BACKGROUND
the cell wall is an important extracellular structure of fungal species.
it is essential for growth and survival as it protects the cell from lysis by its internal turgor pressure.
the cell wall represents a significant investment of the cell as about 30 % of the cellular dry weight consists of this dynamic barrier  <cit> .
the cell wall of filamentous fungi is composed of several different carbohydrate polymers  and glycoproteins   <cit> .
the galactose in galactomannan and galactomannoproteins in filamentous fungi is present in the form of galactofuranose , the five-membered ring form of this hexose.
in contrast, the galactose found in galactomannoproteins in yeasts such as saccharomyces cerevisiae and schizosaccharomyces pombe and galactosaminogalactan in aspergilli is the more common galactopyranose   <cit> .
because the presence of galf is often associated with virulence in pathogenic bacteria, fungi and protozoan, it is considered as an important virulence factor and its biosynthesis as a target for antibiotics .
many of the cell wall polymers  are synthesized by plasma membrane localized enzymes that use nucleotide sugars udp-n-acetylglucosamine , udp-glucose ) as sugar donors .
in contrast, galactomannan, including its galf side-chain, is stepwise assembled during their transit through the secretory pathway  <cit> .
the localization of the biosynthesis of galactomannan is not known in detail but is likely to involve the synthesis of a galactomannan-gpi-anchor precursor in the er, whereafter it is further elongated in the golgi by specific mannosyltransferases and galf transferases  <cit> .
the mannosyltransferases involved in the incorporation of mannose residues on galactomannan in the er or the golgi are currently not known.
the galf transferase genes  have recently been identified in a. fumigatus and a. nidulans  <cit> .
transfer of activated sugar-nucleotides from the cytoplasm to the lumen of the golgi is an essential requirement for glycosylation of glycoproteins and carried out by sugar-nucleotide specific transporters for gdp-mannose  and udp-galactose  <cit> , respectively.
golgi-localized nucleotide sugar transporters are structurally conserved type iii transmembrane proteins, but little is known regarding their exact structure due to the difficulty to crystallize membrane proteins.
in general, nucleotide sugar transporters contain an even number of transmembrane domains  with both the n-terminus and the c-terminus present on the cytosolic side .
the gdp-mannose transporter is relatively well characterized in s. cerevisiae  <cit>  as well as in aspergilli  <cit> .
gfp-tagging of gdp-mannose transporters in a. niger and a. nidulans have shown the typical golgi localization of the gdp-mannose transporter  <cit> .
an udp-galf transporter was first identified and characterized in a. fumigatus  <cit>  and subsequently also in a. nidulans  <cit> .
fractionation studies indicated golgi localization of the transporter, which was further confirmed in a. nidulans by flag-tagging  <cit> .
in this paper, we show that a. niger possesses not a single, but two genes encoding udp-galf transporters.
both genes are functional and the two proteins have redundant functions as deletion of both genes is required to block galactofuranosylation.
methods
strains and growth conditions
the a. niger strains used in this study are listed in table  <dig>  strains were grown on minimal medium   <cit>  containing 1 %  glucose as carbon source or on complete medium  containing  <dig>  %  yeast extract and  <dig>  %  casamino acids in addition to mm.
when required, plates were supplemented with 10 mm uridine.
5’foa selection for the selection of pyrg− strains was performed as described previously  <cit> .
for the plate growth assays, strains were grown on cm or mm plates supplemented with  <dig>  % sds or cfw as described  <cit> .table  <dig> strain used in this study
cspa
Δkusa::amds, pyrg
−
Δkusa::dr-amds-dr pyrg
−
Δkusa::dr-amds-dr pyrg
+
Δugma
Δugta
Δugtb
ΔugtaΔugtb
ugta-cfp in ma <dig>
ugta-cfp
ugtb-cfp in ma <dig>
ugtb-cfp
ugta-cfp, pyrg-
ugtb-cfp, pyrg-
Δugta, pyrg-
ugtb-cfp in Δugta
ugta-cfp in Δugtb
ugta-cfp, ugtb-yfp
Δugtb, pyrg-
ugta-cfp, gmta-yfp
ugtb-cfp, gmta-yfp
general molecular techniques
escherichia coli dh5α strains were transformed by electroporation for propagation and amplification of the plasmids.
amplification of plasmid dna was performed using the xl1-blue strain, which was transformed using the heat-shock protocol as described by inoue  <cit> .
transformation of a. niger and isolation of genomic dna was performed as described by meyer  <cit> .
dctp-labeled probes for southern blots were synthesized using the rediprime ii dna labeling system  according to the instructions of the manufacturer.
all molecular techniques including cloning and pcr amplifications and southern blotting were carried out as described by sambrook  <cit> .
dna sequencing was performed by macrogen europe .
construction of ugta and ugtb deletion and fluorescent protein tagged strains
for the construction of the ugta and ugtb deletion strains deletion cassettes were made using the multisite gateway® three-fragment vector construction kit.
as a marker for deleting the ugtb gene, the pyrg marker of a. oryzae was used.
to facilitate removal of the aopyrg marker, a. nidulans ttrpc repeats were included around the pyrg gene  <cit> .
the hygromycin b  selection marker was used for the deletion of the ugta gene.
all flanking regions of ugta and ugtb as well as the selection markers were pcr amplified using the primers listed in supplemental table  <dig> and cloned in appropriate pdonr vectors.
for all the amplifications phusion™ high-fidelity dna polymerase was used  and gdna from a. niger strain n <dig> was used as template dna to amplify the fragments.
the subsequent lr reaction was performed using the three pdonr vectors and pdest r4-r <dig> vector  <dig> to create the ugta and ugtb deletion plasmids.
the final constructs were verified by restriction analysis and sequencing.
the construction plasmids expressing fp-labeled transporters  were also made using the gateway® three-fragment vector construction kit.
fragments containing the ugta 5’ orf , the ugta 3’flank , ugtb 5’ orf  and ugtb 3’ flank  were amplified using primers listed in table  <dig>  the amplified fragments used in a bp-reaction to give various donor vectors.
the resulting pdonr vectors were: pdonr-ugta5’, pdonr-ugta3’, pdonr-ugtb5’ and pdonr-ugtb3’.
pdonr-cfp-tpt and pdonr-yfp-tpt  bearing the fluorescent protein and the ttrpc-pyrg-ttrpc  selection marker fused to either cfp or yfp were used in the lr reaction.
appropriate pdonr fragments were recombined in the lr reaction to give pdest vectors mj <dig>  , mj <dig>   and mj <dig>  .
the pdest vectors were sequenced and subsequently transformed in a. niger strain ma <dig>  .
transformants were purified on mm and checked in a southern blot.
transformants containing a single copy of ugta-cfp, ugtb-cfp or ugtb-yfp at the ugta or ugtb locus were selected for further analysis.
for the construction of double mutants, the pyrg marker of removed by isolating 5’foa resistant strains as described  <cit> .
