RESULTS

Phenotypic analysis of acrB2, acrB14, and acrB15: As selected, the three acrB mutant strains, acrB2, acrB14, and acrB15, were resistant to the presence of acriflavine in complete medium, with similar levels of resistance between acrB mutant strains and the creD34 strain (Figure 1). Since the creD34 mutation leads to sensitivity to molybdate, the three acrB mutant strains were tested and found to be more sensitive than the wild-type strain to the presence of 11 mm molybdate in synthetic complete medium, but the alleles were heterogeneous, with the acrB2 strain being the most sensitive to the presence of molybdate and the acrB15 strain showing lesser sensitivity. Thus the effects of the acrB mutant alleles when the toxic compounds acriflavine and molybdate are added to the medium are the same as those for creD34 and the opposite to the effects of creB and creC mutant alleles, which confer increased sensitivity to acriflavine and resistance to molybdate added to complete medium (Hynes and Kelly 1977; Kelly and Hynes 1977; Arst 1981).

When tested for their ability to utilize various carbon sources, acrB mutant strains were found to have pleiotropic phenotypes with respect to carbon source utilization. They show decreased ability to utilize a number of different sugars as sole carbon sources in comparison to both the wild type and the creD34 mutant strain, including fructose, cellobiose, raffinose, and starch (Figure 1; Table 2). In general, the acrB14 strain showed a slightly more extreme phenotype than that showed by the acrB2 and acrB15 strains. The reduced growth on various sugars may indicate a failure of uptake or a failure to express genes encoding enzymes required for their utilization. The ability to utilize other alternate carbon sources such as quinate and glycerol was only slightly affected by the acrB mutations, with the exception of ethanol where the acrB mutants grew considerably less well than wild type (Table 2).

Test medium containing 10 mm allyl alcohol in the presence of glucose was used to investigate the expression of the alcohol dehydrogenase encoded by alcA. All the acrB-containing strains, like the wild-type and creD34-containing strains, were resistant, indicating that they showed correct glucose repression of alcA. The acrB mutant strains were hypersensitive on medium containing glucose plus 10 mg/ml fluoroacetamide, indicating an increased level of the acetamidase expressed from the amdS gene compared to the wild-type strain, which is the opposite of the phenotype conferred by the creD34 mutation. The lack of growth of the acrB mutant strains on medium containing glucose plus 10 mm acrylamide as a nitrogen source indicates that this increased level of amdS expression is not due to constitutive expression, as acrylamide is a substrate of the acetamidase but not an inducer of amdS (Hynes and Pateman 1970). Further, wild-type growth of the acrB mutant strains on acetamide as a carbon, nitrogen, or carbon and nitrogen source indicates that the amdS gene is fully inducible. Thus there is a defect in the glucose-mediated repression of amdS but no other effect on expression.

Growth on media containing the ω-amino acids 10 mm β-alanine, 10 mm GABA, or 10 mm pyrrolidinone as nitrogen sources was significantly decreased in the acrB mutant strains compared to the wild-type strain, as was the case for a strain containing creD34. Similarly, growth of the mutant acrB strains on media containing 50 mm GABA and 50 mm pyrrolidinone acting as both a carbon and a nitrogen source was also decreased compared to wild type and the creD34 mutant strain, but this effect was not obvious on media containing 50 mm β-alanine. The acrB mutant alleles affected growth to various degrees on media that had 10 mm pyrrolidinone as a nitrogen source, with the acrB15 strain having strong, almost wild-type-like growth, compared to the intermediate growth of the acrB14 strain and the weaker growth of the acrB2 strain, and this heterogeneity between alleles indicates that they do not all represent complete loss-of-function alleles.

Growth on media containing other amino acids as nitrogen sources, such as 10 mm proline and 10 mm glutamate, was unaffected by the acrB mutations. However, decreased utilization of 50 mm proline and 50 mm glutamate when they were used as both carbon and nitrogen sources was observed for the acrB mutant strains in comparison to the wild-type strain.

Partial suppression by acrB2 of creB and creC mutant phenotypes: The creD34 mutation was identified as a suppressor of the hypersensitivity to fluoroacetamide conferred by the creC27 mutation. The effects of the creD34 mutation are pleiotropic in that it also leads to suppression of the effects of creC27 on other enzymes subject to carbon catabolite repression, such as alcohol dehydrogenase I, but it does not suppress the effects of creC27 that are apparent under derepressing conditions, such as the poor growth on d-quinate medium (Kelly and Hynes 1977; Kelly 1980). The creD34 mutation suppresses not only some of the phenotypes conferred by the creC27 mutation, but also some of the phenotypic effects of the creB15 mutation, and, weakly, of the creA204 mutation (Kelly and Hynes 1977; Kelly 1980). Similarities between the phenotypes of creD and acrB mutant strains led us to test whether the effects of mutation in acrB are also epistatic for some of the phenotypes conferred by creB and creC mutations.

