Construction of otherwise isogenic serotype 6B, 7F, 14, and 19F capsular variants of Streptococcus pneumoniae strain TIGR4

The polysaccharide capsule is the primary virulence factor in Streptococcus pneumoniae. There are at least 90 serotypes of S. pneumoniae, identified based on the immunogenicity of different capsular sugars. The aim of this study was to construct pneumococcal strains that are isogenic except for capsular type. Serotype 4 strain TIGR4 was rendered unencapsulated by recombinational replacement of the capsular polysaccharide synthesis (cps) locus with the bicistronic Janus cassette (C. K. Sung, J. P. Claverys, and D. A. Morrison, Appl. Environ. Microbiol. 67:5190-5196, 2001). In subsequent transformation with chromosomal DNA, the cassette was replaced by the cps locus derived from a strain of a different serotype, either 6B, 7F, 14, or 19F. To minimize the risk of uncontrolled recombinational replacements in loci other than cps, the TIGRcps::Janus strain was "backcross" transformed three times with chromosomal DNA of subsequently constructed capsular type transformants. Capsular serotypes were confirmed in all new capsule variants by the Quellung reaction. Restriction fragment length polymorphism (RFLP) analysis of the cps locus confirmed the integrity of the cps region transformed into the TIGR strain, and RFLP of the flanking regions confirmed their identities with the corresponding regions of the recipient. Transformants had in vitro growth rates greater than or equal to that of TIGR4. All four strains were able to colonize C57BL/6 mice (female, 6 weeks old) for at least 7 days when mice were intranasally inoculated with 6 x 10(6) to 8 x 10(6) CFU. The constructed capsular variants of TIGR4 are suitable for use in studies on the role of S. pneumoniae capsular polysaccharide in immunity, colonization, and pathogenesis.     

Molecular and genetic characterization of the capsule biosynthesis locus of Streptococcus pneumoniae type 19B.

We have previously reported the nucleotide sequence of the Streptococcus pneumoniae type 19F capsular polysaccharide synthesis locus (cps19f), which consists of 15 open reading frames (ORFs) designated cps19fA to -O. Hybridization analysis indicated that close homologs for cps19fA to -H and cps19fK to -O were found in type 19B, but there were no homologs for cps19fI and -J. In this study we used long-range PCR to amplify and clone a 10.5-kb section of the S. pneumoniae type 19B capsule locus (cps19b) between cps19bH and cps19bK. This region of the cps19b locus is 4 kb larger than that in the cps19f locus and replaces cps19fI and cps19fJ with five new ORFs, designated cps19bP, -I, -Q, -R, and -J. We have proposed functions for four of the protein products, including functional homologs of Cps19fI and Cps19fJ. Transformation of a S. pneumoniae mutant containing an interrupted type 19F capsule locus with the 10.5-kb cps19b PCR product converted the recipient strain to type 19B. Southern hybridization analysis indicated that cps19bP, -I, -Q, -R, and -J are unique to type 19B and the closely related type 19C.     

Construction of Otherwise Isogenic Serotype 6B, 7F, 14, and 19F Capsular Variants of Streptococcus pneumoniae Strain TIGR4

The polysaccharide capsule is the primary virulence factor in Streptococcus pneumoniae. There are at least 90 serotypes of S. pneumoniae, identified based on the immunogenicity of different capsular sugars. The aim of this study was to construct pneumococcal strains that are isogenic except for capsular type. Serotype 4 strain TIGR4 was rendered unencapsulated by recombinational replacement of the capsular polysaccharide synthesis (cps) locus with the bicistronic Janus cassette (C. K. Sung, J. P. Claverys, and D. A. Morrison, Appl. Environ. Microbiol. 67:5190-5196, 2001). In subsequent transformation with chromosomal DNA, the cassette was replaced by the cps locus derived from a strain of a different serotype, either 6B, 7F, 14, or 19F. To minimize the risk of uncontrolled recombinational replacements in loci other than cps, the TIGRcps::Janus strain was “backcross” transformed three times with chromosomal DNA of subsequently constructed capsular type transformants. Capsular serotypes were confirmed in all new capsule variants by the Quellung reaction. Restriction fragment length polymorphism (RFLP) analysis of the cps locus confirmed the integrity of the cps region transformed into the TIGR strain, and RFLP of the flanking regions confirmed their identities with the corresponding regions of the recipient. Transformants had in vitro growth rates greater than or equal to that of TIGR4. All four strains were able to colonize C57BL/6 mice (female, 6 weeks old) for at least 7 days when mice were intranasally inoculated with 6 × 10⁶ to 8 × 10⁶ CFU. The constructed capsular variants of TIGR4 are suitable for use in studies on the role of S. pneumoniae capsular polysaccharide in immunity, colonization, and pathogenesis.     

