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ABSTRACTDehydration of cells by acute hyperosmotic stress has profound effects upon cell structure and function. Interphase chromatin and mitotic chromosomes collapse (“congelation”). HL-60/S4 cells remain ~100% viable for, at least, 1 hour, exhibiting shrinkage to ~2/3 their original volume, when placed in 300mM sucrose in tissue culture medium. Fixed cells were imaged by immunostaining confocal and STED microscopy. At a “global” structural level (μm), mitotic chromosomes congeal into a residual gel with apparent (phase) separations of Ki67, CTCF, SMC2, RAD21, H1 histones and HMG proteins. At an “intermediate” level (sub-μm), radial distribution analysis of STED images revealed a most probable peak DNA density separation of ~0.16 μm, essentially unchanged by hyperosmotic stress. At a “local” structural level (~1-2 nm), in vivo crosslinking revealed essentially unchanged crosslinked products between H1, HMG and inner histones. Hyperosmotic cellular stress is discussed in terms of concepts of mitotic chromosome structure and liquid-liquid phase separation.

ABSTRACTThe polyene antifungal amphotericin B (AmB) is widely used to treat life-threatening fungal infections. Even though AmB resistance is exceptionally rare in fungi, most Aspergillus terreusisolates exhibit an intrinsic resistance against the drug in vivoand in vitro. Heat shock proteins perform a fundamental protective role against a multitude of stress responses, thereby maintaining protein homeostasis in the organism. In this study, we elucidated the role of heat shock protein 70 (Hsp70) family members and compared resistant and susceptible A. terreusclinical isolates. The upregulation of cytoplasmic Hsp70 members at the transcriptional as well as translational levels was significantly higher with AmB treatment than without AmB treatment, particularly in resistant A. terreusisolates, thereby indicating a role of Hsp70 proteins in the AmB response. We found that Hsp70 inhibitors considerably increased the susceptibility of resistant A. terreusisolates to AmB but exerted little impact on susceptible isolates. Also, in in vivoexperiments, using the Galleria mellonellainfection model, cotreatment of resistant A. terreusstrains with AmB and the Hsp70 inhibitor pifithrin-μ resulted in significantly improved survival compared with that achieved with AmB alone. Our results point to an important mechanism of regulation of AmB resistance by Hsp70 family members in A. terreusand suggest novel drug targets for the treatment of infections caused by resistant fungal isolates.

We present the first comprehensive capillary electrophoresis electrospray ionization mass spectrometry (CESI-MS) analysis of post-translational modifications derived from H1 and core histones. Using a capillary electrophoresis system equipped with a sheathless high-sensitivity porous sprayer and nano–liquid chromatography electrospray ionization mass spectrometry (nano-LC-ESI-MS) as two complementary techniques, we characterized H1 histones isolated from rat testis. Without any pre-separation of the perchloric acid extraction, a total of 70 different modified peptides, including 50 phosphopeptides, were identified in the rat linker histones H1.0, H1a-H1e, and H1t. Out of the 70 modified H1 histone peptides, 27 peptides could be identified with CESI-MS only, and 11 solely with LC-ESI-MS. Immobilized metal-affinity chromatography enrichment prior to MS analysis yielded a total of 55 phosphopeptides; 22 of these peptides could be identified only by CESI-MS, and 19 only by LC-ESI-MS, showing the complementarity of the two techniques. We mapped 42 H1 modification sites, including 31 phosphorylation sites, of which 8 were novel sites. For the analysis of core histones, we chose a different strategy. In a first step, the sulfuric-acid-extracted core histones were pre-separated using reverse-phase high-performance liquid chromatography. Individual rat testis core histone fractions obtained in this way were digested and analyzed via bottom-up CESI-MS. This approach yielded the identification of 42 different modification sites including acetylation (lysine and Nα-terminal); mono-, di-, and trimethylation; and phosphorylation. When we applied CESI-MS for the analysis of intact core histone subtypes from butyrate-treated mouse tumor cells, we were able to rapidly detect their degree of modification, and we found this method very useful for the separation of isobaric trimethyl and acetyl modifications. Taken together, our results highlight the need for additional techniques for the comprehensive analysis of post-translational modifications. CESI-MS is a promising new proteomics tool as demonstrated by this, the first comprehensive analysis of histone modifications, using rat testis as an example.

