Precursor-directed biosynthesis for the generation of novel glycopetides.
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Actinomycetales↗
Biomedical subjects
R Süssmuth
Publications and source records attributed to R Süssmuth.
Analysis of the proliferative responses to peptides in individuals with vigorous Gag protein-specific proliferation.
Proliferative responses to recombinant HIV proteins in infected individuals may represent a correlate of protection from disease progression. In this study, the proliferative responses to HIV p24, p55 and gp120 were evaluated in infected subjects. Whereas, vigorous proliferative responses directed at the Gag proteins were detected in several individuals, Env-specific proliferation was observed in only one subject. Epitope mapping using overlapping peptides demonstrated proliferative responses of PBMC to Gag peptides. Responses were broadly directed at multiple peptides in some subjects. Although several of the peptides that induced proliferative responses also contain CTL epitopes potentially relevant in the particular individuals, many additional Gag T cell epitopes were present in each subject. This finding may be relevant for the design and testing of HIV candidate vaccines.
AIDS Vaccines↗
Isolation from Cussonia barteri of 1'-O-chlorogenoylchlorogenic acid and 1'-O-chlorogenoylneochlorogenic acid, a new type of quinic acid esters.
1'-O-Chlorogenoylchlorogenic acid and 1'-O-chlorogenoylneochlorogenic acid, a new type of quinic acid esters, have been isolated, in addition to six known quinic acid esters, rutin, and a mixture of saponins, from the methanol extract of Cussonia barteri Seemann (Araliaceae) leaves collected in Cameroon. Structure determination was achieved by NMR, mass, IR, and UV spectroscopy. All compounds were tested for inhibitory activity on 5-lipoxygenase and cyclooxygenase-1, for antimicrobial activity against Bacillus subtilis, Pseudomonas fluorescens, and Cladosporium cucumerinum, and for haemolytic activity.
Anti-Infective Agents↗
Two-step fast protein liquid chromatographic purification of the Serratia marcescens hemolysin and peptide mapping with mass spectrometry.
The pore forming toxin of Serratia marcescens (ShlA) is secreted and activated by an outer membrane protein (ShlB). Activation of inactive ShlA (termed ShlA*) by ShlB is dependent on phosphatidylethanolamine (PE). Activation may be a covalent modification of ShlA. To test this hypothesis, the responsible activation domain (in the N-terminal 255 amino acids of ShlA) was isolated from whole bacteria with 8 M urea in an inactive form (ShlA-255*) and from the culture supernatant in an active form (ShlA-255), followed by a two-step purification by anion-exchange chromatography and gel permeation chromatography. Comparison of a tryptic peptide map of both forms with subsequent electrospray mass spectrometry (ES-MS) and sequencing by tandem ES-MS revealed no modification. These data imply that ShlB presumably imposes a conformation on ShlA-255 that triggers activity.
Amino Acid Sequence↗
Polyphasic characterization of poly-3-hydroxybutyrate-co-3-hydroxyvalerate (p(HB-co-HV)) metabolizing and denitrifying Acidovorax sp. strains.
For the purpose of denitrification in small drinking water plants, a bacterial mixed population was isolated from a packed bed column bioreactor with poly-3-hydroxybutyrate-co-3-hydroxyvalerate (P(HB-co-HV)) as a substrate for the denitrification of ground water (10 degrees C). Isolates 2nIII from the mixed culture, with the ability to denitrify and metabolize P(HB-co-HV), were used as starter cultures for the elimination of nitrate in ground water. The strains were characterized by diverse techniques. Classical phenotypic studies lead to rRNA group III of the genus Pseudomonas. Results obtained by molecular techniques demonstrated that the 2nIII strains are members of the Comamonadaceae and shows similarities to the genus Acidovorax. However, an integration of the 2nIII isolates within one of the known Acidovorax species is not possible for the moment. The 2nIII starter cultures clustered close to Av. temperans according to their whole cell proteins and fatty acids, whereas in DNA/DNA hybridization no significant DNA binding (< 25%) was found. In contrast a significant but low degree of DNA/DNA hybridization was found between the 2nIII strains and Av. facilis and Av. delafieldii. Our polyphasic results lead to the conclusion that the 2nIII strains may constitute a separate Acicdovorax species.
Bacterial Typing Techniques↗
A putative enolpyruvyl transferase gene involved in nikkomycin biosynthesis.