Vaccine escape recombinants emerge after pneumococcal vaccination in the United States

The heptavalent pneumococcal conjugate vaccine (PCV7) was introduced in the United States (US) in 2000 and has significantly reduced invasive pneumococcal disease; however, the incidence of nonvaccine serotype invasive disease, particularly due to serotype 19A, has increased. The serotype 19A increase can be explained in part by expansion of a genotype that has been circulating in the US prior to vaccine implementation (and other countries since at least 1990), but also by the emergence of a novel "vaccine escape recombinant" pneumococcal strain. This strain has a genotype that previously was only associated with vaccine serotype 4, but now expresses a nonvaccine serotype 19A capsule. Based on prior evidence for capsular switching by recombination at the capsular locus, the genetic event that resulted in this novel serotype/genotype combination might be identifiable from the DNA sequence of individual pneumococcal strains. Therefore, the aim of this study was to characterise the putative recombinational event(s) at the capsular locus that resulted in the change from a vaccine to a nonvaccine capsular type. Sequencing the capsular locus flanking regions of 51 vaccine escape (progeny), recipient, and putative donor pneumococci revealed a 39 kb recombinational fragment, which included the capsular locus, flanking regions, and two adjacent penicillin-binding proteins, and thus resulted in a capsular switch and penicillin nonsusceptibility in a single genetic event. Since 2003, 37 such vaccine escape strains have been detected, some of which had evolved further. Furthermore, two new types of serotype 19A vaccine escape strains emerged in 2005. To our knowledge, this is the first time a single recombinational event has been documented in vivo that resulted in both a change of serotype and penicillin nonsusceptibility. Vaccine escape by genetic recombination at the capsular locus has the potential to reduce PCV7 effectiveness in the longer term.     

Evolutionary genetics of the capsular locus of serogroup 6 pneumococci

The evolution of the capsular biosynthetic (cps) locus of serogroup 6 Streptococcus pneumoniae was investigated by analyzing sequence variation within three serotype-specific cps genes from 102 serotype 6A and 6B isolates. Sequence variation within these cps genes was related to the genetic relatedness of the isolates, determined by multilocus sequence typing, and to the inferred patterns of recent evolutionary descent, explored using the eBURST algorithm. The serotype-specific cps genes had a low percent G+C, and there was a low level of sequence diversity in this region among serotype 6A and 6B isolates. There was also little sequence divergence between these serotypes, suggesting a single introduction of an ancestral cps sequence, followed by slight divergence to create serotypes 6A and 6B. A minority of serotype 6B isolates had cps sequences (class 2 sequences) that were approximately 5% divergent from those of other serotype 6B isolates (class 1 sequences) and which may have arisen by a second, more recent introduction from a related but distinct source. Expression of a serotype 6A or 6B capsule correlated perfectly with a single nonsynonymous polymorphism within wciP, the rhamnosyl transferase gene. In addition to ample evidence of the horizontal transfer of the serotype 6A and 6B cps locus into unrelated lineages, there was evidence for relatively frequent changes from serotype 6A to 6B, and vice versa, among very closely related isolates and examples of recent recombinational events between class 1 and 2 cps serogroup 6 sequences.     

The encapsulated strain TIGR4 of Streptococcus pneumoniae is phagocytosed but is resistant to intracellular killing by mouse microglia