5’foa resistant mutants in which the pyrg gene had been looped out were used to construct ugta-cfp/ugtb-yfp, ugta-cfp/gmta-yfp, and ugtb-cfp/gmta-yfp double fluorescent strains .
targeted integration of gdp-mannose transporter  at the pyrg locus using the pyrg* allele was done using the construct described previously  <cit> .table  <dig> primers used in this study
a nucleotides in lowercase indicate attb recombination sites
fluorescent microscopy
light and fluorescence microscopic pictures were captured with a 63x c-apochromatic objectives on an inverted lsm  <dig> microscope equipped with a laser scanning-disk confocal system .
for life imaging of fungal hyphae, conidia were pre-grown on mm agar plate at 30 °c for 1 day.
an agar piece containing mycelium was cut out and placed, upside down, onto an objective glass.
to prevent drying-out of the agar/mycelium piece, 50 μl of mm was applied between the colony and the objective glass.
after cells resumed growth  images were captured.
isolation and analysis of cell wall galactomannan and secreted galactomannoproteins
the isolation of cell wall mannan was performed essentially as described by bardalaye and nordin  <cit>  with minor modifications  <cit> .
monosaccharide analysis of the isolated mannan fractions was performed by high-performance anion exchange chromatography  as described recently  <cit> .
the platelia assay  was carried out as described  <cit> .
a dot blot analysis using the l <dig> monoclonal anti-galf-antibody   <cit>  or cona-labeled peroxidase   were performed as described previously  <cit> .
RESULTS
identification of two putative udp-galf transporters in a. niger genome
in both a. fumigatus and a. nidulans udp-galf transporter proteins have recently been identified  <cit> .
udp-galf transporters belong to the family of nucleotide sugar transporters  and the genomes of aspergilli contain at least  <dig> subfamilies of nsts  <cit> .
the indication for a possible role in udp-galf biosynthesis of this class of nst was the chromosomal clustering with ugma.
ugma  is a udp-galp mutase which is required for the formation of udp-galf  <cit> .
the clustering of ugma homologs with ugta homologs is conserved among pezizomycotina whose genome has been fully sequenced .
also in a. niger, ugma  is clustered with a putative udp-galf transporter  that we named ugta.
interestingly, the genome of a. niger contains an additional close homolog of ugta that we named ugtb .
ugtb homologs were identified in genomes of nine other aspergilli  .
the genome of a. wentii contains also a third homolog.
as all these nine aspergilli also contain an ugta homolog, , it means that all these nine species have two ugt paralogs .
phylogenetically, ugta orthologs and ugtb orthologs cluster separately .
note that a. nidulans and a. fumigatus only possess a single udp-galf transporter, and deletion of this gene resulted in a galf-negative phenotype  <cit> .
a. niger ugta and ugtb are proteins consisting of  <dig> and  <dig> amino acids, respectively, and share 67 % sequence identity between them.
ugta and ugtb are structurally conserved proteins belonging to the superfamily of nucleotide sugar transporters and predicted to contain  <dig> and  <dig> transmembrane domains, respectively .
ugta and ugtb have redundant function in biosynthesis of galf-glycoconjugates
to determine the function of ugta and ugtb, deletion cassettes were constructed to delete the ugta and ugtb genes individually, and to create the ΔugtaΔugtb double mutants described in methods.
putative deletion mutants were subjected to southern blot analysis and mutants with the proper deletion of the corresponding gene were selected for further analysis .fig.
<dig> gene deletion of ugta and ugtb in a. niger.
a and c schematic representation of the strategy to disrupt ugta  and ugtb  using the hygromycinb selection marker  or the pyrg selection marker flanker by repeats of the trpc terminator regions .
the 5’ugta and the 3’ugtb probes used for hybridisation are indicated.
genomic dna was digested with bamhi  or xbai  and the length of the expected fragments is indicated.
b and d southern blots of genomic dna after digestion and hybridisation with 32p-labelled probes.
approximate sizes of the bands based on dna ladder  are indicated
the Δugta and Δugtb strains displayed no obvious phenotype when grown on plate, whereas the ΔugtaΔugtb double mutant showed a reduced growth and reduced sporulation phenotype .
quantification of the numbers of spores indicated a ten-times reduction for both the Δugma and the ΔugtaΔugtb mutant .
the growth phenotype of the ΔugtaΔugtb double mutant is similar to that of the a. niger Δugma mutant .
like those of the Δugma mutant, the hyphae of the ΔugtaΔugtb mutant are irregular in shape, and hyphal compartments are reduced in length .fig.
<dig> phenotypic analysis of ugt mutants.
ten thousand spores of the indicated strains were spotted in the centre of a 9 cm petri dish containing complete medium-agar supplemented with  <dig>  % sds, or supplemented with 100 μg/ml cfw and incubated for three days at 30 °c.
the mutant strains were also grown on complete medium-agar at 42 °c for 3 days
deletion of ugma results in an increased sensitivity towards calcofluor white  and sds.
both compounds are indicative for a compromised integrity of the cell wall  <cit> .
similar to the Δugma mutant, the ΔugtaΔugtb mutant displays an increased sensitivity towards cfw and sds .
like the Δugma mutant, the ΔugtaΔugtb mutant also displays a strong growth defect at 42 °c .
to assess galf biosynthesis in the ΔugtaΔugtb double mutant, medium samples of the wild-type strain n <dig> and the Δugma, Δugta, Δugtb and ΔugtaΔugtb mutants were analysed for the presence of galf by a dot blot analysis using anti-galf antibody l <dig>  <cit> .
as shown in fig.
3a, no reactivity of l <dig> was detected towards the medium samples of the ΔugtaΔugtb double mutant, similar to that of the Δugma mutant.
in contrast, galf was clearly present in the medium samples of both Δugta and Δugtb strains.
to determine the galf content quantitatively, galactomannan of the various strains was isolated  <cit>  and their galf content established by detection with the anti-galf antibody eb-a <dig> in the platelia assay .
the data indicated that the galactomannan of both single ugt mutants have a slightly lower galf-content compared to the wild-type strain, whereas the ΔugtaΔugtb mutant has no detectable galf, similar as the Δugma mutant.
the isolated mannan fractions were also subjected to hydrolysis and their monosaccharide content was subsequently determined by hpaec , which confirmed the absence of galf in the polysaccharide fraction of the ΔugtaΔugtb and the Δugma mutants.
these phenotypic analyses indicate that the transport activity of either ugta or ugtb is sufficient to produce a galactomannan with wild-type properties, but that the lack of both transporters will prevent galactofuranosylation of the mannan backbone.fig.
