Sulfate transport in Penicillium chrysogenum: cloning and characterization of the sutA and sutB genes

In industrial fermentations, Penicillium chrysogenum uses sulfate as the source of sulfur for the biosynthesis of penicillin. By a PCR-based approach, two genes, sutA and sutB, whose encoded products belong to the SulP superfamily of sulfate permeases were isolated. Transformation of a sulfate uptake-negative sB3 mutant of Aspergillus nidulans with the sutB gene completely restored sulfate uptake activity. The sutA gene did not complement the A. nidulans sB3 mutation, even when expressed under control of the sutB promoter. Expression of both sutA and sutB in P. chrysogenum is induced by growth under sulfur starvation conditions. However, sutA is expressed to a much lower level than is sutB. Disruption of sutB resulted in a loss of sulfate uptake ability. Overall, the results show that SutB is the major sulfate permease involved in sulfate uptake by P. chrysogenum.     

Nitrate assimilation genes of the marine diazotrophic, filamentous cyanobacterium Trichodesmium sp. strain WH9601

A 4.0-kb DNA fragment of Trichodesmium sp. strain WH9601 contained gene sequences encoding the nitrate reduction enzymes, nirA and narB. A third gene positioned between nirA and narB encodes a putative membrane protein with similarity to the nitrate permeases of Bacillus subtilis (NasA) and Emericella nidulans (CrnA). The gene was shown to functionally complement a DeltanasA mutant of B. subtilis and was assigned the name napA (nitrate permease). NapA was involved in both nitrate and nitrite uptake by the complemented B. subtilis cells. napA is distinct from the nrt genes that encode the nitrate transporter of freshwater cyanobacteria.     

Functional characterization of a maize purine transporter by expression in Aspergillus nidulans

We have characterized the function of Leaf Permease1 (LPE1), a protein that is necessary for proper chloroplast development in maize, by functional expression in the filamentous fungus Aspergillus nidulans. The choice of this ascomycete was dictated by the similarity of its endogenous purine transporters to LPE1 and by particular genetic and physiological features of purine transport and metabolism in A. nidulans. When Lpe1 was expressed in a purine transport-deficient A. nidulans strain, the capacity for uric acid and xanthine transport was acquired. This capacity was directly dependent on Lpe1 copy number and expression level. Interestingly, overexpression of LPE1 from >10 gene copies resulted in transformants with pleiotropically reduced growth rates on various nitrogen sources and the absolute inability to transport purines. Kinetic analysis established that LPE1 is a high-affinity (K(m) = 30 +/- 2.5 microM), high-capacity transporter specific for the oxidized purines xanthine and uric acid. Competition studies showed that high concentrations of ascorbic acid (>30 mM) competitively inhibit LPE1-mediated purine transport. This work defines the biochemical function of LPE1, a plant representative of a large and ubiquitous transporter family. In addition, A. nidulans is introduced as a novel model system for the cloning and/or functional characterization of transporter genes.     

A putative high affinity hexose transporter, hxtA, of Aspergillus nidulans is induced in vegetative hyphae upon starvation and in ascogenous hyphae during cleistothecium formation

Fungi employ different carbohydrate uptake systems to adapt to certain environmental conditions and to different carbon source concentrations. The hydrolysis of polymeric carbohydrates and the subsequent uptake of monomeric forms may also play a role in development. Aspergillus nidulans accumulates cell wall components during vegetative growth and degrades them during sexual development. We have identified the hxtA (high affinity hexose transporter) gene in a differential library, which was enriched for sexual-specific genes. The hxtA gene is disrupted by 6 introns and predicted to encode a 531 amino acid protein with high similarity to major facilitator superfamily members including the high affinity hexose transporter Gtt1 from Trichoderma harzianum. A. nidulans HxtA contains the 12 predicted transmembrane domains characteristic for this family. Deletion of hxtA did not impair growth of A. nidulans on a variety of carbon sources nor did it inhibit sexual development suggesting redundant sugar uptake systems. We found at least 17 putative hexose transporters in the genome of A. nidulans. Despite the high similarity of HxtA to fungal high affinity glucose transporters, the hxtA gene did not restore growth on glucose of a Saccharomyces cerevisiae mutant, in which all hexose transporters were deleted. Northern blot analysis revealed that the A. nidulans hxtA gene was repressed under high glucose conditions and expressed in vegetative hyphae upon carbon starvation and during sexual development. We found hxtA(p)::sgfp expression in developing cleistothecia specifically in ascogenous hyphae and propose that HxtA is a high affinity glucose transporter involved in sugar metabolism during sexual development.     