The polysaccharide capsule is a major virulence factor of Streptococcus pneumoniae as it confers resistance to phagocytosis. The encapsulated serotype 4 TIGR4 strain was shown to be efficiently phagocytosed by the mouse microglial cell line BV2, whereas the type 3 HB565 strain resisted phagocytosis. Comparing survival after uptake of TIGR4 or its unencapsulated derivative FP23 in gentamicin protection and phagolysosome maturation assays, it was shown that TIGR4 was protected from intracellular killing. Pneumococcal capsular genes were up-regulated in intracellular TIGR4 bacteria recovered from microglial cells. Actual presence of bacteria inside BV2 cells was confirmed by transmission electron microscopy (TEM) for both TIGR4 and FP23 strains, but typical phagosomes/phagolysosomes were detected only in cells infected with the unencapsulated strain. In a mouse model of meningitis based on intracranic inoculation of pneumococci, TIGR4 caused lethal meningitis with an LD₅₀ of 2 × 10² CFU, whereas the LD₅₀ for the unencapsulated FP23 was greater than 10⁷ CFU. Phagocytosis of TIGR4 by microglia was also demonstrated by TEM and immunohistochemistry on brain samples from infected mice. The results indicate that encapsulation does not protect the TIGR4 strain from phagocytosis by microglia, while it affords resistance to intracellular killing.     

Changes in capsular serotype alter the surface exposure of pneumococcal adhesins and impact virulence

We examined the contribution of serotype on Streptococcus pneumoniae adhesion and virulence during respiratory tract infection using a panel of isogenic TIGR4 (serotype 4) mutants expressing the capsule types 6A (+6A), 7F (+7F) and 23F (+23F) as well as a deleted and restored serotype 4 (+4) control strain. Immunoblots, bacterial capture assays with immobilized antibody, and measurement of mean fluorescent intensity by flow cytometry following incubation of bacteria with antibody, all determined that the surface accessibility, but not total protein levels, of the virulence determinants Pneumococcal surface protein A (PspA), Choline binding protein A (CbpA), and Pneumococcal serine-rich repeat protein (PsrP) changed with serotype. In vitro, bacterial adhesion to Detroit 562 pharyngeal or A549 lung epithelial cells was modestly but significantly altered for +6A, +7F and +23F. In a mouse model of nasopharyngeal colonization, the number of +6A, +7F, and +23F pneumococci in the nasopharynx was reduced 10 to 100-fold versus +4; notably, only mice challenged with +4 developed bacteremia. Intratracheal challenge of mice confirmed that capsule switch strains were highly attenuated for virulence. Compared to +4, the +6A, +7F, and +23F strains were rapidly cleared from the lungs and were not detected in the blood. In mice challenged intraperitoneally, a marked reduction in bacterial blood titers was observed for those challenged with +6A and +7F versus +4 and +23F was undetectable. These findings show that serotype impacts the accessibility of surface adhesins and, in particular, affects virulence within the respiratory tract. They highlight the complex interplay between capsule and protein virulence determinants.     

The serotype of type Ia and III group B streptococci is determined by the polymerase gene within the polycistronic capsule operon

Streptococcus agalactiae is a primary cause of neonatal morbidity and mortality. Essential to the virulence of this pathogen is the production of a type-specific capsular polysaccharide (CPS) that enables the bacteria to evade host immune defenses. The identification, cloning, sequencing, and functional characterization of seven genes involved in type III capsule production have been previously reported. Here, we describe the cloning and sequencing of nine additional adjacent genes, cps(III)FGHIJKL, neu(III)B, and neu(III)C. Sequence comparisons suggested that these genes are involved in sialic acid synthesis, pentasaccharide repeating unit formation, and oligosaccharide transport and polymerization. The type III CPS (cpsIII) locus was comprised of 16 genes within 15.5 kb of contiguous chromosomal DNA. Primer extension analysis and investigation of mRNA from mutants with polar insertions in their cpsIII loci supported the hypothesis that the operon is transcribed as a single polycistronic message. The translated cpsIII sequences were compared to those of the S. agalactiae cpsIa locus, and the primary difference between the operons was found to reside in cps(III)H, the putative CPS polymerase gene. Expression of cps(III)H in a type Ia strain resulted in suppression of CPS Ia synthesis and in production of a CPS which reacted with type III-specific polyclonal antibody. Likewise, expression of the putative type Ia polymerase gene in a type III strain reduced synthesis of type III CPS with production of a type Ia immunoreactive capsule. Based on the similar structures of the oligosaccharide repeating units of the type Ia and III capsules, our observations demonstrated that cps(Ia)H and cps(III)H encoded the type Ia and III CPS polymerases, respectively. Additionally, these findings suggested that a single gene can confer serotype specificity in organisms that produce complex polysaccharides.     

