Specific phosphatidylethanolamine dependence of Serratia marcescens cytotoxin activity

The cytolytic and haemolytic activity of Serratia marcescens is determined by the ShlA protein, which is secreted across the outer membrane with the aid of the ShlB protein. In the absence of ShlB, inactive ShlA* remains in the periplasm of Escherichia coli transformed with an shlA-encoding plasmid, which indicates that ShlB converts ShlA* to active ShlA. ShlA* in a periplasmic extract and partially purified ShlA* were activated in vitro by partially purified ShlB. When both proteins were highly purified, ShlA* was only activated by ShlB when phosphatidylethanolamine (PE) or phosphatidylserine was added to the assay, while phosphatidylglycerol contributed little to ShlA* activation. Lyso-PE, cardiolipin, phosphatidylcholine, phosphatidic acid, lipopolysaccharide and various detergents could not substitute for PE. Although radioactively labelled PE was so tightly associated with ShlA that it remained bound to ShlA after heating and SDS–PAGE, it was not covalently linked to ShlA as PE could be removed by thin-layer chromatography with organic solvents. The number of PE molecules associated per molecule of ShlA was 3.9 ± 2.2. Active ShlA was inactivated by treatment with phospholipase A₂, which indicated that PE is also required for ShlA activity. ShlA-255 (containing the 255 N-terminal amino acids of ShlA) reversibly complemented ShlA* to active ShlA and was inactivated by phospholipase A₂, which demonstrated that PE binds to the N-terminal portion of ShlA; this region has previously been found to be involved in ShlA secretion and activation. Electrospray mass spectroscopy of ShlA-255 determined a molar mass that corresponded to that of unmodified ShlA-255. An E. coli mutant that synthesized only minute amounts of PE did not secrete ShlA but contained residual cell-bound haemolytic activity. Since PE binds strongly to ShlA* in the absence of ShlB without converting ShlA* to haemolytic ShlA, ShlB presumably imposes a conformation on ShlA that brings PE into a position to mediate interaction of the hydrophilic haemolysin with the lipid bilayer of the eukaryotic membrane.     

The Serratia marcescens hemolysin is secreted but not activated by stable protoplast-type L-forms of Proteus mirabilis

The outer-membrane protein ShlB of Serratia marcescens activates and secretes hemolytic ShlA into the culture medium. Without ShlB, inactive ShlA (termed ShlA*) remains in the periplasm. Since Proteus mirabilis L-form cells lack an outer membrane and a periplasm, it was of interest to determine in which compartment recombinant ShlA* and ShlB are localized and whether ShlB activates ShlA*. The cloned shlB and shlA genes were transcribed in P. mirabilis stable L-form cells by the temperature-inducible phage T7 RNA polymerase. Radiolabeling, Western blotting, and complementation with C-terminally truncated ShlA (ShlA255) identified inactive ShlA* in the culture supernatant. ShlB remained cell-bound and did not activate ShlA without integration in an outer membrane. Although hemolytic ShlA added to L-form cells had access to the cytoplasmic membrane, it did not affect L-form cells. Synthesis of the large ShlA protein (165 kDa) in P. mirabilis L-form cells under phage T7 promoter control demonstrates that L-form cells are suitable for the synthesis and secretion of large recombinant proteins. This property and the easy isolation of released proteins make L-form cells suitable for the biotechnological production of proteins. Received: 17 February 1998 / Accepted: 30 June 1998     

In vitro activation of the Serratia marcescens hemolysin through modification and complementation.

