organized in 15 tandem repeats of 13 aa length, and a mature protein of 246 aa. Prolysostaphin is processed in the culture supernatant of S. simulans biovar staphylolyticus by an extracellular cysteine protease. Although prolysostaphin was staphylolytically active, the mature lysostaphin was about 4.5-fold more active. The controlled expression in Staphylococcus carnosus of lss and lss with deletions in the prepropeptide region indicated that the tandem repeats of the propeptide are not necessary for protein export or activation of Lss, but keep Lss in a less active state. Intracellularly expressed pro- and mature lysostaphin exert staphylolytic activity in cell-free extracts, but do not affect growth of the corresponding clones. We characterized a lysostaphin immunity factor gene (lif) which is located in the opposite direction to lss. The expression of lif in S. carnosus led to an increase in the serine/glycine ratio of the interpeptide bridges of peptidoglycan from 2 to 35%, suggesting that lysostaphin immunity depends on serine incorporation into the interpeptide bridge. If, in addition to lif, lss is co-expressed the serine/glycine ratio is further increased to 58%, suggesting that Lss selects for optimal serine incorporation. Lif shows similarity to FemA and FemB proteins, which are involved in the biosynthesis of the glycine interpeptide bridge of staphylococcal peptidoglycan. In contrast to that of Lif, the production of FemA and FemB in S. carnosus does not cause lysostaphin immunity. The putative tRNASer gene located downstream of lss had no recognizable influence on lysostaphin immunity. lss and lif are flanked by insertion sequences, suggesting that S. simulans biovar staphylolyticus received lif and lss by horizontal gene transfer.

Amino Acid Sequence↗

PubMed1997

Evidence for autolysin-mediated primary attachment of Staphylococcus epidermidis to a polystyrene surface.

Biofilm formation on a polymer surface which involves initial attachment and accumulation in multilayered cell clusters (intercellular adhesion) is proposed to be the major pathogenicity factor in Staphylococcus epidermidis foreign-body-associated infections. We have characterized two distinct classes of biofilm-negative Tn917 mutants in S. epidermidis affected in initial attachment (class A) or intercellular adhesion (class B). mut1 (class A mutant) lacks five surface-associated proteins with molecular masses of 120, 60, 52, 45 and 38 kDa and could be complemented by transformation with a 16.4 kb wild-type DNA fragment. The complemented mutant was able to attach to a polystyrene surface, to form a biofilm, and produced all of the proteins missing from mut1. Subcloning experiments revealed that the 60 kDa protein is sufficient for initial attachment. Immunofluorescence microscopy using an antiserum raised against the 60 kDa protein showed that this protein is located at the cell surface. DNA-sequence analysis of the complementing region revealed a single open reading frame which consists of 4005 nucleotides and encodes a deduced protein of 1335 amino acids with a predicted molecular mass of 148kDa. The amino acid sequence exhibits a high similarity (61% identical amino acids) to the atl gene product of Staphylococcus aureus, which represents the major autolysin; therefore the open reading frame was designated atlE. By analogy with the S. aureus autolysin, AtlE is composed of two bacteriolytically active domains, a 60 kDa amidase and a 52 kDa glucosaminidase domain, generated by proteolytic processing. The 120 kDa protein missing from mut1 presumably represents the unprocessed amidase and glucosaminidase domain after proteolytic cleavage of the signal- and propeptide. The 45 and 38kDa proteins are probably the degradation products of the 60 and 52 kDa proteins, respectively. Additionally, AtlE was found to exhibit vitronectin-binding activity, indicating that AtlE plays a role in binding of the cells not only to a naked polystyrene surface during early stages of adherence, but also to plasma protein-coated polymer surfaces during later stages of adherence. Our findings provide evidence for a new function of an autolysin (AtlE) in mediating the attachment of bacterial cells to a polymer surface, representing the prerequisite for biofilm formation.

Adhesins, Bacterial↗

PubMed1997

Genetic and biochemical characterization of a new extracellular lipase from Streptomyces cinnamomeus.