Recombinational exchanges at the capsular polysaccharide biosynthetic locus lead to frequent serotype changes among natural isolates of Streptococcus pneumoniae

Serotype 19F variants of the major Spanish multiresistant serotype 23F clone of Streptococcus pneumoniae have been proposed to have arisen by recombinational exchanges at the capsular biosynthetic locus. Members of the Spanish multiresistant serotype 23F clone and the serotype 19F variants were confirmed to be essentially identical in overall genotype, as they were indistinguishable by REP-PCR, and had identical sequences at three polymorphic housekeeping genes. Eight serotype 19F variants were studied and all had large recombinational replacements at the capsular biosynthetic locus. In all cases, one of the recombinational cross-over points appeared to be upstream of dexB which flanks one end of the capsular locus, and in six of the variants the other cross-over point was downstream of aliA , which flanks the other end of the locus. In two strains a recombinational cross-over point between the introduced serotype 19F capsular region and that of the Spanish serotype 23F clone could be clearly identified, within cpsN in one strain and within cpsM in the other. The differences in the recombinational junctions and sequence polymorphisms within the introduced capsular genes, suggested that the eight serotype 19F variants emerged on at least four separate occasions. Changes in capsular type by recombination may therefore be relatively frequent in pneumococci and this has implications for the long-term efficacy of conjugate pneumococcal vaccines that will protect against only a limited number of serotypes.     

The capsule polysaccharide synthesis locus of streptococcus pneumoniae serotype 14: Identification of the glycosyl transferase gene cps14E.

To identify a chromosomal region of Streptococcus pneumoniae serotype 14 involved in capsule polysaccharide synthesis, two strategies were used: (i) Tn916 mutagenesis, followed by the characterization of four unencapsulated mutants, and (ii) cross-hybridization with a capsule polysaccharide synthesis gene (cps) probe from S. agalactiae, which has a structurally similar capsule. The two approaches detected the same chromosomal region consisting of two adjacent EcoRI fragments. One of these EcoRI fragments was cloned and hybridized with a cosmid library. This resulted in clone cMKO2. A similar cosmid clone was obtained from an unencapsulated Tn916 mutant, Spnl4.H. Sequence analysis of the two cosmid clones revealed that in the Tn916 mutant, a gene, cps14E, which is homologous to other bacterial genes encoding glycosyl transferases, had been inactivated. An open reading frame immediately downstream of cps14E, designated cps14F, shows no significant homology with any known genes or proteins. A functional assay showed that cps14E encodes a glycosyl transferase and that a gene-specific knockout mutant lacks this enzyme activity, whereas inactivation of cps14F does not have this effect.     

Comparative structural and molecular characterization of Streptococcus pneumoniae capsular polysaccharide serogroup 10

Streptococcus pneumoniae serogroup 10 includes four cross-reactive capsular polysaccharide (CPS) serotypes (10F, 10A, 10B, and 10C). In the present study, the structures of CPS10B and CPS10C were determined by chemical and high resolution NMR methods to define the features of each serotype. Both CPS10C and CPS10F had β1-6-linked Galf branches formed from the termini of linear repeating units by wzy-dependent polymerization through the 4-OH of subterminal GalNAc. The only difference between these polysaccharides was the wcrC-dependent α1-2 or wcrF-dependent α1-4 linkages between Gal and ribitol-5-phosphate. The presence of one linkage or the other also distinguished the repeating units of CPS10B and CPS10A. However, whereas these polysaccharides both had β1-3-linked Galf branches linked to GalNAc, only CPS10A had additional β1-6-linked Galp branches. These Galp branches and the reaction of a CPS10A-specific monoclonal antibody were eliminated by deletion of wcrG from the cps10A locus. In contrast, deletion of this gene from the cps10B locus had no effect on the structure of CPS10B, thereby identifying wcrG as a pseudogene in this serotype. The β1-3-linked Galf branches of CPS10A and CPS10B were eliminated by deletion of wcrD from each corresponding cps locus. Deletion of this gene also eliminated wcrG-dependent β1-6-linked Galp branches from CPS10A, thereby identifying WcrG as a branching enzyme that acts on the product of WcrD. These findings provide a complete view of the molecular, structural, and antigenic features of CPS serogroup 10, as well as insight into the possible emergence of new serotypes.     

