Allelic polymorphisms in a glycosyltransferase gene shape glycan repertoire in the O-linked protein glycosylation system of Neisseria.

Glycosylation of multiple proteins via O-linkage is well documented in bacterial species of Neisseria of import to human disease. Recent studies of protein glycosylation (pgl) gene distribution established that related protein glycosylation systems occur throughout the genus including nonpathogenic species. However, there are inconsistencies between pgl gene status and observed glycan structures. One of these relates to the widespread distribution of pglG, encoding a glycosyltransferase that in Neisseria elongata subsp. glycolytica is responsible for the addition of di-N-acetyl glucuronic acid at the third position of a tetrasaccharide. Despite pglG residing in strains of N. gonorrhoeae, N. meningitidis and N. lactamica, no glycan structures have been correlated with its presence in these backgrounds. Moreover, PglG function in N. elongata subsp. glycolytica minimally requires UDP-glucuronic acid (GlcNAcA), and yet N. gonorrhoeae, N. meningitidis and N. lactamica lack pglJ, the gene whose product is essential for UDP-GlcNAcA synthesis. We examined the functionality of pglG alleles from species spanning the Neisseria genus by genetic complementation in N. elongata subsp. glycolytica. The results indicate that select pglG alleles from N. meningitidis and N. lactamica are associated with incorporation of an N-acetyl-hexosamine at the third position and reveal the potential for an expanded glycan repertoire in those species. Similar experiments using pglG from N. gonorrhoeae failed to find any evidence of function suggesting that those alleles are missense pseudogenes. Taken together, the results are emblematic of how allelic polymorphisms can shape bacterial glycosyltransferase function and demonstrate that such alterations may be constrained to distinct phylogenetic lineages.

Genetic determinants of genus-level glycan diversity in a bacterial protein glycosylation system.

The human pathogens N. gonorrhoeae and N. meningitidis display robust intra- and interstrain glycan diversity associated with their O-linked protein glycosylation (pgl) systems. In an effort to better understand the evolution and function of protein glycosylation operating there, we aimed to determine if other human-restricted, Neisseria species similarly glycosylate proteins and if so, to assess the levels of glycoform diversity. Comparative genomics revealed the conservation of a subset of genes minimally required for O-linked protein glycosylation glycan and established those pgl genes as core genome constituents of the genus. In conjunction with mass spectrometric-based glycan phenotyping, we found that extant glycoform repertoires in N. gonorrhoeae, N. meningitidis and the closely related species N. polysaccharea and N. lactamica reflect the functional replacement of a progenitor glycan biosynthetic pathway. This replacement involved loss of pgl gene components of the primordial pathway coincident with the acquisition of two exogenous glycosyltransferase genes. Critical to this discovery was the identification of a ubiquitous but previously unrecognized glycosyltransferase gene (pglP) that has uniquely undergone parallel but independent pseudogenization in N. gonorrhoeae and N. meningitidis. We suggest that the pseudogenization events are driven by processes of compositional epistasis leading to gene decay. Additionally, we documented instances where inter-species recombination influences pgl gene status and creates discordant genetic interactions due ostensibly to the multi-locus nature of pgl gene networks. In summary, these findings provide a novel perspective on the evolution of protein glycosylation systems and identify phylogenetically informative, genetic differences associated with Neisseria species.

Disrupted Synthesis of a Di-N-acetylated Sugar Perturbs Mature Glycoform Structure and Microheterogeneity in the O-Linked Protein Glycosylation System of Neisseria elongata subsp. glycolytica.

The genus Neisseria includes three major species of importance to human health and disease (Neisseria gonorrhoeae, Neisseria meningitidis, and Neisseria lactamica) that express broad-spectrum O-linked protein glycosylation (Pgl) systems. The potential for related Pgl systems in other species in the genus, however, remains to be determined. Using a strain of Neisseria elongata subsp. glycolytica, a unique tetrasaccharide glycoform consisting of di-N-acetylbacillosamine and glucose as the first two sugars followed by a rare sugar whose mass spectrometric fragmentation profile was most consistent with di-N-acetyl hexuronic acid and a N-acetylhexosamine at the nonreducing end has been identified. Based on established mechanisms for UDP-di-N-acetyl hexuronic acid biosynthesis found in other microbes, we searched for genes encoding related pathway components in the N. elongata subsp. glycolytica genome. Here, we detail the identification of such genes and the ensuing glycosylation phenotypes engendered by their inactivation. While the findings extend the conservative nature of microbial UDP-di-N-acetyl hexuronic acid biosynthesis, mutant glycosylation phenotypes reveal unique, relaxed specificities of the glycosyltransferases and oligosaccharyltransferases to incorporate pathway intermediate UDP-sugars into mature glycoforms.IMPORTANCE Broad-spectrum protein glycosylation (Pgl) systems are well recognized in bacteria and archaea. Knowledge of how these systems relate structurally, biochemically, and evolutionarily to one another and to others associated with microbial surface glycoconjugate expression is still incomplete. Here, we detail reverse genetic efforts toward characterization of protein glycosylation mutants of N. elongata subsp. glycolytica that define the biosynthesis of a conserved but relatively rare UDP-sugar precursor. The results show both a significant degree of intra- and transkingdom conservation in the utilization of UDP-di-N-acetyl-glucuronic acid and singular properties related to the relaxed specificities of the N. elongata subsp. glycolytica system.