The hemolytic activity of Serratia marcescens is determined by two polypeptides, termed ShlA and ShlB. ShlA is synthesized as an inactive precursor (ShlA*) and secreted with the help of ShlB, which is located in the outer membrane. In this study, it is shown that a cell lysate containing ShlB as well as partially purified ShlB converted ShlA* to the active ShlA hemolysin. ShlA remained active after removal of ShlB by column chromatography. In contrast to the stable modification of ShlA* by ShlB, a reversible activation was achieved by adding to ShlA* an N-terminal fragment of ShlA (ShlA16), consisting of 269 amino acid residues of ShlA and 18 residues of the vector. The nonhemolytic ShlA16 complemented ShlA* only when it was synthesized in an ShlB-producing cell. A deletion derivative of ShlA*, lacking residues 4 to 117, was complemented by ShlA16 but not activated by ShlB. Activation of ShlA* by ShlB at 4 degrees C proceeded at a much slower rate than complementation by ShlA16. It is concluded that ShlA* is modified by ShlB. ShlA16 modified by ShlB complements the missing modification of ShlA* in trans. Modification by ShlB occurs in the N-terminal part of ShlA*, which is also the reaction in vivo which results in active ShlA hemolysin in the culture supernatant. The HpmA hemolysin of Proteus mirabilis, which is very similar to ShlA, was also activated in vitro by ShlB and complemented by ShlA16.     

Serratia marcescens forms a new type of cytolysin

Most Serratia marcescens strains produce a new type of cytolysin (hemolysin) which is also found in other Serratia species. The hemolytic polypeptide ShlA (M(r) 162 101) is secreted across the outer membrane through the help of the ShlB protein which also involves conversion of an inactive precursor in an hemolytically active form. Both proteins are synthesized with signal sequences which are released during export across the cytoplasmic membrane. Mutants expressing inactive ShlB derivatives are impaired in activation and secretion suggesting a tight coupling between both processes. The region of ShlA for activation and secretion is confined to the N-terminal 16% of the polypeptide which contains the sequence NPNG which is also found in the Proteus hemolysin, the Bordetella pertussis filamentous hemagglutinin and two highly expressed outer membrane proteins of Haemophilus influenzae. Substitution of the first asparagine (N) residue by isoleucine converts the Serratia hemolysin into an inactive secretion incompetent form. It is concluded that this region is recognized by ShlB for activation and secretion of ShlA. The Serratia hemolysin forms defined pores in erythrocyte membranes.     

Amino acid replacements in the Serratia marcescens haemolysin ShlA define sites involved in activation and secretion

The haemolysin of Serratia marcescens (ShlA) is translocated through the cytoplasmic membrane by the signal peptide-dependent export apparatus. Translocation across the outer membrane (secretion) is mediated by the ShIB protein. Only the secreted form of ShlA is haemolytic. ShIB also converts in vitro inactive ShlA (ShlA*), synthesized in the absence of ShIB, into the haemolytic form (a process termed activation). To define regions in ShlA involved in both processes, ShlA derivatives were isolated and tested for secretion and activation. Analysis of C-terminally truncated proteins (ShlA) assigned the secretion signal to the amino-terminal 238 residues of ShlA. Trypsin cleavage of a secreted ShlA' derivative yielded a 15kDa N-terminal fragment, by which a haemolytically inactive ShlA* protein could be activated in vitro. It is suggested that the haemolysin activation site is located in this N-terminal fragment. Replacement of asparagine-69 and asparagine-109 by isoleucine yielded inactive haemolysin derivatives. Both asparagine residues are part of two short sequence motifs, reading Ala-Asn-Pro-Asn, which are critical to both activation and secretion. These point mutants as well as N-terminal deletion derivatives which were not activated by ShIB were activated by adding a non-haemolytic N-terminal fragment synthesized in an ShIB⁺ strain (complementation). Apparently the activated N-terminal fragment substituted for the missing activation of the ShlA derivatives and directed them into the erythrocyte membrane, where they formed pores. It is concluded that activation is only required for initiation of pore formation, and that in vivo activation and secretion are tightly coupled processes. Complementation may also indicate that haemolysin oligomers form the pores.     