The presence of the acrB2 mutation reversed the sensitivity conferred by the creB1937 and creC27 mutations to the presence of acriflavine in complete media (Figure 2c). The creD34 acrB2 double mutant strain was even more resistant to the presence of acriflavine than was either of the individual mutant strains, indicating an additive effect. The resistance of the creB1937 and creC27 strains to the presence of molybdate was also reversed in the relevant acrB2 double mutant strains and to a greater degree than that in the creD34 double mutant strains (Figure 2e). The acrB2 mutation also reversed the toxic effects of allyl alcohol added to d-glucose medium in creC27 and creB1937 backgrounds, the same phenotypic suppression previously seen for the creD34 mutation (Figure 2f).

The acrB2 mutation results in reduced growth compared to wild type on sole carbon sources such as fructose, maltose, starch, cellobiose, ethanol, and acetate, a phenotype not seen for the creD34 mutation (Figure 1; Table 2). This reduced growth is also seen in the acrB2creB1937 and acrB2creC27 double mutant strains compared to the single creB1937 and creC27 mutant phenotypes, respectively (Figure 2g). The inability of the creB1937 and creC27 mutant strains to utilize d-quinate as an alternate carbon source was partially suppressed by the acrB2 mutation in the double mutant strains, and this effect was more pronounced than that in the creD34 double mutant strains (Figure 2h). Finally, the acrB2creB1937 and acrB2creC27 strains grew as poorly as the acrB2 strain on glucose medium containing 10 mm pyrrolidinone as the nitrogen source (Figure 2i).

Molecular cloning of acrB: We took advantage of the close genetic linkage of acrB and creB (Clutterbuck 1997) to clone the acrB gene. The bacterial artificial chromosome (BAC) clone that contains creB, BAC4B1 (Lockington and Kelly 2001), was tested for complementation of the acrB2 mutation in a transformation assay testing for the reversal of the resistance to acriflavine in complete medium conferred by the acrB2 mutation. This BAC clone was found to complement the acrB2 phenotype. Cosmid clones have been ordered within this BAC (Lockington and Kelly 2001) and the cosmids containing the creB gene, SL10A08 and RW18E04 (Lockington and Kelly 2001), were tested and failed to complement the acrB2 mutation in the transformation assay. The cosmids flanking these cosmids, SL27D09 and SL19A07, which also hybridized to BAC4B1 (Lockington and Kelly 2001), were also tested in a transformation assay, but again neither complemented the acrB2 mutation. Other cosmids within the region that also hybridized to BAC4B1, SW21D03, SW22F07, SW23H02, and SW23H06, were tested, and SW22F07, SW23H02, and SW23H06 were found to complement the acrB2 mutation. Thus the order of genes in this region is, from centromeric to telomeric, adCadD-acoB-atrC-acrB-creB with acrB ∼30 kb from creB.

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Figure 2.

—Epistatic effects of the acrB2 mutation on creB and creC mutant phenotypes. (a) Key to strain placement. Indicated strains were grown at 37° for 2–3 days on the following media: (b) complete medium, (c) complete medium plus 0.001% acriflavine, (d) 1% d-glucose plus 10 mm ammonium l-tartrate, (e) 1% d-glucose plus 10 mm ammonium l-tartrate plus 33 mm molybdate, (f) 1% d-glucose plus 10 mm ammonium l-tartrate plus 10 mm allyl alcohol, (g) 1% d-maltose plus 10 mm ammonium l-tartrate, (h) 50 mm (d)-quinate plus 10 mm ammonium l-tartrate, and (i) 1% d-glucose plus 10 mm 2-pyrrolidinone.

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Figure 3.

—Location of the acrB mutations within the acrB gene. The complete sequence of the acrB genomic region is shown (GenBank accession no. AF485329). The single intron of 63 bp is depicted in lowercase letters and the 5′ and 3′ splice junction and lariat sequences are underlined. A putative TATA box and a putative CCAAT box are underlined and in boldface letters. The major start point of transcription is indicated above the DNA by the # symbol. A single theoretical CreA binding site in the 5′ untranslated region is double underlined, and a single theoretical AreA binding site in the 5′ untranslated region is underlined with a dashed line. The amino acid sequence is shown in single letter code beneath the DNA sequence, with numbered amino residues indicated on the left-hand side and numbered nucleotide positions indicated on the right-hand side, with the first nucleotide of the start codon as +1. The position and nature of the DNA sequence changes in the acrB2, acrB14, and acrB15 alleles are shown, with (_) indicating a deletion of the base directly below and (+) indicating an addition of a base at the position below. The stop codon is indicated by an asterisk.

Cosmid SW23H06 was selected for further analysis. An EcoRI cutdown that contained a 10-kb insert, c24F6-14, complemented acrB2 in the transformation assay. c24F6-16 contained three ClaI fragments, which were cloned into pCB1004 (p4AcrB, p7AcrB, and p10AcrB), and p4AcrB, containing a 6-kb insert, was found to complement the acrB2 mutation, while p7AcrB and p10AcrB did not. Plasmids containing ∼2-kb SacI or KpnI deletions of each end of p4AcrB (p4AcrBSacIΔ and p4AcrB-KpnIΔ) failed to complement acrB2 in the transformation assay. Thus acrB2 was located in the middle of the 6-kb insert of p4AcrB.