<dig> analysis of galf-containing glycoconjugates in wildtype and galf mutants.
a dot blot assay to detect the presence of galf residues on secreted glycoconjugates from a. niger mutants.
a. niger wild-type strain and galf mutants were grown to early stationary phase and cell-free medium was spotted on nitrocellulose filter paper.
the blots were incubated with the anti-galf antibody  to detect the presence of galf or incubated with cona-po to detect mannoproteins.
b platelia assay with anti-galf antibody eb-a <dig> was performed on indicated amounts of purified mannan from indicated strains.
c the percentage of the monosaccharides  detected on hpaec after hydrolysis of mannan from indicated strains is shown.
figures shown are representative for at least two independent experiments
cellular localization of ugta and ugtb
to localize ugta and ugtb in a. niger, both proteins were c-terminally tagged with cfp or yfp using a gateway based strategy.
both fusion genes were expressed from and integrated at their endogenous locus.
the a. oryzae pyrg selection marker was flanked by a. nidulans trpc repeats inserted downstream of the ugta or ugtb coding region.
this strategy allowed integration of the fusion gene at the endogenous locus via homologous recombination and also allowed reuse of the pyrg marker because the marker can be efficiently removed by 5’foa counter selection  <cit> .
the constructs containing the cfp-tagged galf transporters  were transformed to a. niger ku <dig> strain ma <dig>  and uridine prototrophic transformants were purified.
proper recombination at either the ugta or ugtb locus of the respective cassette was confirmed by southern blot analysis .
to prove functionality of the fusion proteins, the ugta-cfp and ugtb-cfp fusion constructs were also transformed to the Δugtb and the Δugta strains, respectively.
again, proper integration of the tagged transporters was confirmed by southern blot analysis .
analysis of these transformants in which only one of the galf-transporters is present in a fluorescently-labelled form showed that the fusion proteins are fully functional as their growth was identical to the growth of the control strains .
microscopic analysis of growing hyphal cells using confocal fluorescent microscopy confirmed the expected golgi localization of both proteins.
in both cases, a punctuated localization in the hyphal cells was observed, which is indicative for localization in golgi equivalents in a. niger and other fungi  <cit> .
comparison of the fluorescence patterns of ugta and ugtb to marker strains in which er  <cit> , secretory vesicles  <cit>  or vacuoles  were labelled clearly showed a different pattern.
comparison of the intensity signals of both ugta-cfp and ugtb-cfp suggested higher amounts of ugtb-cfp in the golgi membranes compared to ugta.
in general, the signals from strains expressing ugta-cfp were weaker and more diffuse than these from strains expressing ugtb-cfp.
to allow visualization of ugta-cfp, the fluorescence picture shown in fig.
4a was enhanced.fig.
<dig> subcellular localization of ugta and ugtb in a. niger.
strain mh <dig>   and mh <dig>   were grown on mm-agar medium and analysed by fluorescence microscopy during vegetative growth  early conidiostalk formation  and during conidiospore formation .
bars represent 10 μm
the expression and localization of ugta-cfp and ugtb-cfp was also examined during asexual development.
as shown in fig.
4c to f, ugta-cfp and ugtb-cfp are present in golgi-like structure in conidiostalks and in young conidiospores.
enhancement of ugta-cfp fluorescence was not necessary during conidiophores formation, indicating that the expression of ugta is relative higher during conidiophore formation.
to analyse the possible co-localization of ugta and ugtb and to look at co-localization of galf transporters  with a golgi-localized gdp-mannose transporter , several strains were constructed in which two nucleotide sugar transporters ugta-cfp/ugtb-yfp; ugta-cfp/gmta-yfp; ugtb-cfp/gmta-yfp were labelled with two different fluorescent proteins for analysis by fluorescence microscopy.
previously, we have shown the functionality of the yfp-tagged gdp-mannose transporter gmta in a. niger  <cit> .
it should be noted that the ugta-cfp/gmta-yfp and the ugtb-cfp/gmta-yfp also contain the endogenous ugtb and ugta genes, respectively, as well as the gmta gene.
in the ugta-cfp/ugtb-yfp transformant, the signal of ugtb-yfp was stronger and quenched less quickly compared to the ugta-cfp signal.
the difference in intensity and quenching made it difficult to draw strong conclusions about the co-localization of both proteins.
in general the ugtb-yfp and ugta-cfp signals fluorescent images overlap, indicating co-localization of ugta and ugtb in the golgi.
however, the presence of some clear ugta spots  indicating that some golgi equivalent seems to exist with a differential spatial distribution of ugta and ugtb.fig.
<dig> co-localization studies of strain expressing differentially labeled nucleotide sugar transporters.
strains expressing ugta::cfp and ugtb::yfp , ugta::cfp and gmta::yfp  and ugtb::cfp and gmta::yfp  were grown and mm-agar plugs and imaged by confocal microscopy.
arrows point at ugta spots.
images were false colored to red  and green  to improve contrast.
bars represent 10 μm
discussion
over the past few years, genes and proteins involved in the biosynthesis of galactofuranose -containing glycoconjugates such as galactomannan, glycoproteins and glycolipids have been identified in fungal species.
at least four essential enzymatic or transport steps are required; two enzymes for the synthesis of the sugar donor , one transporter step for the translocation of the sugar donor from the cytosol, and finally the transfer of golgi-localized udp-galf to glycoconjugates via a glycosyltransferase  <cit> .
the first step is the conversion of udp-glucose to udp-gal which requires the presence of a udp-glucose epimerase .
the second step involves the conversion of udp-gal into udp-galf and is carried out by the enzyme named udp-galf mutase .
transport of the udp-galf from the cytosol to the golgi requires specific udp-galf transporters .
the transferase activity is mediated by golgi-localized udp-galftransferases .
the genes encoding the different enzymes or transporters have been identified in three aspergillus species , based on reverse genetic approaches  <cit> , genomics- and blast-based searches  <cit>  or mutant screens  <cit> .
studies in these aspergillus species have shown that galf biosynthesis is important for maintaining cell wall integrity and virulence   <cit> .
previous research in our group related to the identification of genes involved in galf biosynthesis was based on a screen for cell wall mutants with an induced expression of alpha-glucan synthase a   <cit> .
agsa is specifically induced in response to cell wall stress conditions and both the ugea and the ugma genes encoding the udp-glucose-4- epimerase and the udp-galf mutase, respectively, were identified via this screen  <cit> .