The haemolysin-secreting ShlB protein of the outer membrane of Serratia marcescens : determination of surface-exposed residues and formation of ion-permeable pores by ShlB mutants in artificial lipid bilayer membranes

The ShlB protein in the outer membrane of Serratia marcescens is the only protein known to be involved in secretion of the ShlA protein across the outer membrane. At the same time, ShlB converts ShlA into a haemolytic and a cytolytic toxin. Surface-exposed residues of ShlB were determined by reaction of an M2 monoclonal antibody with the M2 epitope DYKDDDDK inserted at 25 sites along the entire ShlB polypeptide. The antibody bound to the M2 epitope at 17 sites in intact cells, which indicated surface exposure of the epitope, and to 23 sites in isolated outer membranes. Two insertion mutants contained no ShlB(M2) protein in the outer membrane. The ShlB derivatives activated and/or secreted ShlA. To gain insights into the secretion mechanism, we studied whether highly purified ShlB and ShlB deletion derivatives formed pores in artificial lipid bilayer membranes. Wild-type ShlB formed channels with very low single channel conductance that rarely assumed an open channel configuration. In contrast, open channels with a considerably higher single channel conductance were observed with the deletion mutants ShlB(Delta65-186), ShlB(Delta87-153), and ShlB(Delta126-200). ShlB(Delta126-200) frequently formed permanently open channels, whereas the conductance caused by ShlB(Delta65-186) and ShlB(Delta87-153) did not assume a stationary value, but fluctuated rapidly between open and closed configurations. The results demonstrate the orientation of large portions of ShlB in the outer membrane and suggest that ShlB may function as a specialized pore through which ShlA is secreted.     

Integration of the Serratia marcescens haemolysin into human erythrocyte membranes

The haemolytic activity of Serratia marcescens is determined by two proteins, ShlA and ShlB. ShlA integrates into the erythrocyte membrane and causes osmotic lysis through channel formation. The conformation of ShlA and its interaction with erythrocyte membranes were studied by determining the cleavage of ShlA by added trypsin. Our results suggest that the conformation of inactive ShlA (from an ShlB- strain) differs from the active ShlA, and that in a hydrophobic environment (detergent or membrane) active ShlA assumes a conformation distinct from that in buffer. Only active haemolysin adsorbed to erythrocytes. ShlA was firmly integrated into the erythrocyte membrane since it was only released under conditions which also dissolved the integral erythrocyte membrane proteins. Moreover, ShlA integrated into 'ghosts' remained there and was not haemolytic when incubated with erythrocytes. From the trypsin cleavage pattern obtained with haemolysin and C-terminally truncated, but still active, haemolysin derivatives integrated into erythrocytes, and sealed and unsealed erythrocyte 'ghosts', we conclude that ShlA is preferentially cleaved by trypsin at a few sites but only from the inside of the erythrocyte. Haemolysin in the erythrocyte membrane forms a water-filled channel and is resistant to trypsin and other proteases.     

Superlytic hemolysin mutants of Serratia marcescens.

Hemolysis by Serratia marcescens is caused by two proteins, ShlA and ShlB. ShlA is the hemolysin proper, and ShlB transports ShlA through the outer membrane, whereby ShlA is converted into a hemolysin. Superhemolytic ShlA derivatives that displayed 7- to 20-fold higher activities than wild-type ShlA were isolated. ShlA80 carried the single amino acid replacement of G to D at position 326 (G326D), ShlA87 carried S386N, and ShlA80III carried G326D and N236D. Superhemolysis was attributed to the greater stability of the mutant ShlA derivatives because they aggregated less than the wild-type hemolysin, which lost activity within 3 min at 20 degrees C. In contrast to the highly hemolytic wild-type ShlA at 0 degrees C, the hyperlytic hemolysins were nonhemolytic at 0 degrees C, suggesting that the hyperlytic derivatives differed from wild-type ShlA in adsorption to and insertion into the erythrocyte membrane. However, the size of the pores formed at 20 degrees C by superhemolytic hemolysins could not be distinguished from that of wild-type ShlA. In addition to the N-terminal sequence up to residue 238, previously identified to be important for activation and secretion, sites 326 and 386 contribute to hemolysin activity since they are contained in regions that participate in hemolysin inactivation through aggregation.     