Convergent evolution and orphan genes in the Fur4p-like family and characterization of a general nucleoside transporter in Aspergillus nidulans

The function of seven paralogues phylogenetically related to the Saccharomyces cerevisiae Fur4p together with a number of functionally related transporters present in Aspergillus nidulans has been investigated. After deletion of the cognate genes we checked the incorporation of radiolabelled substrates, utilization of nitrogen sources, resistance to toxic analogues and supplementation of auxotrophies. FurA and FurD encode allantoin and uracil transporters respectively. No function was found for FurB, FurC, FurE, FurF and FurG. As we failed to identify Fur-related transporters for uridine, pyridoxine or thiamine, we deleted other possible candidates for these functions. A FCY2 -like gene carrying in its 5' UTR a putative thiamine pyrophosphate riboswitch, and which encodes a protein similar to the pyridoxine transporter of yeast (Tpn1p), does not encode either a major thiamine or a pyridoxine transporter. CntA, a member of the concentrative nucleoside transporter family, is a general nucleoside permease, while no function was found for PnpA, a member of the equilibrative transporter family. Phylogenetic analysis shows that within the ascomycetes, the same transport activity could be catalysed by totally unrelated proteins and that within the Fur subfamily convergent evolution towards uracil and allantoin transport activity has occurred at least three and two independent times respectively.     

Increased sulfate uptake by E. coli overexpressing the SLC26-related SulP protein Rv1739c from Mycobacterium tuberculosis

Growth and virulence of mycobacteria requires sulfur uptake. The Mycobacterium tuberculosis genome contains, in addition to the ABC sulfate permease cysTWA, three SLC26-related SulP genes of unknown function. We report that induction of Rv1739c expression in E. coli increased bacterial uptake of sulfate, but not Cl(-), formate, or oxalate. Uptake was time-dependent, maximal at pH 6.0, and exhibited a K(1/2) for sulfate of 4.0 muM. Na(+)-independent sulfate uptake was not reduced by bicarbonate, nitrate, or phosphate, but was inhibited by sulfite, selenate, thiosulfate, N-ethylmaleimide and carbonyl cyanide 3-chloro-phenylhydrazone. Sulfate uptake was also increased by overexpression of the Rv1739c transmembrane domain, but not of the cytoplasmic C-terminal STAS domain. Mutation to serine of the three cysteine residues of Rv1739c did not affect magnitude, pH-dependence, or pharmacology of sulfate uptake. Expression of Rv1739c in a M. bovis BCG strain lacking the ABC sulfate permease subunit CysA could not complement sulfate auxotrophy. Moreover, inducible expression of Rv1739c in an E. coli strain lacking CysA did not increase sulfate uptake by intact cells. Our data show that facilitation of bacterial sulfate uptake by Rv1739c requires CysA and its associated sulfate permease activity, and suggest that Rv1739c may be a CysTWA-dependent sulfate transporter.     

Increased sulfate uptake by E. coli overexpressing the SLC26-related SulP protein Rv1739c from Mycobacterium tuberculosis.

Growth and virulence of mycobacteria requires sulfur uptake. The Mycobacterium tuberculosis genome contains, in addition to the ABC sulfate permease cysTWA, three SLC26-related SulP genes of unknown function. We report that induction of Rv1739c expression in E. coli increased bacterial uptake of sulfate, but not Cl(-), formate, or oxalate. Uptake was time-dependent, maximal at pH 6.0, and exhibited a K(1/2) for sulfate of 4.0 muM. Na(+)-independent sulfate uptake was not reduced by bicarbonate, nitrate, or phosphate, but was inhibited by sulfite, selenate, thiosulfate, N-ethylmaleimide and carbonyl cyanide 3-chloro-phenylhydrazone. Sulfate uptake was also increased by overexpression of the Rv1739c transmembrane domain, but not of the cytoplasmic C-terminal STAS domain. Mutation to serine of the three cysteine residues of Rv1739c did not affect magnitude, pH-dependence, or pharmacology of sulfate uptake. Expression of Rv1739c in a M. bovis BCG strain lacking the ABC sulfate permease subunit CysA could not complement sulfate auxotrophy. Moreover, inducible expression of Rv1739c in an E. coli strain lacking CysA did not increase sulfate uptake by intact cells. Our data show that facilitation of bacterial sulfate uptake by Rv1739c requires CysA and its associated sulfate permease activity, and suggest that Rv1739c may be a CysTWA-dependent sulfate transporter.     