the screen for cell wall mutants with increased expression of agsa yielded  <dig> mutants which were all tested in detail for defects in galf biosynthesis as it was expected that also other genes involved in galf biosynthesis would lead to agsa induction.
however, no other mutants besides the ugea and ugma mutants were identified.
this study together with some unpublished results  explain why additional mutants  were not identified in the cell wall mutant screen.
the present study clearly shows that a. niger contains two udp-galf transporter genes with redundant functions.
inactivation by targeted deletion of either one of them did not result in reduced galf levels and consequently did not result in agsa induction.
genetic redundancy is also the reason why the gene encoding the udp-galf transferase was not identified.
the genome of a. niger contains three gfs homologs and deletion of either gfsa, gfsb or gfsc did not result in agsa induction .
it should be noticed however that deletion of gfsa resulted in reduced levels of galf in a dot blot analysis , but this reduction was not sufficient to induce agsa expression.
the redundancy of galf transporters was noticed in nine aspergillus species in addition to a. niger that are present in the aspgd database .
six of these species belong to the aspergillus section nigri , but a. zonatus, a. wentii dto 134e <dig> and a. glaucus cbs  <dig>  are more distantly related and belong to other phylogenetic groups  <cit> .
the presence of these homologs in distantly related groups that also show high level of synteny, suggests that the duplication has been a rather early event in evolution and that the second gene has been lost in many species also in the nigri section.
e.g.
closely related species like a. terreus have only one copy.
it is also interesting to note that the presumed loss of one of two genes is not random.
the ugta homologs  are always present while the ugmb gene can be absent from the genome.
the reason for this preferential presence of ugta is not clear as we could not detect an effect on growth of the deletion of ugta in a. niger.
the ugta and ugtb proteins are predicted to be  <dig> and  <dig> amino acid residues long, respectively.
we slightly modified the gene model which is present in aspgd for the ugtb gene.
the first predicted intron is likely to be  <dig> nucleotides shorter thereby including six amino acids that are also predicted to be present in all ugtb homologs.
in addition, the addition of these six amino acids also improved alignment with ugta homologs.
both ugta and ugtb are predicted to be nucleotide sugar transporters based on the presence of pfam motif <dig> .
alignment of the protein sequences of ugta and ugtb revealed that the difference in length between ugta and ugtb is mainly caused by a shorter c-terminal region of the ugtb protein .
the membrane topology of nucleotide sugar transporters  has been predicted to comprise between  <dig>   <dig> or  <dig> transmembrane domains, linked by hydrophilic loops at both sites of the membrane.
to data, all nst are predicted to have an even number of transmembrane domains in which the n and c-termini of the nst are located in the cytosol.
the only exception is the a. fumigatus ugta/glfb protein.
alignment of a. niger ugta and ugtb proteins to the a. fumigatus ugta/glfb protein suggested that the a. niger ugta protein also comprises  <dig> transmembrane domains.
in comparison with other, more distantly related nsts, ugta seems to possess an additional tm domain in the c-terminal part of the protein .
alignment of the a. niger ugtb protein with a. niger ugta and a. fumigatus ugta/glfb suggests that ugtb contains ten tm, and lacks the most c-terminal tm domain present in ugta .
although not experimentally validated, we propose that ugtb has ten tm domains and in comparison to ugta proteins lacks the most c-terminal tm domain.
the localization studies of the two galf transporters and the gdp-mannose transporter suggest that the proteins co-localize in golgi equivalents.
previous data in a. nidulans have indicated that the gdp-mannose transporter in a. nidulans does not co-localize with copa  <cit> .
copa is a conserved component of the coat-protein complex i  coatomer complex required for retrograde transport between golgi compartments and between golgi and er  <cit>  and considered as an early golgi marker.
several additional markers for early golgi compartments  or late-golgi compartments  have recently been identified in a. nidulans  <cit> .
yet another aspect that requires further attention is the mechanism by which these galf transporters are retained in golgi cisternae.
lysine motifs at the c-terminus of the gdp-mannose transporter have been implicated in their golgi-retrieval  <cit> , but such a motif was not detected in ugta or ugtb.
a role for the c-terminal part of ugta or ugtb in a general mechanism for retrieval seems unlikely as the c-terminal parts of both proteins are very different.
the gfp-tagged versions of ugta and ugtb created in this study, provide starting tools for future studies on mutants or mutations in ugta or ugtb that possibly affect their localization in the golgi.
CONCLUSIONS
a. niger possesses not a single, but two genes encoding udp-galf transporters.
both proteins are localized in the golgi and contribute to the transport of udp-galf over the golgi membrane.
deletion of only one of the two transporter genes, did not result in obvious growth defects or reduced levels of galf in the cell wall or on glycoproteins indicating that the two proteins have redundant functions.
the overlapping function of the two proteins was further shown by simultaneous deletion of the two transporter genes which resulted is the absence of galf-containing glycoconjugates.
additional files
additional file 1: figure s <dig>  homology tree of ugta and ugtb homologs in aspergilli.
protein sequence homologous to a. niger ugta or a. niger ugtb were extracted from the aspgd database and aligned using dnaman.
% of amino acid identity is given.
additional file 2: figure s <dig>  amino acid alignment of a. niger ugta and ugtb proteins with ugta/glfb of a. fumigatus.
transmembrane domains predicted in ugta/glfb and homologous region in the a. niger proteins are highlighted.
abbreviations
galpgalactopyranose
galfgalactofuranose
udpuridine diphosphate
gdpguanine diphosphate
ugtudp-galf transporter
gfpgreen fluorescent protein
cfpcyan fluorescent protein
yfpyellow fluorescent protein
erendoplasmic reticulum
hpaechigh-performance anion-exchange chromatography
5’foa5’fluoroorotic acid
competing interest
the authors declare that they have no competing interests.
authors’ contributions
jp and mh carried out the molecular genetic studies.
bt and el carried out the sugar and immunoassays.
jp, bt and ar participated in the sequence alignments.
bt, jp and ar drafted the manuscript.
cvdh, ivd, and ar conceived the study and participated in the coordination.
all authors contributed to the writing.
all authors read and approved the final manuscript.