Molecular characterization of the hemolysin determinant of Serratia marcescens.

The nucleotide sequence of a 7.3-kilobase-pair fragment of DNA encoding a hemolytic activity from Serratia marcescens was determined. Two large open reading frames were identified, designated shlA (Serratia hemolysin) and shlB, capable of encoding polypeptides of 165, 056 and 61,897 molecular weight, respectively. Both reading frames were expressed in vivo. The shlB gene product was localized to the outer membrane of Escherichia coli cells harboring the S. marcescens hemolysin determinant. Consistent with this location, a signallike sequence was identified at the N terminus of the polypeptide predicted from the nucleotide sequence of the shlB gene. Hyperexpression of the shlB locus permitted the identification of two shlB-encoded polypeptides of 65,000 and 62,000 molecular weight, respectively. Determination of the N-terminal amino acid sequence of the purified 62,000-molecular-weight protein confirmed that it was the mature form of the ShlB protein initially synthesized as a precursor (65,000-molecular-weight protein). By using polyclonal antisera raised against the purified proteins, ShlA and ShlB were identified in the outer membrane of S. marcescens. The shlA gene product was shown to interact with erythrocyte membranes, confirming it as the hemolysin proper. Both hemolysis and the interaction of ShlA with erythrocyte membranes did, however, require the ShlB function. Progressive deletion of the C terminus of the ShlA protein gradually reduced hemolytic activity until 37% of the amino acids had been removed. Elimination of 54% of the amino acids produced a nonhemolytic protein which, however, was still capable of associating with erythrocyte membranes.     

Subcellular location and unique secretion of the hemolysin of Serratia marcescens

It is shown that Serratia marcescens exports a hemolysin to the cell surface and secretes it to the extracellular space. Escherichia coli containing the cloned hemolysin genes shlA and shlB exported and secreted the S. marcescens hemolysin. A nonhemolytic secretion-incompetent precursor of the hemolysin, designated ShlA*, was synthesized in a shlB deletion mutant and accumulated in the periplasmic space of E. coli. Immunogold-labeled ultrathin sections revealed ShlA* bound to the outer face of the cytoplasmic membrane and to the inner face of the outer membrane. A number of mutants carrying 3' deletions in the shlA gene secreted truncated polypeptides, the smallest of which contained only 261 of the 1578 amino acids of the mature ShlA hemolysin, showing that the information for export to the cell surface of E. coli and secretion into the culture medium is located in the NH2-terminal segment of the hemolysin. We propose a secretion pathway in which ShlA and ShlB are exported across the cytoplasmic membrane via a signal sequence-dependent mechanism. ShlB is integrated into the outer membrane. ShlA is translocated across the outer membrane with the help of ShlB. During the latter export process or at the cell surface, ShlA acquires the hemolytically active conformation and is released to the extracellular space. The hemolysin secretion pathway appears to be different from any other secretion system hitherto reported and involves only a single specific export protein.     

Selective blockage of Serratia marcescens ShlA by nickel inhibits the pore‐forming toxin‐mediated phenotypes in eukaryotic cells

Serratia marcescens is an opportunistic pathogen with increasing incidence in clinical settings. This is mainly attributed to the timely expression of a wide diversity of virulence factors and intrinsic and acquired resistance to antibiotics, including β‐lactams, aminoglycosides, quinolones, and polypeptides. For these reasons, S. marcescens has been recently categorised by the World Health Organization as one priority to strengthen efforts directed to develop new antibacterial agents. Therefore, it becomes critical to understand the underlying mechanisms that allow Serratia to succeed within the host. S. marcescens ShlA pore‐forming toxin mediates phenotypes that alter homeostatic and signal transduction pathways of host cells. It has been previously demonstrated that ShlA provokes cytotoxicity, haemolysis and autophagy and also directs Serratia egress and dissemination from invaded nonphagocytic cells. However, molecular details of ShlA mechanism of action are still not fully elucidated. In this work, we demonstrate that Ni²⁺ selectively and reversibly blocks ShlA action, turning wild‐type S. marcescens into a shlA mutant strain phenocopy. Combined use of Ni²⁺ and calcium chelators allow to discern ShlA‐triggered phenotypes that require intracellular calcium mobilisation and reveal ShlA function as a calcium channel, providing new insights into ShlA mode of action on target cells.     

