Search

In den vergangenen 20 Jahren wurden peptidbasierte Antibiotika wie Vancomycin, Teicoplanin oder Daptomycin als Reserveantibiotika betrachtet. In den letzten Jahren erschienen jedoch vermehrt Berichte uber Vancomycin-resistente Krankheitserreger, was die Entwicklung neuartiger Leitstrukturen fur neue wirksame Antibiotika motivierte. Hier wird uber die Totalsynthese einer definierten endo-Typ-B-PPAP-Bibliothek und deren antibiotische Aktivitat gegen multiresistente Staphylococcus-aureus- und diverse Vancomycin-resistente Enterococcus-Stamme berichtet. Es wurden vier neue Strukturen identifiziert, die zeigen, dass das PPAP-Grundgerust eine neue nicht-peptidbasierte Leitstruktur in der Antibiotika-Forschung werden konnte, die sich durch die Kombination von hoher Aktivitat und geringer Zytotoxizitat auszeichnet.

Excretion of cytoplasmic proteins (ECP) is a common physiological feature in bacteria and eukaryotes. However, how these proteins without a typical signal peptide are excreted in bacteria is poorly understood. We studied the excretion pattern of cytoplasmic proteins using two glycolytic model enzymes, aldolase and enolase, and show that their excretion takes place mainly during the exponential growth phase in Staphylococcus aureus very similar to that of Sbi, an IgG-binding protein, which is secreted via the Sec-pathway. The amount of excreted enolase is substantial and is comparable with that of Sbi. For localization of the exit site, we fused aldolase and enolase with the peptidoglycan-binding motif, LysM, to trap the enzymes at the cell wall. With both immune fluorescence labeling and immunogold localization on electron microscopic thin sections aldolase and enolase were found apart from the cytoplasmic area particularly in the cross wall and at the septal cleft of dividing cells, whereas the non-excreted Ndh2, a soluble NADH:quinone oxidoreductase, is only seen attached to the inner side of the cytoplasmic membrane. The selectivity, the timing and the localization suggest that ECP is not a result of unspecific cell lysis but is mediated by an as yet unknown mechanism.

Summary Intimin and Invasin are prototypical inverse (Type Ve) autotransporters and important virulence factors of enteropathogenic Escherichia coli and Yersinia spp. respectively. In addition to a C-terminal extracellular domain and a β-barrel transmembrane domain, both proteins also contain a short N-terminal periplasmic domain that, in Intimin, includes a lysin motif (LysM), which is thought to mediate binding to peptidoglycan. We show that the periplasmic domain of Intimin does bind to peptidoglycan both in vitro and in vivo, but only under acidic conditions. We were able to determine a dissociation constant of 0.8 μM for this interaction, whereas the Invasin periplasmic domain, which lacks a LysM, bound only weakly in vitro and failed to bind peptidoglycan in vivo. We present the solution structure of the Intimin LysM, which has an additional α-helix conserved within inverse autotransporter LysMs but lacking in others. In contrast to previous reports, we demonstrate that the periplasmic domain of Intimin mediates dimerisation. We further show that dimerisation and peptidoglycan binding are general features of LysM-containing inverse autotransporters. Peptidoglycan binding by the periplasmic domain in the infection process may aid in resisting mechanical and chemical stress during transit through the gastrointestinal tract.

In Staphylococci maximal induction of nitrate reductase (narGHJI genes) requires anaerobic conditions, the presence of nitrate, and the NreABC regulatory system. Aerobic regulation is effected by the NreB/NreC two-component system. The role of the nitrate receptor NreA in nitrate induction and its relation to aerobic regulation was analysed in Staphylococcus carnosus. Nitrate induction of a narG-lip reporter gene required presence of NreB/NreC. When nreA was deleted, nitrate was no longer required for maximal induction, suggesting that NreA is a nitrate regulated inhibitor of NreB/NreC. In vitro, NreA and mutant NreA(Y95A) decreased NreB phosphorylation in part or completely, which was due to the inhibition of the autophosphorylating activity rather than an increase of phosphatase activity. Inhibition of phosphorylation was relieved completely when the nitrate-bound NreA was used instead of the nitrate-free form. In the bacterial two-hybrid BACTH system and HPINE interaction assays, NreA interacted with NreB, but not with NreC, and the interaction was diminished by nitrate. In summary, NreA interacts with NreB and controls its phosphorylation level in a nitrate dependent manner. In this way nitrate and NreA modulate the function of the oxygen sensor NreB, resulting in nitrate/oxygen co-sensing by an NreA/NreB sensor unit as part of the NreABC-system.