ABSTRACTHemolytic-uremic syndrome (HUS) is a life-threatening disorder characterized by hemolytic anemia, thrombocytopenia, and renal insufficiency. It is caused mainly by infections with enterohemorrhagic Escherichia coli(EHEC). Recently, Shiga toxin 2, the best-studied virulence factor of EHEC, was reported to interact with complement, implying that complement may be involved in the pathogenesis of EHEC-induced HUS. The aim of the present study was to investigate whether or not the serine protease EspP, an important virulence factor of EHEC, interacts with complement proteins. EspP did not have any effect on the integrity of factor H or factor I. However, EspP was shown to cleave purified C3/C3b and C5. Cleavage of the respective complement proteins also occurred in normal human serum (NHS) as a source of C3/C3b or C5 or when purified complement proteins were added to the supernatant of an EspP-producing wild-type strain. Edman degradation allowed unequivocal mapping of all three main C3b fragments but not of the three main C5 fragments. Complement activation was significantly downregulated in all three pathways for C5-depleted serum to which C5, preincubated with EspP, was added (whereas C5 preincubated with an EspP mutant was able to fully reconstitute complement activation). This indicates that EspP markedly destroyed the functional activity, as measured by a commercial total complement enzyme-linked immunosorbent assay (Wieslab). Downregulation of complement by EspP in vivomay influence the colonization of EHEC bacteria in the gut or the disease severity of HUS.

Hemolytic-uremic syndrome (HUS) is a life-threatening disorder characterized by hemolytic anemia, thrombocytopenia, and renal insufficiency. It is caused mainly by infections with enterohemorrhagic Escherichia coli (EHEC). Recently, Shiga toxin 2, the best-studied virulence factor of EHEC, was reported to interact with complement, implying that complement may be involved in the pathogenesis of EHEC-induced HUS. The aim of the present study was to investigate whether or not the serine protease EspP, an important virulence factor of EHEC, interacts with complement proteins. EspP did not have any effect on the integrity of factor H or factor I. However, EspP was shown to cleave purified C3/C3b and C5. Cleavage of the respective complement proteins also occurred in normal human serum (NHS) as a source of C3/C3b or C5 or when purified complement proteins were added to the supernatant of an EspP-producing wild-type strain. Edman degradation allowed unequivocal mapping of all three main C3b fragments but not of the three main C5 fragments. Complement activation was significantly downregulated in all three pathways for C5-depleted serum to which C5, preincubated with EspP, was added (whereas C5 preincubated with an EspP mutant was able to fully reconstitute complement activation). This indicates that EspP markedly destroyed the functional activity, as measured by a commercial total complement enzyme-linked immunosorbent assay (Wieslab). Downregulation of complement by EspP in vivo may influence the colonization of EHEC bacteria in the gut or the disease severity of HUS.

Fatty aldehyde dehydrogenase (EC 1.2.1.48) converts long-chain fatty aldehydes to the corresponding acids. Deficiency in this enzyme causes the Sjogren Larsson Syndrome, a rare inherited disorder characterized by ichthyosis, spasticity, and mental retardation. Using a fluorescent aldehyde, pyrenedecanal, and HPLC with fluorescence detection, we developed a novel method to monitor fatty aldehyde dehydrogenase activity by quantification of the product pyrenedecanoic acid together with the substrate pyrenedecanal and possible side products, such as aldehyde adducts. As shown with recombinant enzymes, pyrenedecanal showed a high preference for fatty aldehyde dehydrogenase compared with other aldehyde dehydrogenases. The method allowed detection of fatty aldehyde dehydrogenase activity in nanogram amounts of microsomal or tissue protein and microgram amounts of Sjogren Larsson syndrome patients' skin fibroblast protein. It could successfully be adapted for the analysis of fatty aldehyde dehydrogenase activity in gel slices derived from low-temperature SDS-PAGE, showing that fatty aldehyde dehydrogenase activity from solubilized rat liver microsomes migrates as a dimer. Thus, monitoring of pyrenedecanoic acid formation from pyrenedecanal by HPLC with fluorescence detection provides a robust and sensitive method for determination of fatty aldehyde dehydrogenase activity.