 the cell wall is an important extracellular structure of fungal species. it is essential for growth and survival as it protects the cell from lysis by its internal turgor pressure. the cell wall represents a significant investment of the cell as about 30 % of the cellular dry weight consists of this dynamic barrier. the cell wall of filamentous fungi is composed of several different carbohydrate polymers and glycoproteins. the galactose in galactomannan and galactomannoproteins in filamentous fungi is present in the form of galactofuranose, the five-membered ring form of this hexose. in contrast, the galactose found in galactomannoproteins in yeasts such as saccharomyces cerevisiae and schizosaccharomyces pombe and galactosaminogalactan in aspergilli is the more common galactopyranose. because the presence of galf is often associated with virulence in pathogenic bacteria, fungi and protozoan, it is considered as an important virulence factor and its biosynthesis as a target for antibiotics. many of the cell wall polymers are synthesized by plasma membrane localized enzymes that use nucleotide sugars udp-n-acetylglucosamine, udp-glucose ) as sugar donors. in contrast, galactomannan, including its galf side-chain, is stepwise assembled during their transit through the secretory pathway. the localization of the biosynthesis of galactomannan is not known in detail but is likely to involve the synthesis of a galactomannan-gpi-anchor precursor in the er, whereafter it is further elongated in the golgi by specific mannosyltransferases and galf transferases. the mannosyltransferases involved in the incorporation of mannose residues on galactomannan in the er or the golgi are currently not known. the galf transferase genes have recently been identified in a. fumigatus and a. nidulans. transfer of activated sugar-nucleotides from the cytoplasm to the lumen of the golgi is an essential requirement for glycosylation of glycoproteins and carried out by sugar-nucleotide specific transporters for gdp-mannose and udp-galactose, respectively. golgi-localized nucleotide sugar transporters are structurally conserved type iii transmembrane proteins, but little is known regarding their exact structure due to the difficulty to crystallize membrane proteins. in general, nucleotide sugar transporters contain an even number of transmembrane domains with both the n-terminus and the c-terminus present on the cytosolic side. the gdp-mannose transporter is relatively well characterized in s. cerevisiae as well as in aspergilli. gfp-tagging of gdp-mannose transporters in a. niger and a. nidulans have shown the typical golgi localization of the gdp-mannose transporter. an udp-galf transporter was first identified and characterized in a. fumigatus and subsequently also in a. nidulans. fractionation studies indicated golgi localization of the transporter, which was further confirmed in a. nidulans by flag-tagging. in this paper, we show that a. niger possesses not a single, but two genes encoding udp-galf transporters. both genes are functional and the two proteins have redundant functions as deletion of both genes is required to block galactofuranosylation. methods strains and growth conditions the a. niger strains used in this study are listed in table  strains were grown on minimal medium containing 1 % glucose as carbon source or on complete medium containing  % yeast extract and  % casamino acids in addition to mm. when required, plates were supplemented with 10 mm uridine. 5’foa selection for the selection of pyrg− strains was performed as described previously. for the plate growth assays, strains were grown on cm or mm plates supplemented with  % sds or cfw as described.table strain used in this study cspa Δkusa::amds, pyrg − Δkusa::dr-amds-dr pyrg − Δkusa::dr-amds-dr pyrg + Δugma Δugta Δugtb ΔugtaΔugtb ugta-cfp in ma ugta-cfp ugtb-cfp in ma ugtb-cfp ugta-cfp, pyrg- ugtb-cfp, pyrg- Δugta, pyrg- ugtb-cfp in Δugta ugta-cfp in Δugtb ugta-cfp, ugtb-yfp Δugtb, pyrg- ugta-cfp, gmta-yfp ugtb-cfp, gmta-yfp general molecular techniques escherichia coli dh5α strains were transformed by electroporation for propagation and amplification of the plasmids. amplification of plasmid dna was performed using the xl1-blue strain, which was transformed using the heat-shock protocol as described by inoue. transformation of a. niger and isolation of genomic dna was performed as described by meyer. dctp-labeled probes for southern blots were synthesized using the rediprime ii dna labeling system according to the instructions of the manufacturer. all molecular techniques including cloning and pcr amplifications and southern blotting were carried out as described by sambrook. dna sequencing was performed by macrogen europe. construction of ugta and ugtb deletion and fluorescent protein tagged strains for the construction of the ugta and ugtb deletion strains deletion cassettes were made using the multisite gateway® three-fragment vector construction kit. as a marker for deleting the ugtb gene, the pyrg marker of a. oryzae was used. to facilitate removal of the aopyrg marker, a. nidulans ttrpc repeats were included around the pyrg gene. the hygromycin b selection marker was used for the deletion of the ugta gene. all flanking regions of ugta and ugtb as well as the selection markers were pcr amplified using the primers listed in supplemental table and cloned in appropriate pdonr vectors. for all the amplifications phusion™ high-fidelity dna polymerase was used and gdna from a. niger strain n was used as template dna to amplify the fragments. the subsequent lr reaction was performed using the three pdonr vectors and pdest r4-r vector to create the ugta and ugtb deletion plasmids. the final constructs were verified by restriction analysis and sequencing. the construction plasmids expressing fp-labeled transporters were also made using the gateway® three-fragment vector construction kit. fragments containing the ugta 5’ orf, the ugta 3’flank, ugtb 5’ orf and ugtb 3’ flank were amplified using primers listed in table  the amplified fragments used in a bp-reaction to give various donor vectors. the resulting pdonr vectors were: pdonr-ugta5’, pdonr-ugta3’, pdonr-ugtb5’ and pdonr-ugtb3’. pdonr-cfp-tpt and pdonr-yfp-tpt bearing the fluorescent protein and the ttrpc-pyrg-ttrpc selection marker fused to either cfp or yfp were used in the lr reaction. appropriate pdonr fragments were recombined in the lr reaction to give pdest vectors mj, mj and mj. the pdest vectors were sequenced and subsequently transformed in a. niger strain ma. transformants were purified on mm and checked in a southern blot. transformants containing a single copy of ugta-cfp, ugtb-cfp or ugtb-yfp at the ugta or ugtb locus were selected for further analysis. for the construction of double mutants, the pyrg marker of removed by isolating 5’foa resistant strains as described. 5’foa resistant mutants in which the pyrg gene had been looped out were used to construct ugta-cfp/ugtb-yfp, ugta-cfp/gmta-yfp, and ugtb-cfp/gmta-yfp double fluorescent strains. targeted integration of gdp-mannose transporter at the pyrg locus using the pyrg* allele was done using the construct described previously.table primers used in this study a nucleotides in lowercase indicate attb recombination sites fluorescent microscopy light and fluorescence microscopic pictures were captured with a 63x c-apochromatic objectives on an inverted lsm microscope equipped with a laser scanning-disk confocal system. for life imaging of fungal hyphae, conidia were pre-grown on mm agar plate at 30 °c for 1 day. an agar piece containing mycelium was cut out and placed, upside down, onto an objective glass. to prevent drying-out of the agar/mycelium piece, 50 μl of mm was applied between the colony and the objective glass. after cells resumed growth images were captured. isolation and analysis of cell wall galactomannan and secreted galactomannoproteins the isolation of cell wall mannan was performed essentially as described by bardalaye and nordin with minor modifications. monosaccharide analysis of the isolated mannan fractions was performed by high-performance anion exchange chromatography as described recently. the platelia assay was carried out as described. a dot blot analysis using the l monoclonal anti-galf-antibody or cona-labeled peroxidase were performed as described previously.  