Structural and genetic analyses of glycan O-acetylation in a bacterial protein glycosylation system: evidence for differential effects on glycan chain length.

O-acetylation is a common modification of bacterial glycoconjugates. By modifying oligosaccharide structure and chemistry, O-acetylation has important consequences for biotic and abiotic recognition events and thus bacterial fitness in general. Previous studies of the broad-spectrum O-linked protein glycosylation in pathogenic Neisseria species (including N. gonorrhoeae and N. meningitidis) have revealed O-acetylation of some of their diverse glycoforms and identified the committed acetylase, PglI. Herein, we extend these observations by using mass spectrometry to examine a complete set of all glycan variants identified to date. Regardless of composition, all glycoforms and all sugars in the oligosaccharide are subject to acetylation in a PglI-dependent fashion with the only exception of di-N-acetyl-bacillosamine. Moreover, multiple sugars in a single oligosaccharide could be simultaneously modified. Interestingly, O-acetylation status was found to be correlated with altered chain lengths of oligosaccharides expressed in otherwise isogenic backgrounds. Models for how this unprecedented phenomenon might arise are discussed with some having potentially important implications for the membrane topology of glycan O-acetylation. Together, the findings provide better insight into how O-acetylation can both directly and indirectly govern glycoform structure and diversity.

Characterization of a Unique Tetrasaccharide and Distinct Glycoproteome in the O-Linked Protein Glycosylation System of Neisseria elongata subsp. glycolytica.

UNLABELLED: Broad-spectrum O-linked protein glycosylation is well characterized in the major Neisseria species of importance to human health and disease. Within strains of Neisseria gonorrhoeae, N. meningitidis, and N. lactamica, protein glycosylation (pgl) gene content and the corresponding oligosaccharide structure are fairly well conserved, although intra- and interstrain variability occurs. The status of such systems in distantly related commensal species, however, remains largely unexplored. Using a strain of deeply branching Neisseria elongata subsp. glycolytica, a heretofore unrecognized tetrasaccharide glycoform consisting of di-N-acetylbacillosamine-glucose-di-N-acetyl hexuronic acid-N-acetylhexosamine (diNAcBac-Glc-diNAcHexA-HexNAc) was identified. Directed mutagenesis, mass spectrometric analysis, and glycan serotyping confirmed that the oligosaccharide is an extended version of the diNAcBac-Glc-based structure seen in N. gonorrhoeae and N. meningitidis generated by the successive actions of PglB, PglC, and PglD and glucosyltransferase PglH orthologues. In addition, a null mutation in the orthologue of the broadly conserved but enigmatic pglG gene precluded expression of the extended glycoform, providing the first evidence that its product is a functional glycosyltransferase. Despite clear evidence for a substantial number of glycoprotein substrates, the major pilin subunit of the endogenous type IV pilus was not glycosylated. The latter finding raises obvious questions as to the relative distribution of pilin glycosylation within the genus, how protein glycosylation substrates are selected, and the overall structure-function relationships of broad-spectrum protein glycosylation. Together, the results of this study provide a foundation upon which to assess neisserial O-linked protein glycosylation diversity at the genus level. IMPORTANCE: Broad-spectrum protein glycosylation systems are well characterized in the pathogenic Neisseria species N. gonorrhoeae and N. meningitidis. A number of lines of evidence indicate that the glycan components in these systems are subject to diversifying selection and suggest that glycan variation may be driven in the context of glycosylation of the abundant and surface-localized pilin protein PilE, the major subunit of type IV pili. Here, we examined protein glycosylation in a distantly related, nonpathogenic neisserial species, Neisseria elongata subsp. glycolytica. This system has clear similarities to the systems found in pathogenic species but makes novel glycoforms utilizing a glycosyltransferase that is widely conserved at the genus level but whose function until now remained unknown. Remarkably, PilE pilin is not glycosylated in this species, a finding that raises important questions about the evolutionary trajectories and overall structure-function relationships of broad-spectrum protein glycosylation systems in bacteria.