A pore‐forming toxin enables Serratia a nonlytic egress from host cells

Several pathogens co‐opt host intracellular compartments to survive and replicate, and they thereafter disperse progeny to prosper in a new niche. Little is known about strategies displayed by Serratia marcescens to defeat immune responses and disseminate afterwards. Upon invasion of nonphagocytic cells, Serratia multiplies within autophagosome‐like vacuoles. These Serratia‐containing vacuoles (SeCV) circumvent progression into acidic/degradative compartments, avoiding elimination. In this work, we show that ShlA pore‐forming toxin (PFT) commands Serratia escape from invaded cells. While ShlA‐dependent, Ca²⁺ local increase was shown in SeCVs tight proximity, intracellular Ca²⁺ sequestration prevented Serratia exit. Accordingly, a Ca²⁺ surge rescued a ShlA‐deficient strain exit capacity, demonstrating that Ca²⁺ mobilization is essential for egress. As opposed to wild‐type‐SeCV, the mutant strain‐vacuole was wrapped by actin filaments, showing that ShlA expression rearranges host actin. Moreover, alteration of actin polymerization hindered wild‐type Serratia escape, while increased intracellular Ca²⁺ reorganized the mutant strain‐SeCV actin distribution, restoring wild‐type‐SeCV phenotype. Our results demonstrate that, by ShlA expression, Serratia triggers a Ca²⁺ signal that reshapes cytoskeleton dynamics and ends up pushing the SeCV load out of the cell, in an exocytic‐like process. These results disclose that PFTs can be engaged in allowing bacteria to exit without compromising host cell integrity.     

Effect of the chitin binding domain deletion from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> subsp. <Emphasis Type="Italic">kurstaki</Emphasis> chitinase Chi255 on its stability in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Bacillus thuringiensis subsp. kurstaki BUPM255 secretes a chitobiosidase Chi255 having an expected molecular weight of 70.665 kDa. When the corresponding gene, chi255, was expressed in E. coli, the active form, extracted from the periplasmic fraction of E. coli/pBADchi255, was of about 54 kDa, which suggested that Chi255 was excessively degraded by the action of E. coli proteases. Therefore, in vitro progressive C-terminal Chi255 deleted derivatives were constructed in order to study their stability and their activity in E. coli. Interestingly, when the chitin binding domain (CBD) was deleted from Chi255, an active form (Chi2555Δ5) of expected size of about 60 kDa was extracted from the E. coli periplasmic fraction, without the observation of any proteolytic degradation. Compared to Chi255, Chi255Δ5 exhibited a higher chitinase activity on colloidal chitin. Both of the enzymes exhibit activities at broad pH and temperature ranges with maximal enzyme activities at pH 5 and pH 6 and at temperatures 50°C and 40°C, respectively for Chi255 and Chi255Δ5. Thus, it was concluded that the C-terminal deletion of Chi255 CBD might be a nice tool for avoiding the excessive chitinase degradation, observed in the native chitinase, and for improving its activity.     

Characterization of glutathione conjugates of pyrrolylated amino acids and peptides by liquid chromatography-mass spectrometry and tandem mass spectrometry with electrospray ionization