The disruption of JEN1 from Candida albicans impairs the transport of lactate

A lactate permease was biochemically identified in Candida albicans RM1000 presenting the following kinetic parameters at pH 5.0: Km 0.33+/-0.09 mM and Vmax 0.85+/-0.06 nmol s(-1) mg dry wt(-1). Lactate uptake was competitively inhibited by pyruvic and propionic acids; acetic acid behaved as a non-competitive substrate. An open reading frame (ORF) homologous to Saccharomyces cerevisiae gene JEN1 was identified (CaJEN1). Deletions of both CaJEN1 alleles of C. albicans (resulting strain CPK2) resulted in the loss of all measurable lactate permease activity. No CaJEN1 mRNA was detectable in glucose-grown cells neither activity for the lactate transporter. In a medium containing lactic acid, CaJEN1 mRNA was detected in the RM1000 strain, and no expression was found in cells of CPK2 strain. In a strain deleted in the CaCAT8 genes the expression of CaJEN1 was significantly reduced, suggesting the role of this gene as an activator for CaJEN1 expression. Both in C. albicans and in S. cerevisiae cells CaJEN1-GFP fusion was expressed and targeted to the plasma membrane. The native CaJEN1 was not functional in a S. cerevisiae jen1delta strain. Changing ser217-CTG codon (encoding leucine in S. cerevisiae) to a TCC codon restored the permease activity in S. cerevisiae, proving that the CaJEN1 gene codes for a monocarboxylate transporter.     

The Maltose Permease Encoded by the Mal61 Gene of Saccharomyces Cerevisiae Exhibits Both Sequence and Structural Homology to Other Sugar Transporters

The MAL61 gene of Saccharomyces cerevisiae encodes maltose permease, a protein required for the transport of maltose across the plasma membrane. Here we report the nucleotide sequence of the cloned MAL61 gene. A single 1842 bp open reading frame is present within this region encoding the 614 residue putative MAL61 protein. Hydropathy analysis suggests that the secondary structure consists of two blocks of six transmembrane domains separated by an approximately 71 residue intracellular region. The N-terminal and C-terminal domains of 100 and 67 residues in length, respectively, also appear to be intracellular. Significant sequence and structural homology is seen between the MAL61 protein and the Saccharomyces high-affinity glucose transporter encoded by the SNF3 gene, the Kluyveromyces lactis lactose permease encoded by the LAC12 gene, the human HepG2 glucose transporter and the Escherichia coli xylose and arabinose transporters encoded by the xylE and araE genes, indicating that all are members of a family of sugar transporters and are related either functionally or evolutionarily. A mechanism for glucose-induced inactivation of maltose transport activity is discussed.     

Characterization of the Amdr-Controlled Lama and Lamb Genes of Aspergillus Nidulans

Four Aspergillus nidulans genes are known to be under the control of the trans-acting regulatory gene amdR. We describe the isolation and initial characterization of one of these amdR-regulated genes, lamA. The lam locus, however, was found to consist of two divergently transcribed genes, the lamA gene, and a new gene, also under amdR control, which we have designated lamB. Using recombinant DNA techniques we have constructed a strain of A. nidulans lacking a functional lamB gene. Experiments conducted with this strain demonstrate that lamB, like lamA, is involved in utilization of 2-pyrrolidinone in A. nidulans. Metabolism of a related compound, gamma-amino butyric acid (GABA) is not affected. We also provide evidence that the conversion of exogenous 2-pyrrolidinone to endogenous GABA requires a functional lamB gene. The expression of both lamA and lamB is subject to carbon and nitrogen metabolite repression in addition to amdR-mediated induction by omega-amino acids.     