Cytochrome c-based domain modularity governs genus-level diversification of electron transfer to dissimilatory nitrite reduction.

The genus Neisseria contains two pathogenic species (N. meningitidis and N. gonorrhoeae) in addition to a number of commensal species that primarily colonize mucosal surfaces in man. Within the genus, there is considerable diversity and apparent redundancy in the components involved in respiration. Here, we identify a unique c-type cytochrome (cN ) that is broadly distributed among commensal Neisseria, but absent in the pathogenic species. Specifically, cN supports nitrite reduction in N. gonorrhoeae strains lacking the cytochromes c5 and CcoP established to be critical to NirK nitrite reductase activity. The c-type cytochrome domain of cN shares high sequence identity with those localized c-terminally in c5 and CcoP and all three domains were shown to donate electrons directly to NirK. Thus, we identify three distinct but paralogous proteins that donate electrons to NirK. We also demonstrate functionality for a N. weaverii NirK variant with a C-terminal c-type heme extension. Taken together, modular domain distribution and gene rearrangement events related to these respiratory electron carriers within Neisseria are concordant with major transitions in the macroevolutionary history of the genus. This work emphasizes the importance of denitrification as a selectable trait that may influence speciation and adaptive diversification within this largely host-restricted bacterial genus.

Type IV pilus assembly proficiency and dynamics influence pilin subunit phospho-form macro- and microheterogeneity in Neisseria gonorrhoeae.

The PilE pilin subunit protein of the gonococcal Type IV pilus (Tfp) colonization factor undergoes multisite, covalent modification with the zwitterionic phospho-form modification phosphoethanolamine (PE). In a mutant lacking the pilin-like PilV protein however, PilE is modified with a mixture of PE and phosphocholine (PC). Moreover, intrastrain variation of PilE PC modification levels have been observed in backgrounds that constitutively express PptA (the protein phospho-form transferase A) required for both PE and PC modification. The molecular basis underlying phospho-form microheterogeneity in these instances remains poorly defined. Here, we examined the effects of mutations at numerous loci that disrupt or perturb Tfp assembly and observed that these mutants phenocopy the pilV mutant vis a vis phospho-form modification status. Thus, PC modification appears to be directly or indirectly responsive to the efficacy of pilin subunit interactions. Despite the complexity of contributing factors identified here, the data favor a model in which increased retention in the inner membrane may act as a key signal in altering phospho-form modification. These results also provide an alternative explanation for the variation in PilE PC levels observed previously and that has been assumed to be due to phase variation of pptA. Moreover, mass spectrometry revealed evidence for mono- and di-methylated forms of PE attached to PilE in mutants deficient in pilus assembly, directly implicating a methyltransferase-based pathway for PC synthesis in N. gonorrhoeae.

Allelic variation in a simple sequence repeat element of neisserial pglB2 and its consequences for protein expression and protein glycosylation.

Neisseria species express an O-linked glycosylation system in which functionally distinct proteins are elaborated with variable glycans. A major source of glycan diversity in N. meningitidis results from two distinct pglB alleles responsible for the synthesis of either N,N'-diacetylbacillosamine or glyceramido-acetamido trideoxyhexose that occupy the reducing end of the oligosaccharides. Alternative modifications at C-4 of the precursor UDP-4-amino are attributable to distinct C-terminal domains that dictate either acetyltransferase or glyceramidotransferase activity, encoded by pglB and pglB2, respectively. Naturally occurring alleles of pglB2 have homopolymeric tracts of either 7 or 8 adenosines (As) bridging the C-terminal open reading frame (ORF) and the ORF encompassing the conserved N-terminal domain associated with phosphoglycosyltransferase activity. In the work presented here, we explored the consequences of such pglB2 allele variation and found that, although both alleles are functional vis-à-vis glycosylation, the 7A form results in the expression of a single, multidomain protein, while the 8A variant elicits two single-domain proteins. We also found that the glyceramidotransferase activity-encoding domain is essential to protein glycosylation, showing the critical role of the C-4 modification of the precursor UDP-4-amino in the pathway. These findings were further extended and confirmed by examining the phenotypic consequences of extended poly(A) tract length variation. Although ORFs related to those of pglB2 are broadly distributed in eubacteria, they are primarily found as two distinct, juxtaposed ORFs. Thus, the neisserial pglB2 system provides novel insights into the potential influence of hypermutability on modular evolution of proteins by providing a unique snapshot of the progression of ongoing gene fusion.