Sequencing of the insert in p4AcrB revealed a 3111-bp open reading frame (GenBank accession no. AF485329; Figure 3). The acrB gene contained a single intron, which was determined by sequence analysis of PCR amplified cDNAs (see materials and methods), and the intronic sequence conforms to the consensus 5′ and 3′ junction and lariat sequences for fungal introns (Gurret al. 1987). To determine the transcription start point (tsp) of the gene, 5′ RACE of RNA purified from d-glucose-grown mycelium was carried out using nested primers. Direct sequencing of the nested PCR product and of several subclones of this product mapped a single major tsp –221 bp from the translation start (Figure 3). A TATA-like sequence is located 33 bp upstream of the tsp, and a CCAAT box is located 155 bp upstream of the tsp. The acrB gene is not present in the A. nidulans expressed sequence tag (EST) database (containing EST information for cDNA clones from a mixed vegetative and 24-hr asexual development culture of A. nidulans strains FGSCA26 constructed in lambda Zap by R. Aramayo at Texas A&M University; http://www.genome.ou.edu/asperg.html), indicating a low level of expression under these conditions. The results of a Northern analysis using acrB as a probe were consistent with this, and acrB mRNA was below the level of detection in total RNA extracted from glucose-grown wild-type mycelia, but was present in total RNA extracted from either maltose- or fructose-grown mycelia (data not shown). This expression pattern indicates transcriptional control in response to glucose, although only a single theoretical CreA binding site is present in the 312 bases of sequence determined 5′ to the major start point of transcription.

The acrB gene encodes a 1015-amino-acid polypeptide. AcrB is predicted to contain three transmembrane domains near the N terminus, at amino acid residues 158–180 (I), 214–236 (II), and 257–274 (III), and a coiled-coil region, at amino acid residues 596–753 (SMART protein motif analysis program; Schultzet al. 1998). The transmembrane domain prediction program, TMHMM2 (Kroghet al. 2001), predicts the most probable location and orientation of transmembrane helices from sequence data, and this program indicated that the N terminus of the AcrB protein and the region between the second and third transmembrane domains are most likely cytoplasmic, with the C-terminal region of the protein predicted to be external.

A hypothetical protein is in contig 1168 of the TIGR Aspergillus fumigatus Genome Database, which shows 76% identity with the A. nidulans sequence, but these are very closely related ascomycete species. Database searches (SwissProt/SpTrEMBL/PDB) using the BlastP program (Altschulet al. 1990) revealed that the AcrB protein was not highly similar to any protein in these databases. AcrB shows most sequence similarity with hypothetical proteins, one from Neurospora crassa, Q9C2R3 (31% identity over 1015 amino acids), and one from Saccharomyces cerevisiae, YG2K (24% identity over 861 amino acids). The hypothetical N. crassa protein contains three putative transmembrane domains and a coiled-coil domain, and although it shows low similarity along the length of the protein with AcrB, specific regions such as the transmembrane domains share higher sequence identity (55% identity over 24 amino acids). The S. cerevisiae hypothetical protein has a protein structure similar to that of AcrB, with three transmembrane domains and a coiled-coil region, but the sequence similarity is uniformly low throughout the protein.

Molecular analysis of the acrB mutant alleles: Three mutant alleles of acrB were sequenced to identify functional regions of the AcrB protein and to show that acrB rather than a suppressor of acrB2 had been isolated. We used a PCR approach using primers spanning the acrB gene followed by direct sequencing of the PCR products (see materials and methods). The acrB2 mutation is a single base pair deletion at nucleotide (nt) +1734 at the 5′ splice site of the only intron (Figure 3). This would be predicted to disrupt the splicing of the intron, resulting in a frameshift after amino acid 577 and the additional residues, GRYSFYGFRFVSMWSNLCLP, before terminating. The acrB15 mutant allele contains a 2-bp deletion at nt +1815, which results in a frameshift after amino acid 584, with an addition amino acid sequence of ATLISTILPNNHAEELDHQC before truncating. Both acrB2 and acrB15 gene products contain all three transmembrane domains but lack the coiled-coil region. The acrB14 allele contains a C-to-T transition plus a 1-bp insertion (T) at nt +627 that results in a frameshift after amino acid 209 and truncates 84 amino acids later. The additional amino acids are FARYHD CNGWFLPACMGPLYVDVGPEFCSRFSPCPGCHHSGR WRCREKWWCQCALRRYCSDSASHTQQRNTGFCRRP SCFSKNH. The acrB14 mutant gene product lacks two of the three transmembrane domains and the coiled-coil region and, since it is also recessive to the wild-type allele, probably represents a null allele.