Fatty aldehyde dehydrogenase (EC 1.2.1.48) converts long-chain fatty aldehydes to the corresponding acids. Deficiency in this enzyme causes the Sjogren Larsson Syndrome, a rare inherited disorder characterized by ichthyosis, spasticity, and mental retardation. Using a fluorescent aldehyde, pyrenedecanal, and HPLC with fluorescence detection, we developed a novel method to monitor fatty aldehyde dehydrogenase activity by quantification of the product pyrenedecanoic acid together with the substrate pyrenedecanal and possible side products, such as aldehyde adducts. As shown with recombinant enzymes, pyrenedecanal showed a high preference for fatty aldehyde dehydrogenase compared with other aldehyde dehydrogenases. The method allowed detection of fatty aldehyde dehydrogenase activity in nanogram amounts of microsomal or tissue protein and microgram amounts of Sjogren Larsson syndrome patients' skin fibroblast protein. It could successfully be adapted for the analysis of fatty aldehyde dehydrogenase activity in gel slices derived from low-temperature SDS-PAGE, showing that fatty aldehyde dehydrogenase activity from solubilized rat liver microsomes migrates as a dimer. Thus, monitoring of pyrenedecanoic acid formation from pyrenedecanal by HPLC with fluorescence detection provides a robust and sensitive method for determination of fatty aldehyde dehydrogenase activity.

Antiangiogenic effects of the proteasome inhibitor bortezomib were analyzed on tumor xenografts in vivo. Bortezomib strongly inhibited angiogenesis and vascularization in the chicken chorioallantoic membrane. Bortezomib's inhibitory effects on chorioallantoic membrane vascularization were abrogated in the presence of distinct tumor xenografts, thanks to a soluble factor secreted by tumor cells. Through size-exclusion and ion-exchange chromatography as well as mass spectroscopy, we identified GRP-78, a chaperone protein of the unfolded protein response, as being responsible for bortezomib resistance. Indeed, a variety of bortezomib-resistant solid tumor cell lines (PC-3, HRT-18), but not myeloma cell lines (U266, OPM-2), were able to secrete high amounts of GRP-78. Recombinant GRP-78 conferred bortezomib resistance to endothelial cells and OPM-2 myeloma cells. Knockdown of GRP78 gene expression in tumor cells and immunodepletion of GRP-78 protein from tumor cell supernatants restored bortezomib sensitivity. GRP-78 did not bind or complex bortezomib but induced prosurvival signals by phosphorylation of extracellular signal–related kinase and inhibited p53-mediated expression of proapoptotic Bok and Noxa proteins in endothelial cells. From our data, we conclude that distinct solid tumor cells are able to secrete GRP-78 into the tumor microenvironment, thus demonstrating a hitherto unknown mechanism of resistance to bortezomib.

Antiangiogenic effects of the proteasome inhibitor bortezomib were analyzed on tumor xenografts in vivo. Bortezomib strongly inhibited angiogenesis and vascularization in the chicken chorioallantoic membrane. Bortezomib's inhibitory effects on chorioallantoic membrane vascularization were abrogated in the presence of distinct tumor xenografts, thanks to a soluble factor secreted by tumor cells. Through size-exclusion and ion-exchange chromatography as well as mass spectroscopy, we identified GRP-78, a chaperone protein of the unfolded protein response, as being responsible for bortezomib resistance. Indeed, a variety of bortezomib-resistant solid tumor cell lines (PC-3, HRT-18), but not myeloma cell lines (U266, OPM-2), were able to secrete high amounts of GRP-78. Recombinant GRP-78 conferred bortezomib resistance to endothelial cells and OPM-2 myeloma cells. Knockdown of GRP78gene expression in tumor cells and immunodepletion of GRP-78 protein from tumor cell supernatants restored bortezomib sensitivity. GRP-78 did not bind or complex bortezomib but induced prosurvival signals by phosphorylation of extracellular signal–related kinase and inhibited p53-mediated expression of proapoptotic Bok and Noxa proteins in endothelial cells. From our data, we conclude that distinct solid tumor cells are able to secrete GRP-78 into the tumor microenvironment, thus demonstrating a hitherto unknown mechanism of resistance to bortezomib.

Proteome analysis has emerged as a valuable tool for the study of large-scale protein expression profiles. Here, we applied this novel technology to identify specific biomarkers for acute cardiac allograft rejection.