Mutation and functional analysis of the Aspergillus nidulans ammonium permease MeaA and evidence for interaction with itself and MepA

The movement of ammonium across biological membranes is mediated in both prokaryotic and eukaryotic systems by ammonium transport proteins which constitute a family of related sequences (called the AMT/MEP family). Interestingly, recent evidence suggests that human and mouse Rhesus proteins which display significant relatedness to AMT/MEP sequences may function as ammonium transporters. To add to the functional understanding of ammonium transport proteins, the sequence changes in 37 loss-of-function mutations within the Aspergillus nidulans ammonium permease gene, meaA, were characterized. Together with the identification of conserved AMT/MEP residues and regions, the mutational analysis predicted regions important for uptake activity. Specifically, a major facilitator superfamily like motif (161-GAVAERGR-168 in MeaA) may be important for the translocation of ammonium across the membrane as may the conserved Pro186 residue. A specific Gly447 to Asp mutation was introduced into MeaA and this mutant protein was found to trans-inhibit the activity of endogenous MeaA and the other A. nidulans ammonium transporter, MepA. These results suggest that MeaA may interact with itself and with MepA, although any hetero-interaction is not required for ammonium transport function. In addition, cross-feeding studies showed that MeaA and to a lesser extent MepA are also required for the retention of intracellular ammonium.     

Cloning and functional expression of a gene encoding a P1 type nucleoside transporter from Trypanosoma brucei.

Nucleoside transporters are likely to play a central role in the biochemistry of the parasite Trypanosoma brucei, since these protozoa are unable to synthesize purines de novo and must salvage them from their hosts. Furthermore, nucleoside transporters have been implicated in the uptake of antiparasitic and experimental drugs in these and other parasites. We have cloned the gene for a T. brucei nucleoside transporter, TbNT2, and shown that this permease is related in sequence to mammalian equilibrative nucleoside transporters. Expression of the TbNT2 gene in Xenopus oocytes reveals that the permease transports adenosine, inosine, and guanosine and hence has the substrate specificity of the P1 type nucleoside transporters that have been previously characterized by uptake assays in intact parasites. TbNT2 mRNA is expressed in bloodstream form (mammalian host stage) parasites but not in procyclic form (insect stage) parasites, indicating that the gene is developmentally regulated during the parasite life cycle. Genomic Southern blots suggest that there are multiple genes related in sequence to TbNT2, implying the existence of a family of nucleoside transporter genes in these parasites.     

Tat1, a novel sulfate transporter specifically expressed in human male germ cells and potentially linked to rhogtpase signaling

RhoGTPases (Rho, Rac, and Cdc42) are known to regulate multiple functions, including cell motility, adhesion, and proliferation; however, the signaling pathways underlying these pleiotropic effects are far from fully understood. We have recently described a new RhoGAP (GTPase activating protein for RhoGTPases) gene, MgcRacGAP, primarily expressed in male germ cells, at the spermatocyte stage. We report here the isolation, through two-hybrid cloning, of a new partner of MgcRacGAP, very specifically expressed in the male germ line and showing structural features of anion transporters. This large protein (970 amino acids and a predicted size of 109 kDa), we provisionally designated Tat1 (for testis anion transporter 1), is closely related to a sulfate permease family comprising three proteins in human (DRA, Pendrin, and DTD); it is predicted to be an integral membrane protein with 14 transmembrane helices and intracytoplasmic NH(2) and COOH termini. In situ hybridization studies demonstrate that Tat1 and MgcRacGAP genes are coexpressed in male germ cells at the spermatocyte stage. On testis sections, Tat1 protein can be immunodetected in spermatocytes and spermatids associated with plasma membrane. Two-hybrid and in vitro binding assays demonstrate that MgcRacGAP stably interacts through its NH(2)-terminal domain with the Tat1 COOH-terminal region. Expression of Tat1 protein in COS7 cells generates a 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene and chloride-sensitive sulfate transport. Therefore we conclude that Tat1 is a novel sulfate transporter specifically expressed in spermatocytes and spermatids and interacts with MgcRacGAP in these cells. These observations raise the possibility of a new regulatory pathway linking sulfate transport to Rho signaling in male germ cells.