Streptomyces cinnamomeus Tü89 secretes a 30-kDa esterase and a 50-kDa lipase. The lipase-encoding gene, lipA, was cloned from genomic DNA into Streptomyces lividans TK23 with plasmid vector pIJ702. Two lipase-positive clones were identified; each recombinant plasmid had a 5.2-kb MboI insert that contained the complete lipA gene. The two plasmids differed in the orientation of the insert and the degree of lipolytic activity produced. The lipA gene was sequenced; lipA encodes a proprotein of 275 amino acids (29,213 Da) with a pI of 5.35. The LipA signal peptide is 30 amino acids long, and the mature lipase sequence is 245 amino acids long (26.2 kDa) and contains six cysteine residues. The conserved catalytic serine residue of LipA is in position 125. Sequence similarity of the mature lipases (29% identity, 60% similarity) was observed mainly in the N-terminal 104 amino acids with the group II Pseudomonas lipases; no similarity to the two Streptomyces lipase sequences was found. lipA was also expressed in Escherichia coli under the control of lacZ promoter. In the presence of the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG), growth of the E. coli clone was severely affected, and the cells lysed in liquid medium. Lipase activity in the E. coli clone was found mainly in the pellet fraction. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, three additional protein bands of 50, 29, and 27 kDa were visible. The 27-kDa protein showed lipolytic activity and represents the mature lipase; the 29- and 50-kDa forms showed no activity and very probably represent the unprocessed form and a dimeric misfolded form, respectively. For higher expression of lipA in S. lividans, the gene was cloned next to the strong aphII promoter. In contrast to the lipA-expressing E. coli clone, S. cinnamomeus and the corresponding S. lividans clone secreted only an active protein of 50 kDa. The lipase showed highest activity with C6 and C18 triglycerides; no activity was observed with phospholipids, Tween 20, or p-nitrophenylesters. Upstream of lipA and in the same orientation, an open reading frame, orfA, is found whose deduced protein sequence (519 amino acids) shows similarity to various membrane-localized transporters. Downstream of lipA and in the opposite orientation, an open reading frame, orfB (encoding a 199-amino-acid protein) is found, which shows no conspicuous sequence similarity to known proteins, other than an NAD and flavin adenine dinucleotide binding-site sequence.

Amino Acid Sequence↗

PubMed1997

Detection of the intercellular adhesion gene cluster (ica) and phase variation in Staphylococcus epidermidis blood culture strains and mucosal isolates.

Staphylococcus epidermidis is a common cause of catheter-associated infections and septicemia in immunocompromised patients. To answer the question whether S. epidermidis skin isolates differ from isolates causing septicemic diseases, 51 strains obtained from blood cultures, 1 strain from shunt-associated meningitis, and 36 saprophytic isolates were characterized. The study demonstrates that most of the blood culture strains formed a multilayered biofilm on plastic material, whereas skin and mucosal isolates did not. Moreover, biofilm-producing strains were found to generate large bacterial autoaggregates in liquid culture. Autoaggregation and biofilm formation on polymer surfaces was associated with the presence of a DNA sequence encoding an intercellular adhesion gene cluster (ica) that mediates the production of a polysaccharide intercellular adhesin. The presence of the intercellular adhesion genes in blood culture isolates was also found to be correlated with the exhibition of black colonies on Congo red agar, whereas the adhesin-negative strains formed red colonies. Upon subcultivation on Congo red agar, the black colony forms of the blood culture strains exhibited red colony variants which were biofilm and autoaggregation negative and occurred at a frequency of 10(-5). The DNA analysis of these S. epidermidis variants by pulsed-field gel electrophoresis and Southern hybridization with an ica-specific gene probe revealed no detectable difference between the black and red colony types. Moreover, after repeated passage, the phenotype of the parent strain could be restored. Therefore, these colony forms were regarded as phase variants. This phenotypic change was observed exclusively in adhesin-positive clinical isolates and not in adhesin-negative saprophytic strains of S. epidermidis.

Bacteremia↗

PubMed1997

A site-directed Staphylococcus aureus hemB mutant is a small-colony variant which persists intracellularly.