identification of two putative udp-galf transporters in a. niger genome in both a. fumigatus and a. nidulans udp-galf transporter proteins have recently been identified. udp-galf transporters belong to the family of nucleotide sugar transporters and the genomes of aspergilli contain at least subfamilies of nsts. the indication for a possible role in udp-galf biosynthesis of this class of nst was the chromosomal clustering with ugma. ugma is a udp-galp mutase which is required for the formation of udp-galf. the clustering of ugma homologs with ugta homologs is conserved among pezizomycotina whose genome has been fully sequenced. also in a. niger, ugma is clustered with a putative udp-galf transporter that we named ugta. interestingly, the genome of a. niger contains an additional close homolog of ugta that we named ugtb. ugtb homologs were identified in genomes of nine other aspergilli. the genome of a. wentii contains also a third homolog. as all these nine aspergilli also contain an ugta homolog,, it means that all these nine species have two ugt paralogs. phylogenetically, ugta orthologs and ugtb orthologs cluster separately. note that a. nidulans and a. fumigatus only possess a single udp-galf transporter, and deletion of this gene resulted in a galf-negative phenotype. a. niger ugta and ugtb are proteins consisting of and amino acids, respectively, and share 67 % sequence identity between them. ugta and ugtb are structurally conserved proteins belonging to the superfamily of nucleotide sugar transporters and predicted to contain and transmembrane domains, respectively. ugta and ugtb have redundant function in biosynthesis of galf-glycoconjugates to determine the function of ugta and ugtb, deletion cassettes were constructed to delete the ugta and ugtb genes individually, and to create the ΔugtaΔugtb double mutants described in methods. putative deletion mutants were subjected to southern blot analysis and mutants with the proper deletion of the corresponding gene were selected for further analysis.fig. gene deletion of ugta and ugtb in a. niger. a and c schematic representation of the strategy to disrupt ugta and ugtb using the hygromycinb selection marker or the pyrg selection marker flanker by repeats of the trpc terminator regions. the 5’ugta and the 3’ugtb probes used for hybridisation are indicated. genomic dna was digested with bamhi or xbai and the length of the expected fragments is indicated. b and d southern blots of genomic dna after digestion and hybridisation with 32p-labelled probes. approximate sizes of the bands based on dna ladder are indicated the Δugta and Δugtb strains displayed no obvious phenotype when grown on plate, whereas the ΔugtaΔugtb double mutant showed a reduced growth and reduced sporulation phenotype. quantification of the numbers of spores indicated a ten-times reduction for both the Δugma and the ΔugtaΔugtb mutant. the growth phenotype of the ΔugtaΔugtb double mutant is similar to that of the a. niger Δugma mutant. like those of the Δugma mutant, the hyphae of the ΔugtaΔugtb mutant are irregular in shape, and hyphal compartments are reduced in length.fig. phenotypic analysis of ugt mutants. ten thousand spores of the indicated strains were spotted in the centre of a 9 cm petri dish containing complete medium-agar supplemented with  % sds, or supplemented with 100 μg/ml cfw and incubated for three days at 30 °c. the mutant strains were also grown on complete medium-agar at 42 °c for 3 days deletion of ugma results in an increased sensitivity towards calcofluor white and sds. both compounds are indicative for a compromised integrity of the cell wall. similar to the Δugma mutant, the ΔugtaΔugtb mutant displays an increased sensitivity towards cfw and sds. like the Δugma mutant, the ΔugtaΔugtb mutant also displays a strong growth defect at 42 °c. to assess galf biosynthesis in the ΔugtaΔugtb double mutant, medium samples of the wild-type strain n and the Δugma, Δugta, Δugtb and ΔugtaΔugtb mutants were analysed for the presence of galf by a dot blot analysis using anti-galf antibody l. as shown in fig. 3a, no reactivity of l was detected towards the medium samples of the ΔugtaΔugtb double mutant, similar to that of the Δugma mutant. in contrast, galf was clearly present in the medium samples of both Δugta and Δugtb strains. to determine the galf content quantitatively, galactomannan of the various strains was isolated and their galf content established by detection with the anti-galf antibody eb-a in the platelia assay. the data indicated that the galactomannan of both single ugt mutants have a slightly lower galf-content compared to the wild-type strain, whereas the ΔugtaΔugtb mutant has no detectable galf, similar as the Δugma mutant. the isolated mannan fractions were also subjected to hydrolysis and their monosaccharide content was subsequently determined by hpaec, which confirmed the absence of galf in the polysaccharide fraction of the ΔugtaΔugtb and the Δugma mutants. these phenotypic analyses indicate that the transport activity of either ugta or ugtb is sufficient to produce a galactomannan with wild-type properties, but that the lack of both transporters will prevent galactofuranosylation of the mannan backbone.fig. analysis of galf-containing glycoconjugates in wildtype and galf mutants. a dot blot assay to detect the presence of galf residues on secreted glycoconjugates from a. niger mutants. a. niger wild-type strain and galf mutants were grown to early stationary phase and cell-free medium was spotted on nitrocellulose filter paper. the blots were incubated with the anti-galf antibody to detect the presence of galf or incubated with cona-po to detect mannoproteins. b platelia assay with anti-galf antibody eb-a was performed on indicated amounts of purified mannan from indicated strains. c the percentage of the monosaccharides detected on hpaec after hydrolysis of mannan from indicated strains is shown. figures shown are representative for at least two independent experiments cellular localization of ugta and ugtb to localize ugta and ugtb in a. niger, both proteins were c-terminally tagged with cfp or yfp using a gateway based strategy. both fusion genes were expressed from and integrated at their endogenous locus. the a. oryzae pyrg selection marker was flanked by a. nidulans trpc repeats inserted downstream of the ugta or ugtb coding region. this strategy allowed integration of the fusion gene at the endogenous locus via homologous recombination and also allowed reuse of the pyrg marker because the marker can be efficiently removed by 5’foa counter selection. the constructs containing the cfp-tagged galf transporters were transformed to a. niger ku strain ma and uridine prototrophic transformants were purified. proper recombination at either the ugta or ugtb locus of the respective cassette was confirmed by southern blot analysis. to prove functionality of the fusion proteins, the ugta-cfp and ugtb-cfp fusion constructs were also transformed to the Δugtb and the Δugta strains, respectively. again, proper integration of the tagged transporters was confirmed by southern blot analysis. analysis of these transformants in which only one of the galf-transporters is present in a fluorescently-labelled form showed that the fusion proteins are fully functional as their growth was identical to the growth