Positive correlation between tyrosine phosphorylation of CpsD and capsular polysaccharide production in Streptococcus pneumoniae

CpsA, CpsB, CpsC, and CpsD are part of a tyrosine phosphorylation regulatory system involved in modulation of capsule synthesis in Streptococcus pneumoniae and many other gram-positive and gram-negative bacteria. Using an immunoblotting technique, we observed distinct laddering patterns of S. pneumoniae capsular polysaccharides of various serotypes and found that transfer of the polymer from the membrane to the cell wall was independent of size. Deletion of cps2A, cps2B, cps2C, or cps2D in the serotype 2 strain D39 did not affect the ability to transfer capsule to the cell wall. Deletion of cps2C or cps2D, which encode two domains of an autophosphorylating tyrosine kinase, resulted in the production of only short-chain polymers. The function of Cps2A is unknown, and the polymer laddering pattern of the cps2A deletion mutants appeared similar to that of the parent, although the total amount of capsule was decreased. Loss of Cps2B, a tyrosine phosphatase and a kinase inhibitor, resulted in an increase in capsule amount and a normal ladder pattern. However, Cps2B mutants exhibited reduced virulence following intravenous inoculation of mice and were unable to colonize the nasopharynx, suggesting a diminished capacity to sense or respond to these environments. In D39 and its isogenic mutants, the amounts of capsule and tyrosine-phosphorylated Cps2D (Cps2D approximately P) correlated directly. In contrast, restoration of type 2 capsule production followed by deletion of cps2B in Rx1, a laboratory passaged D39 derivative containing multiple uncharacterized mutations, resulted in decreased capsule amounts but no alteration in Cps2D approximately P levels. Thus, a factor outside the capsule locus, which is either missing or defective in the Rx1 background, is important in the control of capsule synthesis.     

Phosphorylation of Wzc, a tyrosine autokinase, is essential for assembly of group 1 capsular polysaccharides in Escherichia coli

Wzc proteins are tyrosine autokinases. They are found in some important bacterial pathogens of humans and livestock as well as plant-associated bacteria, and are often encoded within gene clusters determining synthesis and assembly of capsular and extracellular polysaccharides. Autophosphorylation of Wzc(cps) is essential for assembly of the serotype K30 group 1 capsule in Escherichia coli O9a:K30, although a genetically unlinked Wzc(cps)-homologue (Etk) can also participate with low efficiency. While autophosphorylation of Wzc(cps) is required for assembly of high molecular weight K30 capsular polysaccharide, it is not essential for either the synthesis of the K30 repeat units or for activity of the K30 polymerase enzyme. Paradoxically, the cognate phosphotyrosine protein phosphatase for Wzc(cps), Wzb(cps), is also required for capsule expression. The tyrosine-rich domain at the C terminus of Wzc(cps) was identified as the site of phosphorylation and autophosphorylation of Wzc requires a functional Walker A motif. Intermolecular transphosphorylation of Wzc(cps) was detected in strains expressing a combination of mutant Wzc(cps) derivatives. The N- and C-terminal domains of Wzc(cps) were expressed independently to mimic the situation found naturally in Gram-positive bacteria. In this format, both domains were required for phosphorylation of the Wzc(cps) C terminus, and for capsule assembly. Regulation by a post-translational phosphorylation event represents a new dimension in the assembly of bacterial cell-surface polysaccharides.     

Formation of Streptococcus pneumoniae non-phase-variable colony variants is due to increased mutation frequency present under biofilm growth conditions

In this report, we show that biofilm formation by Streptococcus pneumoniae serotype 19 gives rise to variants (the small mucoid variant [SMV] and the acapsular small-colony variant [SCV]) differing in capsule production, attachment, and biofilm formation compared to wild-type strains. All biofilm-derived variants harbored SNPs in cps19F. SCVs reverted to SMV, but no reversion to the wild-type phenotype was noted, indicating that these variants were distinct from opaque- and transparent-phase variants. The SCV-SMV reversion frequency was dependent on growth conditions and treatment with tetracycline. Increased reversion rates were coincident with antibiotic treatment, implicating oxidative stress as a trigger for the SCV-SMV switch. We, therefore, evaluated the role played by hydrogen peroxide, the oxidizing chemical, in the reversion and emergence of variants. Biofilms of S. pneumoniae TIGR4-ΔspxB, defective in hydrogen peroxide production, showed a significant reduction in variant formation. Similarly, supplementing the medium with catalase or sodium thiosulfate yielded a significant reduction in variants formed by wild-type biofilms. Resistance to rifampin, an indicator for mutation frequency, was found to increase approximately 55-fold in biofilms compared to planktonic cells for each of the three wild-type strains examined. In contrast, TIGR4-ΔspxB grown as a biofilm showed no increase in rifampin resistance compared to the same cells grown planktonically. Furthermore, addition of 2.5 and 10 mM hydrogen peroxide to planktonic cells resulted in a 12- and 160-fold increase in mutation frequency, respectively, and gave rise to variants similar in appearance, biofilm-related phenotypes, and distribution of biofilm-derived variants. The results suggest that hydrogen peroxide and environmental conditions specific to biofilms are responsible for the development of non-phase-variable colony variants.     