Although small-colony variants (SCVs) of Staphylococcus aureus have been recognized for many years, this phenotype has only recently been related to persistent and recurrent infections. Clinical S. aureus SCVs are frequently auxotrophic for menadione or hemin, two compounds involved in the biosynthesis of the electron transport chain elements menaquinone and cytochromes, respectively. While this observation as well as other biochemical characteristics of SCVs suggests a link between electron-transport-defective strains and persistent infections, the strains examined thus far have been genetically undefined SCVs. Therefore, we generated a stable mutant in electron transport by interrupting one of the hemin biosynthetic genes, hemB, in S. aureus by inserting an ermB cassette into hemB. We isolated a hemB mutant, due to homologous recombination, by growth at a nonpermissive temperature and selection for erythromycin resistance. This mutant showed typical characteristics of clinical SCVs, such as slow growth, decreased pigment formation, low coagulase activity, reduced hemolytic activity, and resistance to aminoglycosides. Additionally, the mutant was able to persist within cultured endothelial cells due to decreased alpha-toxin production. Northern and Western blot analyses showed that expression of alpha-toxin and that of protein A were markedly reduced, at both the mRNA and the protein level. The SCV phenotype of the hemB mutant was reversed by growth with hemin or by complementation with intact hemB. Hence, a defect in the electron transport system allows S. aureus SCVs to resist aminoglycosides and persist intracellularly.

Aminoglycosides↗

PubMed1996

The lipase from Staphylococcus aureus. Expression in Escherichia coli, large-scale purification and comparison of substrate specificity to Staphylococcus hyicus lipase.

The genes coding for the mature part of the lipases from Staphylococcus aureus NCTC8530 and Staphylococcus hyicus have been cloned and overexpressed in Escherichia coli as fusion proteins with an N-terminal hexa-histidine tag. The enzymes accumulated in the cytoplasm and were purified using sequential precipitation with protamine sulphate and ammonium sulphate, followed by metal-affinity and hydroxyapatite chromatography. The yield of pure lipase was 4.5 mg/g wet cells for S. aureus lipase and 13 mg/g for S. hyicus lipase. The purified enzymes need calcium for activity, albeit with different affinities, and a low residual activity was found in the absence of calcium. In contrast to S. hyicus lipase, not only strontium but also barium can replace calcium with full retention of activity of S. aureus lipase. Whereas S. hyicus lipase is optimally active at pH 8.5, the optimum pH for enzymatic activity for S. aureus lipase was found to be pH 6.5. The S. aureus lipase has a narrow substrate specificity: short-chain triacylglycerols and acyl esters of both p-nitrophenol and umbelliferone are readily degraded, whereas medium- and long-chain lipids, as well as phospholipids, are poor substrates. In contrast, S. hyicus lipase prefers phospholipids as substrate and hydrolyses neutral lipids irrespective of their chain length. The results are discussed in view of the large sequence similarity between both lipases.

Esters↗

PubMed1996

Sequence and analysis of the replication region of the Staphylococcus xylosus plasmid pSX267.

The region of the 29.5-kb plasmid pSX267 from Staphylococcus xylosus DSM 20267 that is required for autonomous replication in staphylococci was isolated on a 1.8-kb DNA fragment. The sequence analysis of the fragment yielded two open reading frames, repA and orf2, encoding proteins of 37.2 and 13.2 kDa, respectively. The deduced amino acid sequence of repA showed similarity to the replication initiator protein of plasmid pAD1 from Enterococcus faecalis, to two proteins of unknown function encoded by the E. faecalis plasmid pCF10 and the lactobacillus helveticus plasmid pLJ1, and surprisingly to the mouse interferon-response element binding factor 1. The repA gene of pSX267 is indispensable for replication suggesting that it encodes the replication initiator protein of pSX267. Introduction of a frameshift mutation into repA or deletion of 26 codons at its 3'-end resulted in nonreplicative plasmids. Removal of sequences required for repA expression also abolished replication. Complementation experiments with repA in trans identified the ori of pSX267 within the repA coding region. The second orf, which is located downstream of repA, could be deleted without affecting plasmid replication. It seems to be a nonfunctional remainder of a rolling circle plasmid of the pC194/pUB110 family, since its deduced amino acid sequence resembles the pC194/pUB110-type replication proteins. The minimal replicon of pSX267 defined so far comprises 1255 bp. Besides repA, no other plasmid-encoded gene is required to mediate replication in staphylococci.

Amino Acid Sequence↗

PubMed1996

[Extracorporeal shockwave therapy in childhood].

In 27 children aged 4-17 years extracorporeal shock wave lithotripsy (ESWL) was performed for renal and ureteral calculi between January 1 1993 and December 31 1994 at the University Medical School of Pécs. A total of 32 calculi was treated requiring 34 ESWL sessions. All treatments were done with the standard Dornier Compact Mobile Lithotripter device. The mean size of the stones was 10.65 mm and the number of shock waves averaged 2607. No serious intraoperative or postoperative complications were encountered. No auxiliary procedures were performed. The stonefree rate was 27/32 stones (84.4%) 3 months after treatment. 5 patient has residual stone fragments. Lithotripsy using Dornier Compact Mobile device in our series of pediatric patients was found safe and effective.