The nikO gene encoding a putative enolpyruvyl transferase has been identified within the Streptomyces tendae Tü901/8c nikkomycin gene cluster. nikO encodes a deduced protein of 471 amino acid residues which exhibits significant sequence similarity to UDP-N-acetylglucosamine enolpyruvyl transferase and 5-enol-pyruvylshikimate 3-phosphate synthase from various origin. The nikO gene was inactivated by inserting a kanamycin resistance cassette; the mutant did not produce biologically active nikkomycins I, J, X, and Z nor the nucleoside moieties, nikkomycins C(x) and C(z), but accumulated the novel component RT 2.0. RT 2.0 has been isolated from culture filtrate and its structure was determined by using mass spectrometry and NMR analyses as ribofuranosyl-4-formyl-4-imidazolone which represents a novel nucleoside. The putative activity of the nikO gene product in nikkomycin biosynthesis will be discussed.
Alkyl and Aryl Transferases↗
Simocyclinones, novel cytostatic angucyclinone antibiotics produced by Streptomyces antibioticus Tü 6040. I. Taxonomy, fermentation, isolation and biological activities.
Two novel angucyclinone-type antibiotics, simocyclinones D4 and D8, were detected in the mycelium extract of Streptomyces antibioticus Tü 6040 by HPLC-diode-array and HPLC-electrospray-mass-spectrometry screening. The compounds show antibiotic activities against Gram-positive bacteria and cytostatic effects on various tumor cell lines.
Anti-Bacterial Agents↗
Inhibition of virulence factor expression in Staphylococcus aureus by the Staphylococcus epidermidis agr pheromone and derivatives.
The agr quorum-sensing system in Staphylococci controls the production of surface proteins and exoproteins. In the pathogenic species Staphylococcus aureus, these proteins include many virulence factors. The extracellular signal of the quorum-sensing system is a thiolactone-containing peptide pheromone, whose sequence varies among the different staphylococcal strains. We demonstrate that a synthetic Staphylococcus epidermidis pheromone is a competent inhibitor of the Staphylococcus aureus agr system. Derivatives of the pheromone, in which the N-terminus or the cyclic bond structure was changed, were synthesized and their biological activity was determined. The presence of a correct N-terminus and a thiolactone were absolute prerequisites for an agr-activating effect in S. epidermidis, whereas inhibition of the S. aureus agr system was less dependent on the original structure. Our results show that effective quorum-sensing blockers that suppress the expression of virulence factors in S. aureus can be designed based on the S. epidermidis pheromone.
Bacterial Proteins↗
Identification of unusual amino acids in peptides using automated sequential Edman degradation coupled to direct detection by electrospray-ionization mass spectrometry.
The determination of the primary structure of peptides and proteins is routine in many laboratories; however, many of the obtained sequences are incomplete or can be misinterpreted when the samples contain unusual amino acids. Here we report the development of an automated peptide sequenator coupled to an electrospray-ionization (ESI) mass spectrometer (MS) that, in conjunction with minor modifications to the sequencing conditions and, in some cases, prior derivatization of amino acids, allows the detection of the phenylthiohydantoin (PTH) derivatives of a number of unusual amino acids. Using the coupled sequenator-ESI-MS system we were able to determine the complete sequence of the lantibiotic gallidermin, a partial sequence of the calcium-dependent peptide antibiotic CDA2 as well as the pool sequence of a mixture of synthetic peptides containing nonproteinogenic amino acids. In addition to the 20 proteinogenic amino acids, the procedure was able to detect PTH derivatives of hydroxyphenylglycine, 2,3-didehydroasparagine, 3-methylglutamic acid, oxytryptophan, ornithine, N-methylglycine, dihydroxyphenylalanine, and alpha-aminoisobutyric acid. Similarly, after a simple derivatization procedure, we were also able to correctly identify educts of 2,3-didehydroalanine, 2,3-didehydrobutyrine, lanthionine, and 3-methyllanthionine.
Amino Acid Sequence↗
A model for the generation of low level chemiluminescence from microbiological growth media and its depletion by bacterial cells.
We present a model for the development of chemiluminescence (CL) in autoclaved liquid growth media, as they are used in microbiology for the culturing of microorganisms, or in other related Maillard systems. The model distinguishes between four different stages consisting of sugar fragmentation during heating, autooxidation of highly reducing fragmentation products, radical chain reactions leading to a peroxidation of the media, and finally the formation of excited states, energy transfer reactions and CL emission. The proposed model is also discussed in regard of a recently reported elimination of this CL in growing cultures of microorganisms and possible pathways for this interference are suggested.