High-performance liquid chromatography (HPLC) coupled with electrospray mass spectrometry (ES-MS) and tandem mass spectrometry (MS-MS) was used to identify the products formed upon reaction of lysine-containing peptides with the neurotoxicant 2,5-hexanedione (2,5-HD). In addition, secondary autoxidative reaction products of the resultant alkylpyrroles with the biological thiol, glutathione, were characterized. ES mass spectra of the HPLC-separated conjugates showed intense [M+H]⁺ ions as well as several ions formed by amide and C-S bond cleavage. The glutathione conjugates of pyrrolylated amino acids and peptides were analyzed by ES ionization and MS-MS, and product-ion spectra showed fragmentation pathways typical of glutathione conjugates. ES-MS-MS analysis of a synthetic nonapeptide modeling a sequence found in neurofilament proteins showed pyrrole formation after incubation with 2,5-HD, and sequence ions were used to assign the position of the pyrrole adduct. Subsequent reaction of the pyrrolylated peptide with reduced glutathione was evidenced by a shift in m/z of the sequence ions of the reaction products with or without prior methylation. The results demonstrate the utility of ES-MS and ES-MS-MS in the characterization of xenobiotic-modified peptides and confirm that stable pyrrole-thiol conjugates are formed by the reaction of biological thils with pyrrolylated peptides.     

Determination of flunarizine in rat brain by liquid chromatography–electrospray mass spectrometry

A rapid liquid chromatography–electrospray mass spectrometry (LC–ES-MS) assay for the determination of flunarizine (FZ) in rat brain has been developed. A C₁₈ column and an isocratic elution were employed for the separation. Using post-column split, 64% of the eluent was introduced into the ES-MS system for detection. The [M+H]⁺ (m/z 406) and a fragmented ion (m/z 203) were detected using selected ion monitoring. The linear range of this assay was good, ranging from 0.05 to 5 μM (r²=0.99). The intra- and inter-day precisions showed relative standard deviations ranging from 1.4% to 2.0% and 1.3% to 2.9%, respectively. The application of this newly developed method was demonstrated by examining the pharmacokinetics of FZ in rat brain.     

Hypoxia/ischemia induces dephosphorylation of rat brain neuromodulin/GAP-43 in vivo

The in vivo state of phosphorylation and the modification of two Cys residues of neuromodulin/ GAP-43 (Nm) were analyzed by electrospray ionization-mass spectrometry (ES-MS). The protein was purified from rat brain with homogenization buffer containing 1% Nonidet P-40, protease inhibitors, protein phosphatase inhibitors, and sulfhydryl reagent, 4-vinylpyridine. Nm was purified by HPLC and ion-exchange chromatography, and the various fractions were identified by ES-MS as unphosphorylated and mono-, di-, tri-, and tetraphosphorylated species. All of these Nm species contained 2 mol of added 4-vinylpyridine per mol of Nm, suggesting that the two Cys residues are in the reduced form in the brain. In vivo, the majority of Nm is in the phosphorylated form (approximately 80%), of which the levels of the mono- and diphospho forms are higher than those of the tri- and tetraphospho species. Four in vivo phosphorylation sites, Ser41, Thr95, Ser142, and Thr172, were identified by amino acid sequencing and tandem ES-MS of the peptides derived from Lys-C endoproteinase digestion. Among these sites, only Ser41 is a known target of PKC, whereas the kinases responsible for the phosphorylation of the other three novel sites are unknown. Hypoxia/ischemia caused a preferential dephosphorylation of Ser41 and Thr172, whereas Thr95 is the least susceptible to dephosphorylation.     

Two-step purification of the outer membrane transporter and activator protein ShlB from Escherichia coli using internally His6-tagged constructs

ShlB from Serratia marcescens was isolated and purified from a porin-deficient Escherichia coli BL21 strain using a combination of detergent extraction, affinity and ion-exchange chromatography. An internal histidine affinity tag was introduced that did not interfere with activity. At each stage of the purification scheme biological activity of the ShlB protein was assessed. Using this scheme, several His(6)-tagged mutants of ShlB were purified to electrophoretic homogeneity.     