Aspergillus fumigatus excretes the fusarinine-type siderophore desferri-triacetylfusarinine C (DF-TafC) to mobilize iron. DF-TafC is a cyclic peptide consisting of three N5-cis-anhydromevalonyl-N5-hydroxy-N2-acetyl-L-ornithine residues linked by ester bonds; these linkages are in contrast to peptide linkages found for ferrichrome-type siderophores. Subsequent to the binding of iron and uptake, triacetylfusarinine C (TafC) is hydrolyzed, the cleavage products are excreted, and the iron is transferred to the metabolism or to the intracellular siderophore desferri-ferricrocin (DF-FC) for iron storage. Here we report the identification and characterization of the TafC esterase EstB, the first eukaryotic siderophore-degrading enzyme to be characterized at the molecular level. The encoding gene, estB, was found to be located in an iron-regulated gene cluster, indicating a role in iron metabolism. Deletion of estB in A. fumigatus eliminated TafC esterase activity of cellular extracts and caused increased intracellular accumulation of TafC and TafC hydrolysis products in vivo. Escherichia coli-expressed EstB displayed specific TafC esterase activity but did not hydrolyze fusarinine C, which has the same core structure as TafC but lacks three N2-acetyl residues. Localization of EstB via enhanced green fluorescent protein tagging suggested that TafC hydrolysis takes place in the cytoplasm. EstB abrogation reduced the intracellular transfer rate of iron from TafC to DF-FC and delayed iron sensing. Furthermore, EstB deficiency caused a decreased radial growth rate under iron-depleted but not iron-replete conditions. Taken together, these data suggest that EstB-mediated TafC hydrolysis optimizes but is not essential for TafC-mediated iron uptake in A. fumigatus.

ABSTRACTAspergillus fumigatusexcretes the fusarinine-type siderophore desferri-triacetylfusarinine C (DF-TafC) to mobilize iron. DF-TafC is a cyclic peptide consisting of three N5-cis-anhydromevalonyl-N5-hydroxy-N2-acetyl-l-ornithine residues linked by ester bonds; these linkages are in contrast to peptide linkages found for ferrichrome-type siderophores. Subsequent to the binding of iron and uptake, triacetylfusarinine C (TafC) is hydrolyzed, the cleavage products are excreted, and the iron is transferred to the metabolism or to the intracellular siderophore desferri-ferricrocin (DF-FC) for iron storage. Here we report the identification and characterization of the TafC esterase EstB, the first eukaryotic siderophore-degrading enzyme to be characterized at the molecular level. The encoding gene, estB, was found to be located in an iron-regulated gene cluster, indicating a role in iron metabolism. Deletion of estBin A. fumigatuseliminated TafC esterase activity of cellular extracts and caused increased intracellular accumulation of TafC and TafC hydrolysis products in vivo. Escherichia coli-expressed EstB displayed specific TafC esterase activity but did not hydrolyze fusarinine C, which has the same core structure as TafC but lacks three N2-acetyl residues. Localization of EstB via enhanced green fluorescent protein tagging suggested that TafC hydrolysis takes place in the cytoplasm. EstB abrogation reduced the intracellular transfer rate of iron from TafC to DF-FC and delayed iron sensing. Furthermore, EstB deficiency caused a decreased radial growth rate under iron-depleted but not iron-replete conditions. Taken together, these data suggest that EstB-mediated TafC hydrolysis optimizes but is not essential for TafC-mediated iron uptake in A. fumigatus.

[^99mTc-EDDA–HYNIC-D-Phe¹,Tyr³]octreotide (^99mTc-EDDA/HYNIC–TOC) is a promising new agent with the potential to replace [¹¹¹In-DTPA-D-Phe¹]-octreotide in somatostatin receptor scintigraphy. This hydrazinonicotinic acid derivatized somatostatin complex contains ethylenediamine N,N' diacetic acid (EDDA) as a coligand resulting in a high in vitro and in vivo stability. Since direct ^99mTc-labelling of HYNIC–TOC with EDDA results in low labelling yields, in this study we describe the preparation of ^99mTc-EDDA/HYNIC-TOC via coligand exchange from Tricine for EDDA. Exchange of coligands is achieved at elevated temperature and under optimized conditions of pH, EDDA concentration and stannous ion. High labelling yields (mean 95.9%) were achieved at high specific activities (>37GBq/µmol). Characterization via HPLC, receptor binding and LC–MS of the resulting complex is described. The formulation developed enables rapid and simple labelling of ^99mTc-EDDA/HYNIC–TOC in a manner suitable for a clinical setting. Copyright © 2003 John Wiley & Sons, Ltd.