Potent melanin production enhancement of human tyrosinase plasmid (pAH7/Tyr, P) in mouse melanoma cells (B16F10) by Tat peptide (T) and an entrapment in elastic cationic niosomes (E) was described. The E composed of Tween 61/cholesterol/dodecyl dimethyl ammonium bromide at 1:1:0.5 molar ratio was prepared by freeze-dried emptying liposomes method. PE at P/E ratio of 1:160 w/w and TPE at T/P/E ratio of 0.125:1:160, 0.25:1:160, and 0.5:1:160 w/w/w were prepared. The final concentration of the plasmid in the study was 4 ng/μL. By sulforhodamine B assay, PE and TPE complexes showed slight or no cytotoxic effect. The cells transfected with TPE (0.5:1:160) exhibited the highest enhancement of tyrosinase enzyme activity of 11.82-, 7.67-, 5.07-, and 6.29-folds of control, P, PE, and TP (0.5:1) and melanin production of 13.03-, 8.46-, 5.36-, and 6.58-folds of control, P, PE, and TP (0.5:1), respectively. The elastic cationic niosomes demonstrated an increase in thermal stability of P at 4 ± 2, 25 ± 2, and 45 ± 2 °C. The vesicular size and the zeta potential values of PE and TPE complexes were slightly increased but still in the range of stable dispersion (out of ±30 mV). These results indicated the high potential application of the TPE complexes for further investigation for vitiligo gene therapy.

Scope: Thymol is a component of several plants with antimicrobial activity. Little is known about the effects of thymol on immune cells of the host. This study addressed the effects of thymol on dendritic cells (DCs), regulators of innate and adaptive immunity. Methods and results: Immunohistochemistry, Western blotting and fluorescence-activated cell sorting analysis were performed in bone marrow-derived DCs either from wild-type mice or from mice lacking acid sphingomyelinase (ASM−/−) treated and untreated for 24 h with thymol (2–100 μg/mL). Thymol treatment resulted in activation of ASM, stimulation of ceramide formation, downregulation of anti-apoptotic Bcl-2 and Bcl-xL proteins, activation of caspase 3 and caspase 8, DNA fragmentation as well as cell membrane scrambling. The effects were dependent on the presence of ASM and were lacking in ASM−/− mice or in wild-type DCs treated with sphingomyelinase inhibitor amitriptyline. Conclusion: Thymol triggers suicidal DC death, an effect mediated by and requiring activation of ASM.

Innate immune sensing of Staphylococcus aureus unravels basic mechanisms leading to either effective antibacterial immune responses or harmful inflammation. The nature and properties of S. aureus-derived pathogen-associated molecular pattern (PAMPs) are still not completely understood. We investigated the innate immune sensing of peptidoglycan (PGN) structures and subsequent immune consequences. Macromolecular PGN (PGN(polymer)) preparations activated NF-κB through human Toll-like receptors 2 (TLR2), as shown by luciferase reporter assays, and induced murine dendritic cell (DC) maturation and cytokine production. In contrast, PGN(polymer) from lgt-mutant S. aureus failed to stimulate human TLR2, demonstrating that lipoproteins within the macromolecular structures of PGN(polymer), but not PGN itself, activate TLR2. Thus, HPLC-purified monomeric PGN (PGN(monomer)) structures were investigated. Strikingly, PGN(monomer) completely lacked NF-κB activation, lacked TLR2 activity, and failed to functionally activate murine DCs. However, PGN(monomer) in concert with various TLR ligands most effectively stimulated DCs to up-regulate IL-12p70 and IL-23 by ≥3- to 5-fold. Consequently, DCs coactivated by PGN(monomer) markedly up-regulated Th1 and Th17 while suppressing Th2 cell priming. Notably, PGN(monomer) failed to coactivate NOD2(-/-) DCs. This demonstrates that PGN(monomer) is a natural ligand of NOD2, which was previously only demonstrated for synthetic compounds like muramyl dipeptide. Interestingly, murine DCs lacking TLR2 remained mute in response to the combinative immune sensing of S. aureus-derived PAMPs, including PGN(monomer), providing for the first time an explanation of why S. aureus can colonize the nasal mucosa in the absence of inflammation. This is very likely based on the lack of TLR2 expression in mucosal epithelial cells under normal conditions, which determines the unresponsiveness to S. aureus PAMPs.