An extended spectrum of target proteins and modification sites in the general O-linked protein glycosylation system in Neisseria gonorrhoeae.

The bacterial human pathogen Neisseria gonorrhoeae expresses a general O-linked protein glycosylation (Pgl) system known to target at least 12 membrane-associated proteins. To facilitate a better understanding of the mechanisms, significance and function of this glycosylation system, we sought to further delineate the target proteome of the Pgl system. To this end, we employed immunoaffinity enrichment of glycoproteins using a monoclonal antibody against the glycan moiety. Enzymatically generated peptides were subsequently analyzed by MS to identify glycopeptides and glycosylation sites. In this way, we increase the total number of known glycoproteins in N. gonorrhoeae to 19. These new glycoproteins are involved in a wide variety of extracytoplasmic functions. By employing collision fragmentation, we mapped nine new glycosylation sites, all of which were serine. No target sequon was readily apparent, although attachment sites were most often localized with regions of low sequence complexity. Moreover, we found that 5 of the proteins were modified with more than one glycan. This work thus confirms and extends earlier observations on the structural features of Neisseria glycoproteins.

Insights into type IV pilus biogenesis and dynamics from genetic analysis of a C-terminally tagged pilin: a role for O-linked glycosylation.

Type IV pili are surface organelles essential for pathogenicity of many Gram-negative bacteria. In Neisseria gonorrhoeae, the major subunit of type IV pili, PilE, is a target of its general O-linked glycosylation system. This system modifies a diverse set of periplasmic and extracellular gonococcal proteins with a variable set of glycans. Here we show that expression of a particular hexa-histidine-tagged PilE was associated with growth arrest. By studying intra- and extragenic suppressors, we found that this phenotype was dependent on pilus assembly and retraction. Based on these results, we developed a sensitive tool to identify factors with subtle effects on pilus dynamics. Using this approach, we found that glycan chain length has differential effects on the growth arrest that appears to be mediated at the level of pilin subunit-subunit interactions and bidirectional remodelling of pilin between its membrane-associated and assembled states. Gonococcal pilin glycosylation thus plays both an intracellular role in pilus dynamics and potential extracellular roles mediated through type IV pili. In addition to demonstrating the effect of glycosylation on pilus dynamics, the study provides a new way of identifying factors with less dramatic effects on processes involved in type IV pilus biogenesis.

Novel protein substrates of the phospho-form modification system in Neisseria gonorrhoeae and their connection to O-linked protein glycosylation.

The zwitterionic phospho-form moieties phosphoethanolamine (PE) and phosphocholine (PC) are important components of bacterial membranes and cell surfaces. The major type IV pilus subunit protein of Neisseria gonorrhoeae, PilE, undergoes posttranslational modifications with these moieties via the activity of the pilin phospho-form transferase PptA. A number of observations relating to colocalization of phospho-form and O-linked glycan attachment sites in PilE suggested that these modifications might be either functionally or mechanistically linked or interact directly or indirectly. Moreover, it was unknown whether the phenomenon of phospho-form modification was solely dedicated to PilE or if other neisserial protein targets might exist. In light of these concerns, we screened for evidence of phospho-form modification on other membrane glycoproteins targeted by the broad-spectrum O-linked glycosylation system. In this way, two periplasmic lipoproteins, NGO1043 and NGO1237, were identified as substrates for PE addition. As seen previously for PilE, sites of PE modifications were clustered with those of glycan attachment. In the case of NGO1043, evidence for at least six serine phospho-form attachment sites was found, and further analyses revealed that at least two of these serines were also attachment sites for glycan. Finally, mutations altering glycosylation status led to the presence of pptA-dependent PC modifications on both proteins. Together, these results reinforce the associations established in PilE and provide evidence for dynamic interplay between phospho-form modification and O-linked glycosylation. The observations also suggest that phospho-form modifications likely contribute biologically at both intracellular and extracellular levels.

Structural alterations in a component of cytochrome c oxidase and molecular evolution of pathogenic Neisseria in humans.