N-glycans are not required for the efficient degradation of the mutant Saccharomyces cerevisiae CPY* in Schizosaccharomyces pombe

In eukaryotic cells, aberrant proteins generated in the endoplasmic reticulum (ER) are degraded by the ER-associated degradation (ERAD) pathway. Here, we report on the ERAD pathway of the fission yeast Schizosaccharomyces pombe. We constructed and expressed Saccharomyces cerevisiae wild-type CPY (ScCPY) and CPY-G255R mutant (ScCPY*) in S. pombe. While ScCPY was glycosylated and efficiently transported to the vacuoles in S. pombe, ScCPY* was retained in the ER and was not processed to the matured form in these cells. Cycloheximide chase experiments revealed that ScCPY* was rapidly degraded in S. pombe, and its degradation depended on Hrd1p and Ubc7p homologs. We also found that Mnl1p and Yos9p, proteins that are essential for ERAD in S. cerevisiae, were not required for ScCPY* degradation in S. pombe. Moreover, the null-glycosylation mutant of ScCPY, CPY*0000, was rapidly degraded by the ERAD pathway. These results suggested that N-linked oligosaccharides are not important for the recognition of luminal proteins for ERAD in S. pombe cells.     

Peptaibiomics: an advanced, rapid and selective analysis of peptaibiotics/peptaibols by SPE/LC-ES-MS

Summary. “Proteomics” and “peptidomics” are used as technical terms to define the analysis and study of all proteins and peptides expressed in an organism or tissue. In analogy we propose the name peptaibiomics for the analysis of a group of fungal peptide antibiotics (peptaibiotics) containing the characteristic amino acid Aib (α-aminoisobutyric acid). In analogy to the peptidome the complete expression of peptaibiotics by fungal multienzyme complexes should be named the peptaibiome. Peptaibiotics are defined as peptides containing Aib and exerting a variety of bioactivities. They comprise the sub-groups of N-acetylated peptaibols, characterized also by a C-terminal amide-linked 2-amino alcohol, and lipopeptaibols having in place of an acetyl group a lipophilic fatty acid acyl group. Furthermore, lipoaminopeptides are also known with long-chain fatty acid on the N-termini, a lipoamino acid in position three and a strongly basic secondary or tertiary amine form a subgroup of mixed forms which could not be integrated in one of these three previously mentioned groups. Here we present a specific and rapid screening method on the peptaibiome applicable directly onto filamentous fungi cultured in a single Petri dish. The method comprises solid-phase extraction (SPE) of peptaibiotics followed by on-line reversed-phase HPLC coupled to an ion trap electrospray tandem mass spectrometer (ES-MS). The presence of these peptides is indicated by characteristic mass differences of Δm = 85.1 Da representing Aib-residues which can be observed in the b-series of acylium fragment ions resulting from ES-MS. Partial sequences can be deduced from the data and compared with structures compiled in electronic peptaibol data bases. The judgement is possible whether or not structures are novel, already known or related to known structures. Suitability of the method is demonstrated with the analysis of strains of Trichoderma and its teleomorph Hypocrea. New sequences of peptaibiotics are presented and those being related to established 10- to 18-residue peptaibols trichovirin, trichogin and trichotoxin, which have been described in the literature.     

AFFINITY MODIFICATION OF EcoRII DNA METHYLTRANSFERASE BY THE DIALDEHYDE-SUBSTITUTED DNA DUPLEXES: MAPPING THE ENZYME REGION THAT INTERACTS WITH DNA

ABSTRACT

Affinity modification of EcoRII DNA methyltransferase (M·EcoRII) by DNA duplexes containing oxidized 2′-O-β-D-ribofuranosylcytidine (Crib*) or 1-(β-D-galactopyranosyl)thymine (Tgal*) residues was performed. Cross-linking yields do not change irrespective of whether active Crib* replaces an outer or an inner (target) deoxycytidine within the EcoRII recognition site. Chemical hydrolysis of M·EcoRII in the covalent cross-linked complex with the Tgal*-substituted DNA indicates the region Gly²⁶⁸-Met³⁹¹ of the methylase that is likely to interact with the DNA sugar-phosphate backbone. Both specific and non-specific DNA interact with the same M·EcoRII region. Our results support the theoretically predicted DNA binding region of M·EcoRII.