Microtechnology enables the design and fabrication of cost-effective novel platforms for bio diagnostics. By employing plasma technologies for the specific adjustment of surface characteristics, custom-designed platforms are feasible. Special test chips have been developed for a fast and reliable adjustment of product properties. The results of this development and the first products are presented in this article.

Die Mikrotechnologie ermoglicht die Auslegung und Herstellung neuartiger Plattformen, die kostengunstig im Bereich der Biodiagnostik eingesetzt werden. Durch den Einsatz der Plasmatechnologie zur gezielten Einstellung der Oberflacheneigenschaften werden die Plattformen anwendungsspezifisch modifiziert. Um die Produkteigenschaften schnell und zuverlassig einstellen und prufen zu konnen, wurden spezielle Testchips entwickelt. Die Entwicklungsergebnisse und erste Produkte werden in diesem Artikel vorgestellt. Microtechnology enables the dimensioning and fabrication of cost-effective novel platforms for bio diagnostics. By the employment of plasma technologies for specific adjustment of surface characteristics, custom-designed platforms are feasible. For a fast and reliable adjustment of product properties, special test chips have been developed. The conclusions for this development and first products are presented in this article.

Staphylococcus species belong to one of the few bacterial genera that are completely lysozyme resistant, which greatly contributes to their persistence and success in colonizing the skin and mucosal areas of humans and animals. In an attempt to discover the cause of lysozyme resistance, we identified a gene, oatA, in Staphylococcus aureus. The corresponding oatA deletion mutant had an increased sensitivity to lysozyme. HPLC and electrospray ionization tandem mass spectrometry analyses of the cell wall revealed that the muramic acid of peptidoglycan of the wild-type strain was O-acetylated at C6-OH, whereas the muramic acid of the oatA mutant lacked this modification. The complemented oatA mutant was lysozyme resistant. We identified the first bacterial peptidoglycan-specific O-acetyltransferase in S. aureus and showed that OatA, an integral membrane protein, is the molecular basis for the high lysozyme resistance in staphylococci.

The genus Staphylococcus consists of an ever-expanding group of gram-positive cocci that have evolved to colonize specific environmental niches, including preferential human tissues and diverse mammalian species. The study of staphylococcal biofilms in vitro has the advantage that in vivo foreign body-associated infections are often colonized by what seems to be single strains or species. The capsular polysaccharide adhesin (PS/A) was isolated from biofilm-forming S. epidermidis and shown to mediate binding of the bacteria to silastic catheter surfaces. More recently identified members of this family of autolysin/adhesins include the homologous Staphylococcus saprophyticus autolysin, Aas, which was shown to bind fibronectin and sheep erythrocytes and to exhibit bacteriolytic properties. The presence of an icaA homolog has also been inferred in S. auricularis, S. capitis, S. intermedius, S. lugdunensis, S. piscifermentans, and S. pasteuri based on DNA cross-hybridization experiments. Some of the environmental stimuli that induce biofilm formation in vitro that have been identified so far are described in this chapter. Examination of temporal and spatial gene expression patterns within a biofilm may reveal a community of cells resembling a multicellular organism, with heterogeneous roles for diverse phenotypic cell types. The problem will remain, however, that a small portion of the population may be able to survive any treatment regimen and effective therapy may have to include a combination of targets. This scenario is all the more likely when one considers the diverse and redundant biofilm-forming mechanisms already known to be present in the staphylococcal arsenal.