Three closely related bacterial species within the genus Neisseria are of importance to human disease and health. Neisseria meningitidis is a major cause of meningitis, while Neisseria gonorrhoeae is the agent of the sexually transmitted disease gonorrhea and Neisseria lactamica is a common, harmless commensal of children. Comparative genomics have yet to yield clear insights into which factors dictate the unique host-parasite relationships exhibited by each since, as a group, they display remarkable conservation at the levels of nucleotide sequence, gene content and synteny. Here, we discovered two rare alterations in the gene encoding the CcoP protein component of cytochrome cbb(3) oxidase that are phylogenetically informative. One is a single nucleotide polymorphism resulting in CcoP truncation that acts as a molecular signature for the species N. meningitidis. We go on to show that the ancestral ccoP gene arose by a unique gene duplication and fusion event and is specifically and completely distributed within species of the genus Neisseria. Surprisingly, we found that strains engineered to express either of the two CcoP forms conditionally differed in their capacity to support nitrite-dependent, microaerobic growth mediated by NirK, a nitrite reductase. Thus, we propose that changes in CcoP domain architecture and ensuing alterations in function are key traits in successive, adaptive radiations within these metapopulations. These findings provide a dramatic example of how rare changes in core metabolic proteins can be connected to significant macroevolutionary shifts. They also show how evolutionary change at the molecular level can be linked to metabolic innovation and its reversal as well as demonstrating how genotype can be used to infer alterations of the fitness landscape within a single host.

Genetic, structural, and antigenic analyses of glycan diversity in the O-linked protein glycosylation systems of human Neisseria species.

Bacterial capsular polysaccharides and lipopolysaccharides are well-established ligands of innate and adaptive immune effectors and often exhibit structural and antigenic variability. Although many surface-localized glycoproteins have been identified in bacterial pathogens and symbionts, it not clear if and how selection impacts associated glycoform structure. Here, a systematic approach was devised to correlate gene repertoire with protein-associated glycoform structure in Neisseria species important to human health and disease. By manipulating the protein glycosylation (pgl) gene content and assessing the glycan structure by mass spectrometry and reactivity with monoclonal antibodies, it was established that protein-associated glycans are antigenically variable and that at least nine distinct glycoforms can be expressed in vitro. These studies also revealed that in addition to Neisseria gonorrhoeae strain N400, one other gonococcal strain and isolates of Neisseria meningitidis and Neisseria lactamica exhibit broad-spectrum O-linked protein glycosylation. Although a strong correlation between pgl gene content, glycoform expression, and serological profile was observed, there were significant exceptions, particularly with regard to levels of microheterogeneity. This work provides a technological platform for molecular serotyping of neisserial protein glycans and for elucidating pgl gene evolution.

Broad spectrum O-linked protein glycosylation in the human pathogen Neisseria gonorrhoeae.

Protein glycosylation is an important element of biologic systems because of its significant effects on protein properties and functions. Although prominent within all domains of life, O-linked glycosylation systems modifying serine and threonine residues within bacteria and eukaryotes differ substantially in target protein selectivity. In particular, well-characterized bacterial systems have been invariably dedicated to modification of individual proteins or related subsets thereof. Here we characterize a general O-linked glycosylation system that targets structurally and functionally diverse groups of membrane-associated proteins in the gram-negative bacterium Neisseria gonorrhoeae, the etiologic agent of the human disease gonorrhea. The 11 glycoproteins identified here are implicated in activities as varied as protein folding, disulfide bond formation, and solute uptake, as well as both aerobic and anaerobic respiration. Along with their common trafficking within the periplasmic compartment, the protein substrates share quasi-related domains bearing signatures of low complexity that were demonstrated to encompass sites of glycan occupancy. Thus, as in eukaryotes, the broad scope of this system is dictated by the relaxed specificity of the glycan transferase as well as the bulk properties and context of the protein-targeting signal rather than by a strict amino acid consensus sequence. Together, these findings reveal previously unrecognized commonalities linking O-linked protein glycosylation in distantly related life forms.

Neisseria gonorrhoeae O-linked pilin glycosylation: functional analyses define both the biosynthetic pathway and glycan structure.