Adolescent↗

PubMed1996

Inducible production and cellular location of the epidermin biosynthetic enzyme EpiB using an improved staphylococcal expression system.

The antimicrobial peptide epidermin is distinguished by thioether amino acids such as meso-lanthionine, 3-methyl-lanthionine, and 2-aminovinylcysteine. The enzyme EpiB, encoded on a plasmid of the producing strain Staphylococcus epidermidis Tü3298, is very likely involved in the formation of these unusual amino acids. In order to obtain high-level production of EpiB, an improved staphylococcal expression vector based on the xylose-inducible xylA promoter of Staphylococcus xylosus was constructed. As shown by the expression of a lipase reporter gene, the new plasmid pTX15 mediated a considerably higher expression level after induction and a lower background expression level in the uninduced state than the previously described vector pCX15. The epiB gene was inserted in pTX15 and expressed in Staphylococcus carnosus. The EpiB protein was detected both in the cytoplasmic and the membrane fraction and was partially purified in three steps.

Anti-Bacterial Agents↗

PubMed1996

Central pontine myelinolysis: clinical presentation and radiologic findings.

Central pontine myelinolysis (CPM) is a neurologic disorder once thought to be uniformly fatal. With the introduction of CT and MRI there was an increasing number of reports on nonfatal cases of CPM. Nearly all reports on nonfatal cases describe severe clinical syndromes with tetraparesis, bulbar palsy, and coma. We reviewed nine patients with CPM and compared the size of the pontine lesion on MRI and CT with the severity of clinical presentation. Clinical presentation of CPM was highly variable: The symptoms ranged from severe neurologic disorders to mild neurologic disturbances only. Two of nine patients died from CPM. The size of the pontine lesion did not correlate with the severity of the neurologic illness or the final outcome. Mild forms of CPM might be difficult to diagnose clinically. This applies even more for patients with underlying diseases such as Wernicke's encephalopathy, which in itself might cause a clinical picture similar to that of CPM. Central Pontine Myelinolysis is a major differential diagnosis in acute neurologic deterioration indicating pontine damage. Magnetic resonance imaging is the decisive diagnostic tool for CPM.

Adult↗

PubMed1996

The biosynthesis of the lantibiotics epidermin, gallidermin, Pep5 and epilancin K7.

Lantibiotics are antibiotic peptides that contain the rare thioether amino acids lanthionine and/or methyllanthionine. Epidermin, Pep5 and epilancin K7 are produced by Staphylococcus epidermidis whereas gallidermin (6L-epidermin) was isolated from the closely related species Staphylococcus gallinarum. The biosynthesis of all four lantibiotics proceeds from structural genes which code for prepeptides that are enzymatically modified to give the mature peptides. The genes involved in biosynthesis, processing, export etc. are found in gene clusters adjacent to the structural genes and code for transporters, immunity functions, regulatory proteins and the modification enzymes LanB, LanC and LanD, which catalyze the biosynthesis of the rare amino acids. LanB and LanC are responsible for the dehydration of the serine and threonine residues to give dehydroalanine and dehydrobutyrine and subsequent addition of cysteine SH-groups to the dehydro amino acids which results in the thioether rings. EpiD, the only LanD enzyme known so far, catalyzes the oxidative decarboxylation of the C-terminal cysteine of epidermin which gives the C-terminal S-aminovinylcysteine after addition of a dehydroalanine residue.

ATP-Binding Cassette Transporters↗

PubMed1996

Post-translational modifications of lantibiotics.

Several newly reported post-translational modification reactions are involved in lantibiotic biosynthesis. A short overview of the present knowledge on the post-translational modifications and on the enzymes involved in lantibiotic biosynthesis is given. The oxidative decarboxylation of the epidermin precursor peptide EpiA is described in detail. The FMN-containing oxidoreductase EpiD is involved in the formation of the C-terminal S-[(Z)-2-aminovinyl]-D-cysteine residue of epidermin: under reducing conditions the side chain of the C-terminal cysteine residue of EpiA is converted to an enethiol. EpiD has no absolute substrate specificity and can be used for modification of peptides having the C-terminal consensus motif [V/I/L/(M)/F/Y/W]-[A/S/V/T/C/(I/L)]-C.