Summary The nreABC ( n itrogen re gulation) operon encodes a new staphylococcal two-component regulatory sys- tem that controls dissimilatory nitrate/nitrite reduc- tion in response to oxygen. Unlike other two- component sensors NreB is a cytosolic protein with four N-terminal cysteine residues. It was shown that both the NreB-cysteine cluster and Fe ions are required for function. Isolated NreB was converted to the active form by incubation with cysteine desul- phurase, ferrous ions and cysteine. This activation is typical for FeS-containing proteins and was reversed by oxygen. During reconstitution an absorption band at 420 nm and a yellow-brownish colour (typical for an FNR-type iron-sulphur cluster formation) devel- oped. After alkylation of thiol groups in NreB and in the cysteine mutant NreB(C62S) almost no iron- sulphur cluster was incorporated; both findings corroborated the importance of the cysteine resi- dues. Comparison of the kinase activity of (i) the reconstituted (ii) the unreconstituted, and (iii) the unreconstituted and deferrated NreB-His indicated that NreB kinase activity depended on iron availabil- ity and was greatly enhanced by reconstitution. NreB is the first direct oxygen-sensing protein described in staphylococci so far. Reconstituted NreB contains 4- 8 acid-labile Fe and sulphide ions per NreB which is in agreement with the presence of 1-2 iron-sulphur (4Fe-4S)

Biofilm formation is an important aspect of the pathogenesis of staphylococcal infections. A beta-1,6-linked N-acetyl glucosamine polysaccharide is critical to biofilm elaboration and is synthesized by proteins encoded by the intercellular adhesion (ica) locus. These studies were undertaken to characterize the mechanism by which transcription of the ica locus in S. aureus is regulated using isogenic S. aureus MN8 and MN8 mucoid (MN8m) strains, the latter of which constitutively overproduces biofilm. Transformation of the ica locus from MN8m to the ica knock-out mutants of two strains, MN8 and NCTC 10833, conferred a strong biofilm-producing phenotype. Sequence analysis revealed a 5-nucleotide deletion within the promoter region of the ica locus in MN8m compared with the sequence in the wild-type locus. Deletion or substitution of these 5 nucleotides within the wild-type ica locus augmented transcription of the ica locus and induced the strong biofilm-producing phenotype. Gel shift analysis demonstrated that a protein(s) within cell-free lysates from strain MN8 bind(s) specifically to oligonucleotides representative of the wild-type ica promoter sequence and that this binding is greatly diminished by the deletion or substitution of the 5 nucleotides. DNase I footprint analysis revealed that purified IcaR, thought to be a regulator of ica transcription, also binds to the ica promoter sequence just upstream of the ica start codon, but its affinity for the ica promoter is unaffected by deletion of the 5-nucleotide motif. These findings identify a 5-nucleotide motif within the ica promoter region that has a functional role in transcriptional regulation of the ica locus that is independent of IcaR, and also show that IcaR binds to the promoter region of the S. aureus ica locus.

Indolicidin is a cationic 13 amino acid peptide amide produced in the granules of bovine neutrophils with the sequence H-ILPWKWPWWPWRR-NH2. Indolicidin is both antimicrobial and, to a lesser extent, haemolytic. In order to systematically investigate structure-function relationships, the solid-phase synthesis of indolicidin and 48 distinct analogues are reported, as well as the characterization of their respective biological properties. Peptides synthesized and characterized include analogues with modified terminal functions, truncations from either terminus, an alanine scan to determine the role of each individual amino acid, specific amino acid exchanges of aromatic, charged and structural residues and several retro-, inverso- and retroinverso-analogues. Together, characterization of these analogues identifies specific residues involved in antimicrobial or haemolytic activity and suggests a core structure that may form a scaffold for the further development of peptidomimetic analogues of indolicidin.