of the control strains. microscopic analysis of growing hyphal cells using confocal fluorescent microscopy confirmed the expected golgi localization of both proteins. in both cases, a punctuated localization in the hyphal cells was observed, which is indicative for localization in golgi equivalents in a. niger and other fungi. comparison of the fluorescence patterns of ugta and ugtb to marker strains in which er, secretory vesicles or vacuoles were labelled clearly showed a different pattern. comparison of the intensity signals of both ugta-cfp and ugtb-cfp suggested higher amounts of ugtb-cfp in the golgi membranes compared to ugta. in general, the signals from strains expressing ugta-cfp were weaker and more diffuse than these from strains expressing ugtb-cfp. to allow visualization of ugta-cfp, the fluorescence picture shown in fig. 4a was enhanced.fig. subcellular localization of ugta and ugtb in a. niger. strain mh and mh were grown on mm-agar medium and analysed by fluorescence microscopy during vegetative growth early conidiostalk formation and during conidiospore formation. bars represent 10 μm the expression and localization of ugta-cfp and ugtb-cfp was also examined during asexual development. as shown in fig. 4c to f, ugta-cfp and ugtb-cfp are present in golgi-like structure in conidiostalks and in young conidiospores. enhancement of ugta-cfp fluorescence was not necessary during conidiophores formation, indicating that the expression of ugta is relative higher during conidiophore formation. to analyse the possible co-localization of ugta and ugtb and to look at co-localization of galf transporters with a golgi-localized gdp-mannose transporter, several strains were constructed in which two nucleotide sugar transporters ugta-cfp/ugtb-yfp; ugta-cfp/gmta-yfp; ugtb-cfp/gmta-yfp were labelled with two different fluorescent proteins for analysis by fluorescence microscopy. previously, we have shown the functionality of the yfp-tagged gdp-mannose transporter gmta in a. niger. it should be noted that the ugta-cfp/gmta-yfp and the ugtb-cfp/gmta-yfp also contain the endogenous ugtb and ugta genes, respectively, as well as the gmta gene. in the ugta-cfp/ugtb-yfp transformant, the signal of ugtb-yfp was stronger and quenched less quickly compared to the ugta-cfp signal. the difference in intensity and quenching made it difficult to draw strong conclusions about the co-localization of both proteins. in general the ugtb-yfp and ugta-cfp signals fluorescent images overlap, indicating co-localization of ugta and ugtb in the golgi. however, the presence of some clear ugta spots indicating that some golgi equivalent seems to exist with a differential spatial distribution of ugta and ugtb.fig. co-localization studies of strain expressing differentially labeled nucleotide sugar transporters. strains expressing ugta::cfp and ugtb::yfp, ugta::cfp and gmta::yfp and ugtb::cfp and gmta::yfp were grown and mm-agar plugs and imaged by confocal microscopy. arrows point at ugta spots. images were false colored to red and green to improve contrast. bars represent 10 μm discussion over the past few years, genes and proteins involved in the biosynthesis of galactofuranose -containing glycoconjugates such as galactomannan, glycoproteins and glycolipids have been identified in fungal species. at least four essential enzymatic or transport steps are required; two enzymes for the synthesis of the sugar donor, one transporter step for the translocation of the sugar donor from the cytosol, and finally the transfer of golgi-localized udp-galf to glycoconjugates via a glycosyltransferase. the first step is the conversion of udp-glucose to udp-gal which requires the presence of a udp-glucose epimerase. the second step involves the conversion of udp-gal into udp-galf and is carried out by the enzyme named udp-galf mutase. transport of the udp-galf from the cytosol to the golgi requires specific udp-galf transporters. the transferase activity is mediated by golgi-localized udp-galftransferases. the genes encoding the different enzymes or transporters have been identified in three aspergillus species, based on reverse genetic approaches, genomics- and blast-based searches or mutant screens. studies in these aspergillus species have shown that galf biosynthesis is important for maintaining cell wall integrity and virulence. previous research in our group related to the identification of genes involved in galf biosynthesis was based on a screen for cell wall mutants with an induced expression of alpha-glucan synthase a. agsa is specifically induced in response to cell wall stress conditions and both the ugea and the ugma genes encoding the udp-glucose-4- epimerase and the udp-galf mutase, respectively, were identified via this screen. the screen for cell wall mutants with increased expression of agsa yielded mutants which were all tested in detail for defects in galf biosynthesis as it was expected that also other genes involved in galf biosynthesis would lead to agsa induction. however, no other mutants besides the ugea and ugma mutants were identified. this study together with some unpublished results explain why additional mutants were not identified in the cell wall mutant screen. the present study clearly shows that a. niger contains two udp-galf transporter genes with redundant functions. inactivation by targeted deletion of either one of them did not result in reduced galf levels and consequently did not result in agsa induction. genetic redundancy is also the reason why the gene encoding the udp-galf transferase was not identified. the genome of a. niger contains three gfs homologs and deletion of either gfsa, gfsb or gfsc did not result in agsa induction. it should be noticed however that deletion of gfsa resulted in reduced levels of galf in a dot blot analysis, but this reduction was not sufficient to induce agsa expression. the redundancy of galf transporters was noticed in nine aspergillus species in addition to a. niger that are present in the aspgd database. six of these species belong to the aspergillus section nigri, but a. zonatus, a. wentii dto 134e and a. glaucus cbs are more distantly related and belong to other phylogenetic groups. the presence of these homologs in distantly related groups that also show high level of synteny, suggests that the duplication has been a rather early event in evolution and that the second gene has been lost in many species also in the nigri section. e.g. closely related species like a. terreus have only one copy. it is also interesting to note that the presumed loss of one of two genes is not random. the ugta homologs are always present while the ugmb gene can be absent from the genome. the reason for this preferential presence of ugta is not clear as we could not detect an effect on growth of the deletion of ugta in a. niger. the ugta and ugtb proteins are predicted to be and amino acid residues long, respectively. we slightly modified the gene model which is present in aspgd for the ugtb gene. the first predicted intron is likely to be nucleotides shorter thereby including six amino acids that are also predicted to be present in all ugtb homologs. in addition, the addition of these six amino acids also improved alignment with ugta homologs. both ugta and ugtb are predicted to be nucleotide sugar transporters based on the presence of pfam motif. alignment of the protein sequences of ugta and ugtb revealed that the difference in length between ugta and ugtb is mainly caused by a shorter c-terminal region of the ugtb protein. the membrane topology of nucleotide sugar transporters has been predicted to comprise between or transmembrane domains, linked by hydrophilic loops at both sites of the membrane. to data, all nst are predicted to have an even number of transmembrane domains in which the n and c-termini of the nst are located