The replacement linker histones H1(0) and H5 are present in frog and chicken erythrocytes, respectively, and their accumulation coincides with cessation of proliferation and compaction of chromatin. These cells have been analyzed for the affinity of linker histones for chromatin with cytochemical and biochemical methods. Our results show a stronger association between linker histones and chromatin in chicken erythrocyte nuclei than in frog erythrocyte nuclei. Analyses of linker histones from chicken erythrocytes using capillary electrophoresis showed H5 to be the subtype strongest associated with chromatin. The corresponding analyses of frog erythrocyte linker histones using reverse-phase high performance liquid chromatography showed that H1(0) dissociated from chromatin at somewhat higher ionic strength than the three additional subtypes present in frog blood but at lower ionic strength than chicken H5. Which of the two H1(0) variants in frog is expressed in erythrocytes has thus far been unknown. Amino acid sequencing showed that H1(0)-2 is the only H1(0) subtype present in frog erythrocytes and that it is 100% acetylated at its N termini. In conclusion, our results show differences between frog and chicken linker histone affinity for chromatin probably caused by the specific subtype composition present in each cell type. Our data also indicate a lack of correlation between linker histone affinity and chromatin condensation.

Methylation of the N-terminal region of histones was first described more than 35 years ago, but its biological significance has remained unclear. Proposed functions range from transcriptional regulation to the higher order packing of chromatin in progress of mitotic condensation. Primarily because of the recent discovery of the SET domain-depending H3-specific histone methyltransferases SUV39H1 and Suv39h1, which selectively methylate lysine 9 of the H3 N terminus, this posttranslational modification has regained scientific interest. In the past, investigations concerning the biological significance of histone methylation were largely limited because of a lack of simple and sensitive analytical procedures for detecting this modification. The present work investigated the methylation pattern of histone H4 both in different mammalian organs of various ages and in cell lines by applying mass spectrometric analysis and a newly developed hydrophilic-interaction liquid chromatographic method enabling the simultaneous separation of methylated and acetylated forms, which obviates the need to work with radioactive materials. In rat kidney and liver the dimethylated lysine 20 was found to be the main methylation product, whereas the monomethyl derivative was present in much smaller amounts. In addition, for the first time a trimethylated form of lysine 20 of H4 was found in mammalian tissue. A significant increase in this trimethylated histone H4 was detected in organs of animals older than 30 days, whereas the amounts of mono- and dimethylated forms did not essentially change in organs from young (10 days old) or old animals (30 and 450 days old). Trimethylated H4 was also detected in transformed cells; although it was present in only trace amounts in logarithmically growing cells, we found an increase in trimethylated lysine 20 in cells in the stationary phase.

We report here on the HPCE separation of two chicken H5 histones, which do not show the heterogeneity (Gln/Arg) at residue 15 first found by Greenaway and Murray [Greenaway and Murray (1971) Nat. New Biol. 229, 233–238]. The two subfractions obtained were identified using reversed-phase HPLC, hydrophilic interaction HPLC, Edman degradation, and MALDI-MS analysis. We found that the two H5 subcomponents differ only by an acetylated (designated H5a) and an unacetylated N-terminus (H5b). In contrast to the N-terminally acetylated form of rat kidney histone H1○, which increased by about 40% with aging of the animal, the corresponding form of chicken H5 did not: the ratio N-terminally acetylated: unacetylated remained constant (30:70) when histone H5 was extracted from erythrocytes of newly hatched chickens and from adult chickens, respectively. The HPCE technique used in this investigation represents a quick and convenient method for analyzing N-terminally acetylated proteins in the presence of unacetylated forms.

Abstract: The composition of the H1° histone subfractions was examined in different rat and mouse tissues. Using reverse-phase HPLC and hydrophilic-interaction liquid chromatography we have found that the relative proportions of all four forms of H1° differ from tissue to tissue and from species to species. In principle, we observed an age-dependent increase in the amount of both the N-terminally acetylated (H1°a Asn-3 and H1°a Asp-3) and the deamidated forms of H1° (H1°a Asp-3 and H1°b Asp-3). Compared with the proportion of N-terminally acetylated H1° forms in liver, kidney and brain of rats and mice 20 days of age, we found an increase in these H1° subfractions of up to 30% in the corresponding organs of 300-day-old animals. The proportion of deamidated H1° forms was 1.6- to 4-fold higher in the livers and 8- to 12-fold higher in the brains of 300-day-old mice and rats, respectively, than in 20-day-old animals. The tissue-specific nature of the ratio of H1° subfractions suggests that the different forms of histone H1° have specific individual functions. The possible biological significance of age-related accumulation of N-terminal acetylated and deamidated histone H1° forms is discussed in the light of our results.