Amino Acid Sequence↗

PubMed1996

Protein engineering of lantibiotics.

Whereas protein engineering of enzymes and structural proteins nowadays is an established research tool for studying structure-function relationships of polypeptides and for improving their properties, the engineering of posttranslationally modified peptides, such as the lantibiotics, is just coming of age. The engineering of lantibiotics is less straightforward than that of unmodified proteins, since expression systems should be developed not only for the structural genes but also for the genes encoding the biosynthetic enzymes, immunity protein and regulatory proteins. Moreover, correct posttranslational modification of specific residues could in many cases be a prerequisite for production and secretion of the active lantibiotic, which limits the number of successful mutations one can apply. This paper describes the development of expression systems for the structural lantibiotic genes for nisin A, nisin Z, gallidermin, epidermin and Pep5, and gives examples of recently produced site-directed mutants of these lantibiotics. Characterization of the mutants yielded valuable information on biosynthetic requirements for production. Moreover, regions in the lantibiotics were identified that are of crucial importance for antimicrobial activity. Eventually, this knowledge will lead to the rational design of lantibiotics optimally suited for fighting specific undesirable microorganisms. The mutants are of additional value for studies directed towards the elucidation of the mode of action of lantibiotics.

Amino Acid Sequence↗

PubMed1996

Cloning, sequencing, and characterization of a gene (narT) encoding a transport protein involved in dissimilatory nitrate reduction in Staphylococcus carnosus.

A Tn917 mutant of Staphylococcus carnosus TM300, nrIII, was isolated and characterized. Mutant nrIII did not take up nitrate or accumulate nitrite when grown in B-medium supplemented with up to 10 mM nitrate under anoxic conditions; however, it displayed wild-type levels of benzyl Delta viologen-linked nitrate reductase activity. Cultivated in B-medium with nitrate under oxic conditions, mutant nrIII accumulated fivefold less nitrite than the wild-type. The mutation in S. carnosus nrIII could be complemented with a 2-kb chromosomal EcoRI-HpaI fragment from the wild-type. The gene affected by transposon insertion in mutant nrIII was cloned and sequenced. Analysis of the deduced amino acid sequence revealed that this gene, designated narT, encodes a highly hydrophobic 42-kDa transmembrane protein of 388 amino acids and shows similarities to transport proteins that play a role in nitrate import or nitrite export. The inability of nrIII to take up nitrate under anoxic conditions and its ability to take up and accumulate nitrite in the presence of benzyl viologen, a nitrate ionophore, under the same conditions suggest that NarT represents a transport protein required for nitrate uptake under anoxic conditions in S. carnosus.

Amino Acid Sequence↗

PubMed1996

Genomic variability of Staphylococcus aureus and the other coagulase-positive Staphylococcus species estimated by macrorestriction analysis using pulsed-field gel electrophoresis.

The genomic DNAs of 95 culture collection and hospital Staphylococcus aureus subsp. aureus strains of various origins, as well as the genomic DNAs of other coagulase-positive Staphylococcus species, were cleaved with restriction endonuclease SmaI and subjected to pulsed-field gel electrophoresis. The levels of similarity of the SmaI restriction patterns of the S. aureus subsp. aureus strains varied from 30 to 100%, which is considered characteristic of this species; thus, these organisms belonged to the same species restriction group. Within this range of similarity values 13 S. aureus intraspecies restriction groups were identified, and each group consisted of strains whose levels of similarity ranged from 65 to 100%. S. aureus subsp. aureus CCM 885T (T = type strain) belonged to the major intraspecies restriction group that comprised 39% of the S. aureus strains which we studied. The strains of the other coagulase-positive staphylococci, including Staphylococcus aureus subsp. anaerobius, Staphylococcus hyicus, Staphylococcus intermedius, Staphylococcus delphini, and Staphylococcus schleiferi subsp. coagulans, clustered with their type strains in separate restriction groups. S. aureus subsp. aureus exhibited almost no similarity to these species. We found 44-kb SmaI fragments in all of the S. aureus subsp. aureus and S. aureus subsp. anaerobius strains studied, and these fragments are considered characteristic of the species S. aureus. The high level of homology of these fragments was confirmed by the results of DNA hybridization experiments in which we used representatives of individual intraspecies restriction groups. Of the other staphylococci studied, only Staphylococcus epidermidis and one strain of S. hyicus contained these fragments. However, the levels of homology between these fragments and the fragments of S. aureus were found to be very low.