in the cytosol. the only exception is the a. fumigatus ugta/glfb protein. alignment of a. niger ugta and ugtb proteins to the a. fumigatus ugta/glfb protein suggested that the a. niger ugta protein also comprises transmembrane domains. in comparison with other, more distantly related nsts, ugta seems to possess an additional tm domain in the c-terminal part of the protein. alignment of the a. niger ugtb protein with a. niger ugta and a. fumigatus ugta/glfb suggests that ugtb contains ten tm, and lacks the most c-terminal tm domain present in ugta. although not experimentally validated, we propose that ugtb has ten tm domains and in comparison to ugta proteins lacks the most c-terminal tm domain. the localization studies of the two galf transporters and the gdp-mannose transporter suggest that the proteins co-localize in golgi equivalents. previous data in a. nidulans have indicated that the gdp-mannose transporter in a. nidulans does not co-localize with copa. copa is a conserved component of the coat-protein complex i coatomer complex required for retrograde transport between golgi compartments and between golgi and er and considered as an early golgi marker. several additional markers for early golgi compartments or late-golgi compartments have recently been identified in a. nidulans. yet another aspect that requires further attention is the mechanism by which these galf transporters are retained in golgi cisternae. lysine motifs at the c-terminus of the gdp-mannose transporter have been implicated in their golgi-retrieval, but such a motif was not detected in ugta or ugtb. a role for the c-terminal part of ugta or ugtb in a general mechanism for retrieval seems unlikely as the c-terminal parts of both proteins are very different. the gfp-tagged versions of ugta and ugtb created in this study, provide starting tools for future studies on mutants or mutations in ugta or ugtb that possibly affect their localization in the golgi.  a. niger possesses not a single, but two genes encoding udp-galf transporters. both proteins are localized in the golgi and contribute to the transport of udp-galf over the golgi membrane. deletion of only one of the two transporter genes, did not result in obvious growth defects or reduced levels of galf in the cell wall or on glycoproteins indicating that the two proteins have redundant functions. the overlapping function of the two proteins was further shown by simultaneous deletion of the two transporter genes which resulted is the absence of galf-containing glycoconjugates. additional files additional file 1: figure s homology tree of ugta and ugtb homologs in aspergilli. protein sequence homologous to a. niger ugta or a. niger ugtb were extracted from the aspgd database and aligned using dnaman. % of amino acid identity is given. additional file 2: figure s amino acid alignment of a. niger ugta and ugtb proteins with ugta/glfb of a. fumigatus. transmembrane domains predicted in ugta/glfb and homologous region in the a. niger proteins are highlighted. abbreviations galpgalactopyranose galfgalactofuranose udpuridine diphosphate gdpguanine diphosphate ugtudp-galf transporter gfpgreen fluorescent protein cfpcyan fluorescent protein yfpyellow fluorescent protein erendoplasmic reticulum hpaechigh-performance anion-exchange chromatography 5’foa5’fluoroorotic acid competing interest the authors declare that they have no competing interests. authors’ contributions jp and mh carried out the molecular genetic studies. bt and el carried out the sugar and immunoassays. jp, bt and ar participated in the sequence alignments. bt, jp and ar drafted the manuscript. cvdh, ivd, and ar conceived the study and participated in the coordination. all authors contributed to the writing. all authors read and approved the final manuscript. 
based on homology with recently identified udp-galf-transporters in a. fumigatus and a. nidulans, two putative udp-galf-transporters in a. niger were found. the two putative udp-galf-transporters in a. niger are homologous to each other and are predicted to contain eleven transmembrane domains or ten transmembrane domains due to a reduced length of the c-terminal part of the ugtb protein. the presence of two putative udp-galf-transporters in the genome was not unique for a. niger. deletion analysis of the single and double mutants in a. niger indicated that the two putative udp-galf-transporters have a redundant function in udp-galf-transport as only the double mutant displayed a galf-negative phenotype. a. niger contains two genes encoding udp-galf-transporters. galactofuranose -containing glycoconjugates are present in numerous microbes, including filamentous fungi where they are important for morphology, virulence and maintaining cell wall integrity. three of the nine species were outside the aspergillus section nigri, indication an early duplication of udp-galf-transporters and subsequent loss of the ugtb copy in several aspergilli. from the twenty aspergillus species analysed, nine species contained two additional putative udp-galf-transporters. deletion and localization studies indicate that ugta and ugtb have redundant functions in the biosynthesis of galf-containing glycoconjugates. both ugt proteins co-localize with each other as well as with the gdp-mannose nucleotide transporter, as was demonstrated by fluorescence microscopy, thereby confirming their predicted localization in the golgi.
 galactofuranose -containing glycoconjugates are present in numerous microbes, including filamentous fungi where they are important for morphology, virulence and maintaining cell wall integrity. the incorporation of galf-residues into galactomannan, galactomannoproteins and glycolipids is carried out by golgi-localized galf transferases. the nucleotide sugar donor used by these transferases is produced in the cytoplasm and has to be transported to the lumen of the golgi by a dedicated nucleotide sugar transporter. methods based on homology with recently identified udp-galf-transporters in a. fumigatus and a. nidulans, two putative udp-galf-transporters in a. niger were found. their function and localization was determined by gene deletions and gfp-tagging studies, respectively.  the two putative udp-galf-transporters in a. niger are homologous to each other and are predicted to contain eleven transmembrane domains or ten transmembrane domains due to a reduced length of the c-terminal part of the ugtb protein. the presence of two putative udp-galf-transporters in the genome was not unique for a. niger. from the twenty aspergillus species analysed, nine species contained two additional putative udp-galf-transporters. three of the nine species were outside the aspergillus section nigri, indication an early duplication of udp-galf-transporters and subsequent loss of the ugtb copy in several aspergilli. deletion analysis of the single and double mutants in a. niger indicated that the two putative udp-galf-transporters have a redundant function in udp-galf-transport as only the double mutant displayed a galf-negative phenotype. the galf-negative phenotype of the double mutant could be complemented by expressing either cfp-ugta or cfp-ugtb fusion proteins from their endogenous promoters, indicating that both cfp-tagged proteins are functional. both ugt proteins co-localize with each other as well as with the gdp-mannose nucleotide transporter, as was demonstrated by fluorescence microscopy, thereby confirming their predicted localization in the golgi.  a. niger contains two genes encoding udp-galf-transporters. deletion and localization studies indicate that ugta and ugtb have redundant functions in the biosynthesis of galf-containing glycoconjugates. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords cell wallgalactomannangalactofuranosesugar nucleotide transportersgolgiissue-copyright-statement© the author 2015 