Analysis of the 5' portion of the type 19A capsule locus identifies two classes of cpsC, cpsD, and cpsE genes in Streptococcus pneumoniae.

Analysis of the sequence data obtained from the 5' portion of the Streptococcus pneumoniae type 19A capsular polysaccharide biosynthesis locus (cps19a) revealed that the first seven genes are homologous to the first seven genes in the type 19F (cps19f) locus. The former genes were designated cps19aA to -G and were 70 to 90% identical to their cps19f counterparts. Southern hybridization analysis of the cps loci from various S. pneumoniae serotypes with probes specific for the cps19aC, cps19aD, and cps19aE genes indicated a hybridization pattern complementary to that previously reported for cps19fC, cps19fD, and cps19fE. That is, all serotypes tested contained high-stringency homologues of either the cps19aC to -E genes or the cps19fC to -E genes, but not both. On this basis S. pneumoniae cps loci can be divided into two distinct classes. Long-range PCR was used to amplify the cps regions between cpsB and aliA from a variety of pneumococcal serotypes. Direct sequencing of the 5' end of these PCR products, and phylogenetic analysis of the sequence data, confirmed the presence of the two distinct classes of cpsC. Whereas members within one class are greater than 95% identical to each other, the DNA sequence identity between the two classes is only approximately 70%.     

Comparative Genetics of Capsular Polysaccharide Biosynthesis in Streptococcus pneumoniae Types Belonging to Serogroup 19

The genetic basis for the structural diversity of capsule polysaccharide (CPS) in Streptococcus pneumoniae serogroup 19 (consisting of types 19F, 19A, 19B, and 19C) has been determined for the first time. In this study, the genetic basis for the 19A and 19C serotypes is described, and the structures of all four serogroup 19 cps loci and their flanking sequences are compared. Transformation studies show that the structural difference between the 19A and 19F CPSs is likely to be a consequence of differences between their respective polysaccharide polymerase genes (cps19aI and cps19fI). The CPS of type 19C differs from that of type 19B by the addition of glucose. We have identified a single gene difference between the two cps loci (cps19cS), which is likely to encode a glucosyl transferase. The arrangement of the genes within the cps19 loci is highly conserved, with 13 genes (cps19A to -H and cps19K to -O) common to all four serogroup 19 members. These cps genes encode functions required for the synthesis of the shared trisaccharide component of the group 19 CPS repeat unit structures. Furthermore, the genetic differences between the group 19 cps loci identified are consistent with the CPS structures of the individual serotypes. Functions have been assigned to nearly all of the cps19 gene products, based on either gene complementation or similarity to other proteins with known functions, and putative biosynthetic pathways for production of all four group 19 CPSs have been proposed.     

Deletion and allelic exchange of the Aspergillus fumigatus veA locus via a novel recyclable marker module

Detailed evaluation of gene functions in an asexual fungus requires advanced methods of molecular biology. For the generation of targeted gene deletions in the opportunistic pathogen Aspergillus fumigatus we designed a novel blaster module allowing dominant selection of transformants due to resistance to phleomycin as well as dominant (counter)selection of a Cre recombinase-mediated marker excision event. For validation purposes we have deleted the A. fumigatus pabaA gene in a wild-type isolate by making use of this cassette. The resulting pabaA::loxP strain served as the recipient for subsequent targeting of the velvet locus. Homologous reconstitution of the deleted gene was performed by an allele whose expression is driven in a nitrogen source-dependent manner, as validated by Northern analyses. Overexpression of the veA locus in A. fumigatus does not result in any obvious phenotype, whereas the sporulation capacities of the veA null mutant are reduced on nitrate-containing medium, a phenotype that is completely restored in the reconstituted strain.