The cytoplasmic B‐type histone acetyltransferase was purified to apparent homogeneity from maize embryos. We established a novel protocol for easy large‐scale preparation of acetylated core histone species, using preparative acetic acid‐urea‐Triton PAGE. The pure maize histone acetyltransferase B was highly specific for histone H4 under various assay conditions, modifying H4 up to the di‐acetylated isoform. Only non‐acetylated H4 isoform was accepted as substrate, whereas mono‐acetylated H4 could not be further acetylated. The enzyme selectively acetylated lysines 12 and 5 in a sequential manner. The same results were obtained with a partially purified cytoplasmic histone acetyltransferase of rat liver. Protein sequencing results were supported by immunological characterization of acetylated H4 subspecies with site‐specific H4‐acetyllysine antibodies.

Histone H10is known to consist of two subfractions named H10a and H10b. The present work was performed with the aim of elucidating the nature of these two subfractions. By using reversed-phase high performance liquid chromatography in combination with hydrophilic interaction liquid chromatography, we fractionated human histone H10into even four subfractions. Hydrophilic interaction liquid chromatographic analysis of the peptide fragments obtained after cleavage with cyanogen bromide and digestion with chymotrypsin suggested that the four H10subfractions differ only in their small N-terminal end of the H10molecule (30 residues). Edman degradation of the N-terminal H10peptide fragments and mass spectra analysis have indicated that human histone H10consists of intact histones H10(named H10Asn-3) and deamidated H10forms (H10Asp-3) having an aspartic acid residue at position 3 instead of asparagine. Moreover, both H10Asn-3 and H10Asp-3 are blocked (H10a Asn-3, H10a Asp-3) and unblocked (H10b Asn-3, H10b Asp-3) on their N terminus. Acid-urea gel electrophoretic analysis has shown that the histone subfraction, in the literature originally named H10a, actually consists of a mixture of H10a Asn-3 and H10a Asp-3, whereas H10b consists of H10b Asn-3 and H10b Asp-3. Furthermore, we found that hydrophilic interaction liquid chromatography separates rat and mouse histone H10just like human H10into four subfractions. Hydrophilic interaction liquid chromatographic analysis of brain and liver histone H10from rats of different ages revealed an age-dependent increase of both the N-terminally acetylated and the deamidated forms of H10. In addition, we found that the relative proportions of the four forms of H10histones differ from tissue to tissue.

The cytoplasmic B-type histone acetyltransferase was purified to apparent homogeneity from maize embryos. We established a novel protocol for easy large-scale preparation of acetylated core histone species, using preparative acetic acid-urea-Triton PAGE. The pure maize histone acetyltransferase B was highly specific for histone H4 under various assay conditions, modifying H4 up to the di-acetylated isoform. Only non-acetylated H4 isoform was accepted as substrate, whereas mono-acetylated H4 could not be further acetylated. The enzyme selectively acetylated lysines 12 and 5 in a sequential manner. The same results were obtained with a partially purified cytoplasmic histone acetyltransferase of rat liver. Protein sequencing results were supported by immunological characterization of acetylated H4 subspecies with site-specific H4-acetyllysine antibodies.

This work reports the discovery of a novel genetic toolbox comprising multiple, endogenous selectable markers for targeted genomic insertions of DNAs of interest (DOIs). Marker genes encode proteins involved in 5-fluorocytosine uptake and pyrimidine salvage activities mediating 5-fluorocytosine deamination as well as 5-fluorouracil phosphoribosylation. The requirement for their genomic replacement by DOIs to confer 5-fluorocytosine or 5-fluorouracil resistance for transformation selection enforces site-specific integrations. Due to the fact that the described markers are endogenously encoded, there is no necessity for the exogenous introduction of commonly employed markers such as auxotrophy-complementing genes or antibiotic resistance cassettes. Importantly, inactivation of the described marker genes had no adverse effects on nutrient requirements, growth, or virulence of the human pathogen Aspergillus fumigatus. Given the limited number and distinct types of selectable markers available for the genetic manipulation of prototrophic strains such as wild-type strains, we anticipate that the proposed methodology will significantly advance genetic as well as metabolic engineering of fungal species.

No relevant conflicts of interest to declare.

Abstract 4805