Biofilm formation of Staphylococcus epidermidis on smooth polymer surfaces has been shown to be mediated by the ica operon. Upon activation of this operon, a polysaccharide intercellular adhesin (PIA) is synthesized that supports bacterial cell-to-cell contacts and triggers the production of thick, multilayered biofilms. Thus, the ica gene cluster represents a genetic determinant that significantly contributes to the virulence of specific Staphylococcus epidermidis strains. PIA synthesis has been reported recently to undergo a phase variation process. In this study, biofilm-forming Staphylococcus epidermidis strains and their PIA-negative phase variants were analysed genetically to investigate the molecular mechanisms of phase variation. We have characterized biofilm-negative variants by Southern hybridization with ica-specific probes, polymerase chain reaction and nucleotide sequencing. The data obtained in these analyses suggested that in approximately 30% of the variants the missing biofilm formation was due to the inactivation of either the icaA or the icaC gene by the insertion of the insertion sequence element IS256. Furthermore, it was shown that the transposition of IS256 into the ica operon is a reversible process. After repeated passages of the PIA-negative insertional mutants, the biofilm-forming phenotype could be restored. Nucleotide sequence analyses of the revertants confirmed the complete excision of IS256, including the initially duplicated 8 bp target sites. These results elucidate, for the first time, a molecular mechanism mediating phase variation in staphylcocci, and they demonstrate that a naturally occurring insertion sequence element is actively involved in the modulation of expression of a Staphylococcus virulence factor.

It is generally assumed that type A lantibiotics primarily kill bacteria by permeabilization of the cytoplasmic membrane. As previous studies had demonstrated that nisin interacts with the membrane-bound peptidoglycan precursors lipid I and lipid II, we presumed that this interaction could play a role in the pore formation process of lantibiotics. Using a thin-layer chromatography system, we found that only nisin and epidermin, but not Pep5, can form a complex with [14C]-lipid II. Lipid II was then purified from Micrococcus luteus and incorporated into carboxyfluorescein-loaded liposomes made of phosphatidylcholine and cholesterol (1:1). Liposomes supplemented with 0.05 or 0.1 mol% of lipid II did not release any marker when treated with Pep5 or epilancin K7 (peptide concentrations of up to 5 mol% were tested). In contrast, as little as 0.01 mol% of epidermin and 0.1 mol% of nisin were sufficient to induce rapid marker release; phosphatidylglycerol-containing liposomes were even more susceptible. Controls with moenomycin-, undecaprenol- or dodecaprenolphosphate-doped liposomes demonstrated the specificity of the lantibiotics for lipid II. These results were correlated with intact cells in an in vivo model. M. luteus and Staphylococcus simulans were depleted of lipid II by preincubation with the lipopeptide ramoplanin and then tested for pore formation. When applied in concentrations below the minimal inhibitory concentration (MIC) and up to 5-10 times the MIC, the pore formation by nisin and epidermin was blocked; at higher concentrations of the lantibiotics the protective effect of ramoplanin disappeared. These results demonstrate that, in vitro and in vivo, lipid II serves as a docking molecule for nisin and epidermin, but not for Pep5 and epilancin K7, and thereby facilitates the formation of pores in the cytoplasmic membrane.

The agr quorum-sensing system is responsible for the regulation of several virulence factors in staphylococci, with an extracellular pheromone peptide as signalling molecule. By monitoring the biological activity of synthetic peptides, it could be demonstrated that the pheromone of the agr system in Staphylococcus epidermidis is an octapeptide containing a thiolester linkage between the central cysteine and the C-terminal carboxyl group. The peptide was active at nanomolar concentrations. The N-terminus of the peptide pheromone, which is encoded as part of a protein precursor, proved to be crucial for biological activity.

Summary 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 biofilmnegative Tn917 mutants in S. epidermidis affected in initial attachment (class A) or intercellular adhesion (class B). mut1 (class A mutant) lacks five surfaceassociated 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 148 kDa. 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 38 kDa 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.