Bacteria↗
Degradation of desferrioxamines by Azospirillum irakense: assignment of metabolites by HPLC/electrospray mass spectrometry.
Based on a recent finding that an Azospirillum isolate ASP-1 possessing high 16S rDNA similarity to Azospirillum irakense was able to degrade desferrioxamine type siderophores (Winkelmann et al. BioMetals 9, 78-83, 1996), various members of the genus Azospirillum were analyzed for their ability to degrade desferrioxamines. While the desferrioxamine-degrading activity was absent or scarcely detectable in strains of A. lipoferum, A. brasilense, A. amazonense, degradation activity seemed to be confined to the species A. irakense (KBC-1, KA3). Also the identity of strain ASP-1 as A. irakense could be confirmed by species-specific oligonucleotide hybridization, Inter-LINE PCR fingerprinting and carbon source utilization pattern (BIOLOG) analysis. Products of desferrioxamine B degradation were analyzed by analytical HPLC and HPLC/electrospray mass spectrometry. Using whole cells and purified enzyme it was shown that the trihydroxamate desferrioxamine B (561 amu) is split at the N-terminal amide bond yielding a monohydroxamate (MH1, 219 amu) and a dihydroxamate (DH1, 361 amu) metabolite. A second monohydroxamate (MH2, 319 amu) resulted from DH1 after splitting the acetylhydroxamate bond. Minor amounts of a further dihydroxamate (DH2, 419 amu) originated from splitting the second amide bond in desferrioxamine B. In addition to desferrioxamine B, several other linear and cyclic desferrioxamines and derivatives were degraded, whereas desferricoprogen and desferri-ferrichrome were not degraded, indicating high substrate specificity of the desferrioxamine hydrolase in A. irakense species. A simple microtiter plate assay was developed which can be used to phenotypically discriminate and identify species of A. irakense from other Azospirillum species by their characteristic feature of desferrioxamine degradation.
Azospirillum↗
Low level chemiluminescence from liquid culture media.
Low level chemiluminescence (CL) can be observed from autoclaved liquid culture media, as used in microbiology. The light emission is oxygen-dependent and arises from reactions following auto-oxidation of reducing Maillard products which are formed during autoclaving. The inhibition of this CL by radical scavengers and antioxidants has been studied. As superoxide radicals and hydrogen peroxide are predominantly involved in the initiation of the CL, the investigation of CL from culture media offers a convenient tool for the detection of exogenous (medium-mediated) oxidative stress being imposed onto micro-organisms in culture. Transition metal ions showed, dependent on concentration, both inhibitory and stimulating effects on the CL, which was also affected by the presence of complexing agents. Iron porphyrins and related complexes displayed a very efficient quenching of the CL, which may be of particular importance, as aerobic micro-organisms have been previously shown to be very efficient in quenching the CL from culture media.
Antioxidants↗
Identification and analysis of the balhimycin biosynthetic gene cluster and its use for manipulating glycopeptide biosynthesis in Amycolatopsis mediterranei DSM5908.
Seven complete genes and one incomplete gene for the biosynthesis of the glycopeptide antibiotic balhimycin were isolated from the producer, Amycolatopsis mediterranei DSM5908, by a reverse-cloning approach and characterized. Using oligonucleotides derived from glycosyltransferase sequences, a 900-bp glycosyltransferase gene fragment was amplified and used to identify a DNA fragment of 9,882 bp. Of the identified open reading frames, three (oxyA to -C) showed significant sequence similarities to cytochrome P450 monooxygenases and one (bhaA) showed similarities to halogenase, and the genes bgtfA to -C showed similarities to glycosyltransferases. Glycopeptide biosynthetic mutants were created by gene inactivation experiments eliminating oxygenase and glycosyltransferase functions. Inactivation of the oxygenase gene(s) resulted in a balhimycin mutant (SP1-1) which was not able to synthesize an antibiotically active compound. Structural analysis by high-performance liquid chromatography-mass spectrometry, fragmentation studies, and amino acid analysis demonstrated that these oxygenases are involved in the coupling of the aromatic side chains of the unusual heptapeptide. Mutant strain HD1, created by inactivation of the glycosyltransferase gene bgtfB, produced at least four different compounds which were not glycosylated but still antibiotically active.