Neisseria gonorrhoeae expresses an O-linked protein glycosylation pathway that targets PilE, the major pilin subunit protein of the Type IV pilus colonization factor. Efforts to define glycan structure and thus the functions of pilin glycosylation (Pgl) components at the molecular level have been hindered by the lack of sensitive methodologies. Here, we utilized a 'top-down' mass spectrometric approach to characterize glycan status using intact pilin protein from isogenic mutants. These structural data enabled us to directly infer the function of six components required for pilin glycosylation and to define the glycan repertoire of strain N400. Additionally, we found that the N. gonorrhoeae pilin glycan is O-acetylated, and identified an enzyme essential for this unique modification. We also identified the N. gonorrhoeae pilin oligosaccharyltransferase using bioinformatics and confirmed its role in pilin glycosylation by directed mutagenesis. Finally, we examined the effects of expressing the PglA glycosyltransferase from the Campylobacter jejuni N-linked glycosylation system that adds N-acetylgalactosamine onto undecaprenylpyrophosphate-linked bacillosamine. The results indicate that the C. jejuni and N. gonorrhoeae pathways can interact in the synthesis of O-linked di- and trisaccharides, and therefore provide the first experimental evidence that biosynthesis of the N. gonorrhoeae pilin glycan involves a lipid-linked oligosaccharide precursor. Together, these findings underpin more detailed studies of pilin glycosylation biology in both N. gonorrhoeae and N. meningitidis, and demonstrate how components of bacterial O- and N-linked pathways can be combined in novel glycoengineering strategies.

A Pseudomonas aeruginosa quorum-sensing molecule influences Candida albicans morphology.

Candida albicans is an opportunistic pathogen that is commonly found as a member of the human microflora. The ability of C. albicans to alter its cellular morphology has been associated with its virulence; yeast cells are more prevalent in commensal interactions whereas filamentous cells appear important in opportunistic infections. C. albicans encounters a multitude of other microbial species in the host environment and it is likely that they impact the C. albicans transition between virulent and non-virulent states. Here, we report that C. albicans morphology is significantly affected by the presence of Pseudomonas aeruginosa, another opportunistic pathogen. In a screen using a C. albicans HWP1-lacZ strain to indicate regions of filamentous growth, we identified P. aeruginosa mutants incapable of inhibiting C. albicans filamentation. Through these studies, we found that 3-oxo-C12 homoserine lactone, a cell-cell signalling molecule produced by P. aeruginosa, was sufficient to inhibit C. albicans filamentation without affecting fungal growth rates. Both microscopic analysis and real-time reverse transcription polymerase chain reaction analysis of morphology-specific markers confirmed that filamentation was suppressed by 200 microM 3-oxo-C12 homoserine lactone. Structurally related compounds with a 12-carbon chain length, e.g. C12-acyl homoserine lactone and dodecanol also affected C. albicans filamentation at similar concentrations. In contrast, other acylated homoserine lactones of different chain lengths did not affect fungal morphology. The activity of 3OC12HSL is compared with that of farnesol, a C. albicans-produced molecule also with a C12-backbone. The effects that bacteria have on the morphology of C. albicans represents one of the ways by which bacteria can influence the behaviour of fungal cells.

The SAT1 flipper, an optimized tool for gene disruption in Candida albicans.

The construction of Candida albicans mutants by targeted gene disruption usually depends on the use of nutritional markers for the selection of prototrophic transformants from auxotrophic host strains, but it is becoming increasingly evident that this strategy may cause difficulties in the interpretation of mutant phenotypes. Here, we describe a new method for inactivating both alleles of a target gene in C. albicans wild-type strains to obtain homozygous null mutants. The SAT1 flipping method relies on the use of a cassette that contains a dominant nourseothricin resistance marker (caSAT1) for the selection of integrative transformants and a C. albicans-adapted FLP gene that allows the subsequent excision of the cassette, which is flanked by FLP target sequences, from the genome. Two rounds of integration/excision generate homozygous mutants that differ from the wild-type parent strain only by the absence of the target gene, and reintegration of an intact gene copy for complementation of mutant phenotypes is performed in the same way. Transformants are obtained after only 1 day of growth on a selective medium, and integration into the target locus occurs with high specificity after adding homologous flanking sequences on both sides of the cassette. FLP-mediated excision of the SAT1 flipper cassette is achieved by simply growing the transformants for a few hours in medium without selective pressure, and nourseothricin-sensitive (NouS) derivatives can easily be identified by their slower growth on indicator plates containing a low concentration of nourseothricin. We demonstrate the use of the system by deleting the OPT1 gene, which encodes an oligopeptide transporter, in the C. albicans model strain SC5314. The null mutants became resistant to the toxic peptide KLLEth, and reintroduction of an intact OPT1 copy restored susceptibility. The SAT1 flipping method provides a highly efficient method for gene disruption in C. albicans wild-type strains, which eliminates currently encountered problems in the genetic analysis of this important human fungal pathogen.