The lipase gene, geh, from Staphylococcus aureus NCTC8530 was cloned in Staphylococcus carnosus. DNA sequencing revealed an open reading frame (ORF) of 2046 nucleotides encoding a 682-amino-acid protein with a molecular mass of 76900 Da. Determination of the transcriptional start site revealed a 203-nucleotide mRNA leader. Expression of geh in the protease-negative S. carnosus (pT181copSA22) resulted in overexpression of a 83-kDa lipase found in the culture supernatant. N-terminal protein sequencing and sequence comparison with three other staphylococcal lipases suggest that this lipase is organised as a pre-pro-enzyme. The substrate specificity of this lipase is different from the Staphylococcus hyicus lipase. The S. hyicus lipase expressed both a high Ca(2+)-dependent phospholipase and lipase activity while the S. aureus lipase lacked this phospholipase activity and its activity with tributyrylglycerol or p-nitrophenyl octanoate is hardly stimulated by Ca2+ ions. A hybrid protein was constructed in which the C-terminal 146 residues of the S. hyicus lipase were substituted by 145 residues of the C-terminal of the S. aureus lipase, which contains the proposed active-site amino acids Asp602 and His641. The hybrid enzyme was still active and revealed an intermediary enzymic activity. The most striking effect was that it had lost the S. hyicus-specific phospholipase activity and that, in contrast to the two parental enzymes, its activity with p-nitrophenyl octanoate became highly sensitive to the presence of Ca2+. These observations suggest that the C-terminal domain of the S. hyicus lipase strongly contributes to the binding pocket of the polar headgroup of phospholipids. The Ca(2+)-binding site seems to be located in the N-terminal fragment of the S. hyicus lipase. The fact that two closely related enzymes differ in the need for Ca2+ underscores the notion that it plays a structural rather than a catalytic role.

We investigated the role of epiQ in the biosynthesis of the lantibiotic epidermin. epiQ was essential for epidermin production. It was shown that EpiQ controls epidermin production by transcriptionally activating the epiA promoter, used for transcription of most of the epidermin biosynthetic genes. Additional copies of epiQ increased epidermin production in the epidermin-producing wild-type strain Staphylococcus epidermidis Tü3298. The epiA promoter region was characterized by primer extension analysis. Two inverted repeats, putative operator sites for EpiQ binding, are located upstream of the -35 region and one is localized downstream of the -10 region. Crude protein extracts from S. epidermidis Tü3298 and epiQ expressing Escherichia coli cells led to gel mobility shifts of a DNA fragment bearing the inverted repeat which is located immediately upstream of the -35 region. DNA fragments bearing the other two inverted repeats were not shifted. The epiQ gene product could be detected by overexpression in the E. coli T7 system using antiserum raised against synthetic peptides of EpiQ. Furthermore, EpiQ, like other DNA-binding proteins, was shown to bind strongly to heparin sepharose.

Epidermin is produced by Staphylococcus epidermidis Tu3298 which harbors the 54-kb plasmid, pTu32. The plasmid contains not only the epidermin structural gene epiA, but also a flanking DNA region which is necessary for epidermin biosynthesis. The DNA sequence of this region revealed, in addition to epiA, five additional open reading frames, epiB, C, D, Q and P [Schnell, N., Engelke, G., Augustin J., Rosenstein, R., Ungermann, V., Gotz, F. & Entian, K.-D. (1992) Eur. J. Biochem. 204, 57-68]. We isolated a number of stable mutants from strain Tu3298 which are unable to produce biologically active epidermin. Complementation studies using the newly constructed staphylococcal plasmid vectors pT181mcs and pCU1 led to their classification as epiA, epiB, epiC or epiD mutants. Furthermore, evidence is presented that epiB lacks its own promoter and is co-transcribed from the epiA promoter. There is evidence that epiC and D possess their own promoters. Although epiQ and epiP mutants were not isolated, it could be shown by heterologous gene expression in S. carnosus and S. xylosus that the corresponding DNA region is involved in epidermin biosynthesis. We can not exclude the possibility that, in addition to the four open reading frames, epiA, B, C, D, and the DNA region comprising epiQ and P, host-encoded functions are necessary for epidermin production. Thus, the genetic information for epidermin biosynthesis in S. carnosus and S. xylosus is located on an 8-kb DNA fragment of pTu32. A further characterization of the two epiA mutants revealed that in both mutants, the preepidermin nucleotide sequence was changed. In one mutant, the mutation led to a substitution of Ser3 by Asn; in the other of Gly10 by Glu.