Coagulase↗

PubMed1996

Molecular basis of intercellular adhesion in the biofilm-forming Staphylococcus epidermidis.

The Staphylococcus epidermidis genes icaABC are involved in the synthesis of the polysaccharide intercellular adhesin (PIA), which is located mainly on the cell surface, as shown by immunofluorescence studies with PIA-specific antiserum. PIA was shown to be a linear beta-1,6-linked glucosaminoglycan composed of at least 130 2-deoxy-2-amino-D-glucopyranosyl residues of which 80-85% are N-acetylated, the rest being non-N-acetylated and positively charged. A transposon insertion in the icaABC gene cluster (ica, intercellular adhesion) led to the loss of several traits, such as the ability to form a biofilm on a polystyrene surface, cell aggregation, and PIA production. The mutant could be complemented by transformation with the icaABC-carrying plasmid pCN27. Transfer of pCN27 into the heterologous host Staphylococcus carnosus led to the formation of large cell aggregates, the formation of a biofilm on a glass surface, and PIA expression. The nucleotide sequence of icaABC suggests that the three genes are organized in an operon and that they are co-transcribed from the mapped icaA promoter. IcaA contains four potential transmembrane helices, indicative of a membrane location. The deduced IcaA sequence shows similarity to those of polysaccharide-polymerizing enzymes, the most pronounced being with a Rhizobium meliloti N-acetylglucosaminyltransferase involved in lipo-chitin biosynthesis (22.5% overall identity and 37.4% overall similarity). This similarity suggests that IcaA has N-acetylglucosaminyltransferase activity in the formation of the beta-1, 6-linked N-acetyl-D-glucosaminyl polymer. IcaB is secreted into the medium and contains a typical signal peptide. IcaC is hydrophobic and contains six predicted transmembrane helices distributed over its entire length, typical for an integral membrane protein. Neither IcaB nor IcaC shares similarity with known proteins, and their function is unknown. Inactivation of icaA, icaB, or icaC in pCN27 led to the complete loss of the intercellular adhesion phenotype in S. carnosus, suggesting that all three genes are involved in intercellular adhesion, PIA expression, and translocation.

Bacterial Adhesion↗

PubMed1996

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 10,000 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 peptidoglycan web of the cell wall. When the lipase was replaced by another enzyme, the Escherichia coli beta-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 normality 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 cell-wall-spanning region and a standard cell-wall sorting signal.

Adhesins, Bacterial↗

PubMed1996

Characterization of Tn917 insertion mutants of Staphylococcus epidermidis affected in biofilm formation.

Biofilm formation is thought to result from the concerted action of primary attachment to a specific surface and accumulation in multilayered cell clusters. Here we describe the isolation and characterization of transposon (Tn917) mutants of Staphylococcus epidermidis O-47 which were biofilm negative in the polystyrene microtiter plate assay. Among 5,000 Tn917 insertion mutants, 4 biofilm-negative mutants were isolated. Each mutant carried one copy of Tn917. The mutants were divided into two phenotypic classes: class A (mut1 and mut1a) and class B (mut2 and mut2a). Mutants of phenotypic class A lacked four cell surface proteins, were less hydrophobic, and were affected in primary attachment to polystyrene, but were still able to form multilayered cell clusters. They were able to form a biofilm on a glass surface, a trait that was even more pronounced than in the wild-type stain O-47. Loss of several surface proteins might have led to the reduced surface hydrophilic structures, thus favoring primary attachment to a glass surface and leading to subsequent biofilm formation. Mutants of phenotype class B were able to attach to polystyrene but were unable to form multilayered cell clusters, had unchanged cell surface proteins and hydrophobicity, and were unable to form a biofilm on a glass surface, mut1 and mut2 could be complemented by wild-type DNA fragments containing the Tn917 insertion sites of mut1 and mut2, respectively. The complemented biofilm-positive clone mut1 (pRC20) produced a 60-kDa protein which is postulated to function as the adhesin for binding to plastic. The traits of binding to polystyrene and the ability to form multilayered cell clusters are phenotypically and genetically distinct.

Animals↗