Anti-Bacterial Agents↗
Production of nikkomycins Bx and Bz by mutasynthesis with genetically engineered Streptomyces tendae Tü901.
The previously described Streptomyces tendae nikC::aph mutant was used to mutasynthesize nikkomycins Bx and Bz. The mutant is deficient in L-lysine 2-aminotransferase, which transaminates lysine to form piperideine 2-carboxylate, the precursor of the peptidyl side chain of the biologically active nikkomycins I, J, X, and Z, and is therefore unable to produce these nikkomycins. The mutant accumulates the biologically inactive biosynthetic nucleoside precursors nikkomycins Cx and Cz. Resting cell cultures of the mutant fed with benzoic acid produced the biologically active nikkomycins Bx and Bz, which contain 2-amino-4-hydroxy-3-methyl-4-(4'-hydroxyphenyl)butanoic acid as the peptidyl side chain. The structures of nikkomycins Bx and Bz were confirmed by mass spectrometry and NMR. Nikkomycins Bx and Bz exhibit significantly higher pH stability than their analogues nikkomycins X and Z.
Aminoglycosides↗
Novel nikkomycins Lx and Lz produced by genetically engineered Streptomyces tendae Tü901.
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Aminoglycosides↗
Characterization of the N-acetylglucosaminyltransferase activity involved in the biosynthesis of the Staphylococcus epidermidis polysaccharide intercellular adhesin.
The polysaccharide intercellular adhesin (PIA) is an important factor in the colonization of medical devices by Staphylococcus epidermidis. The genes encoding PIA production are organized in the icaADBC (intercellular adhesion) operon. To study the function of the individual genes, we have established an in vitro assay with UDP-N-acetylglucosamine, the substrate for PIA biosynthesis, and analyzed the products by thin-layer chromatography and mass spectrometry. IcaA alone exhibited a low N-acetylglucosaminyltransferase activity and represents the catalytic enzyme. Coexpression of icaA with icaD led to a significant increase in activity. The newly identified icaD gene is located between icaA and icaB and overlaps both genes. N-Acetylglucosamine oligomers produced by IcaAD reached a maximal length of 20 residues. Only when icaA and icaD were expressed together with icaC were oligomer chains that react with PIA-specific antiserum synthesized. IcaA and IcaD are located in the cytoplasmic membrane, and IcaC also has all the structural features of an integral membrane protein. These results indicate a close interaction between IcaA, IcaD, and IcaC. Tunicamycin and bacitracin did not affect the in vitro synthesis of PIA intermediates or the complete PIA biosynthesis in vivo, suggesting that a undecaprenyl phosphate carrier is not involved. IcaAD represents a novel protein combination among beta-glycosyltransferases.
Amino Acid Sequence↗
Structure of the pheromone peptide of the Staphylococcus epidermidis agr system.
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.
Amino Acid Sequence↗
Adjuvant lipopeptide interaction with model membranes.
The cationic lipohexapeptide Pam3Cys-Ser-(Lys)4 is a synthetic model for the triacylated N-terminal part of bacterial lipoproteins, and it is used as an adjuvant and macrophage activator. The amphiphilic lipopeptide was injected below a phosphatidylserine monolayer at the air-water interface. It interacted with the interface, as seen by a decrease in the surface potential (deltaV), and it was inserted in the monolayer, until surface charge neutralization was reached, as seen by the parallel increases of deltaV and of the surface pressure. No insertion occurred above 29 mN/m. The interaction kinetics was sensitive to ionic strength and to the nature of acidic phospholipids and of their acyl chains, but the final equilibrium was independent of these factors. Addition of the lipopeptide to large unilamellar vesicles (LUVs) induced their aggregation, and an exchange of lipids between fluorophor-labelled and non-labelled LUVs. However, no fusion was observed, just as reported for polylysine. The lipopeptide strongly inhibited calcium-induced fusion of PS LUVs, in contrast to the published effect of polylysine. This was probably due to inhibition of calcium fixation on liposomes, since it was observed that the lipopeptide efficiently displaced 45Ca2+ from a PS monolayer. In addition, a phospholipid segregation was observed in SUVs for a few ten micromolar of the lipopeptide.
Lipoproteins↗