Monophosphatidylinositol inositol phosphohydrolase (phosphatidylinositol-specific phospholipase C. PtdIns-PLC. EC 3.1.4.10) has been purified from a Bacillus thuringiensis culture supernatant and from the cellular fraction of a recombinant Escherichia coli clone containing the PtdIns-PLC gene from B. thuringiensis. The two-step purification procedure involved ion-exchange chromatography on DEAE-Sepharose followed by separation on a Mono-Q/FPLC-column with yields of 32% and 50%, respectively. The molecular mass was determined to be 34 kDa by SDS/PAGE. The isoelectric point of the enzyme was 5.15. The amino-terminal sequences were shown to be identical for the enzymes purified from both organisms. PtdIns-PLC was inhibited by divalent cations using mixed micelles of Triton X-100 and pure phosphatidylinositol. PtdIns-PLC activity was detectable on polyacrylamide gels by activity staining on phosphatidylinostiol-containing agarose.

Staphylococcus epidermidis ATCC 14990 possesses an l-lactate dehydrogenase dependent on nicotinamide adenine dinucleotide (NAD) which is activated by fructose 1,6-bisphosphate (fructose-1,6-P2). Molecular weight studies with purified l-lactate dehydrogenase showed that fructose-1,6-P2 is necessary to stabilise the tetrameric form, having a molecular weight of about 130000. Dialysation of the enzyme led to a dissociation into subunits which could reasociate with fructose-1,6-P2 to the tetramer. The dialysed enzyme also exhibited a lag-period in the progress curve obtained by plotting the dependence of velocity versus time. This lag-period is absent when the enzyme was preincubated with fructose-1,6-P2. The lactate dehydrogenase exhibited only pyruvate reduction activity; the reverse reaction (l-lactate oxidation) could not be demonstrated in the enzyme assay system. Physiological studies demonstrated that the intracellular concentration of fructose-1,6-P2 influences the lactate dehydrogenase activity. The influence of the intracellular level of fructose-1,6-P2 on the active state of the lactate dehydrogenase could be shown in several ways. A high intracellular fructose-1,6-P2 level, present in cells grown in a glucose-excess medium under anaerobic growth conditions, is correlated with an active state of the lactate dehydrogenase, which means that the lactate dehydrogenase in the cell-free extract was saturated with fructose-1,6-P2. The addition of fructose-1,6-P2 to the assay system could not increase the lactate dehydrogenase activity. In cells grown in a glucose-limited medium under anaerobic conditions, the fructose-1,6-P2 pool was exhausted due to glucose limitation. In this case, the lactate dehydrogenase was not fully saturated with fructose-1,6-P2 because the lactate dehydrogenase activity from the cell-free extract could be increased about 10-fold by addition of fructose-1,6-P2 to the assay system. A similar situation is present with cells grown under aerobic conditions in a glucose-excess medium. Here too, no intracellular fructose-1,6-P2 was detected and the active state of lactate dehydrogenase was markedly reduced. Glucose degradation studies with resting cell cultures under both anaerobic and aerobic conditions showed that only under anaerobic conditions a high intracellular level of fructose-1,6-P2 was present and a high amount of l-lactate was produced.

Fermentation under modified gravity could be of interest in application to (a) increasing productivity of growth and growth linked production with microorganisms at high cell densities and (b) increasing the productivity of highly viscous pseudoplastic polysaccharide fermentation. In both cases, higher oxygen transfer rates in centrifugal fields result in higher productivities since these fermentations are usually oxygen limited. A further aspect of fermentation under increased gravity is the reduction of foam since foam coalescence time decreases with acceleration number. On the other hand, under microgravity, shear reduction would allow growth and production even for very shear sensitive organisms. In order to carry out fermentations under modified gravity, a special type of fermenter–the centrifugal field bioreactor CFBR–has been developed at the Institute of Chemical Engineering (Head: Prof. Mersmann) of the Technical University of Munich. For the first time, exoprotein biosynthesis of lipase with S. carnosus has been carried out under sterile and controlled conditions in this novel bioreactor, in presence of increased mass forces.