A third ADP/ATP translocator gene in yeast

The op1 mutation in yeast is known to be due to a defect in the mitochondrial ADP/ATP translocator. Sequencing of the gene AAC2 revealed that the mutation resulted from a single base change that caused a replacement of arginine 97 by a histidine. The gene encoding AAC2 was also cloned and sequenced from an op1 revertant capable of growth on glycerol as a sole carbon source. Sequence analysis indicates that the reverted gene underwent rearrangement in which a portion of an unknown gene was used to repair the mutation. An oligonucleotide complementary to this insert was used to clone a previously unrecognized gene encoding ADP/ATP translocator in yeast. The newly discovered gene, AAC3, is homologous with the previously known genes AAC1 and AAC2. Gene disruption experiments suggest that AAC2 encodes the majority of the translocator. Expression of AAC1 and AAC2 required derepressed conditions whereas expression of AAC3 occurred almost exclusively under anaerobic conditions. Both the op1 mutant and the strain that contains an interrupted AAC2 were able to grow under anaerobic conditions, suggesting that AAC3 can replace the gene product of AAC2. Indeed, when cloned into multicopy plasmid, AAC3 was able to replace the disrupted AAC2 in the JLY-73 strain. The concomitant disruption of the AAC2 and AAC3, however, results in arrest of cell growth under conditions of low oxygen tension. The discovery of a third gene encoding ADP/ATP translocator helps to clarify certain characteristics of op1 mutants which could not be resolved in the past.     

Heterologous complementation of the Klaac null mutation of Kluyveromyces lactis by the Saccharomyces cerevisiae AAC3 gene encoding the ADP/ATP carrier

The KlAAC gene, encoding the ADP/ATP carrier, has been assumed to be a single gene in Kluyveromyces lactis, an aerobic, petite-negative yeast species. The Klaac null mutation, which causes a respiratory-deficient phenotype, was fully complemented by AAC2, the Saccharomyces cerevisiae major gene for the ADP/ATP carrier and also by AAC1, a gene that is poorly expressed in S. cerevisiae. In this study, we demonstrate that the Klaac null mutation is partially complemented by the ScAAC3 gene, encoding the hypoxic ADP/ATP carrier isoform, whose expression in S. cerevisiae is prevented by oxygen. Once introduced into K. lactis, the AAC3 gene was expressed both under aerobic and under partial anaerobic conditions but did not support the growth of K. lactis under strict anaerobic conditions.     

Separate genes encode functionally equivalent ADP/ATP carrier proteins in Saccharomyces cerevisiae. Isolation and analysis of AAC2

Genetic and biochemical analysis of Saccharomyces cerevisiae containing a disruption of the nuclear gene (AAC1) encoding the mitochondrial ADP/ATP carrier has revealed a second gene for this protein. The second gene, designated AAC2, has been isolated by genetic complementation and sequenced. AAC2 contains a 954-base pair open reading frame coding for a protein of 318 amino acids which is highly homologous to the AAC1 gene product except that it is nine amino acids longer at the NH2 terminus. The two yeast genes are highly conserved at the level of DNA and protein and share identity with the ADP/ATP carriers from other organisms. Both genes complement an ADP/ATP carrier defect (op1 or pet9). However, the newly isolated gene AAC2 need be present only in one or two copies while the previously isolated AAC1 gene must be present in multiple copies to support growth dependent on a functional carrier protein. This gene dosage-dependent complementation combined with the high degree of conservation suggest that these two functionally equivalent genes may be differentially expressed.     

Structure-function studies of adenine nucleotide transport in mitochondria. II. Biochemical analysis of distinct AAC1 and AAC2 proteins in yeast

AAC1 and AAC2 genes in yeast each encode functional ADP/ATP carrier (AAC) proteins of the mitochondrial inner membrane. In the present study, mitochondria harboring distinct AAC proteins and the pet9 Arg96 to HIS mutant (Lawson, J., Gawaz, M., Klingenberg, M., and Douglas, M. G. (1990) J. Biol. Chem. 265, 14195-14201) protein have been characterized. In addition, properties of the different AAC proteins have been defined following reconstitution into proteoliposomes. Deletion of AAC2 but not AAC1 causes a major reduction in the mitochondrial cytochrome content and respiration, and this level remains low even when the level of AAC1 protein is increased to 20% that of the AAC2 gene product. In reconstitution studies, the rate of nucleotide transport by isolated AAC1 protein is approximately 40% that of the AAC2 protein. Thus, the lack of mitochondrial-dependent growth supported by the AAC1 gene product alone may be due to the combination of low abundance and reduced activity. Surprisingly, analysis of the Arg96 to His mutant protein revealed binding and transport activities similar to the functional AAC1 and AAC2 gene products. These observations are discussed in relation to a molecular analysis of this highly conserved small transporter and its function in conjunction with other proteins in the mitochondrial membrane.     

The molecular basis for relative physiological functionality of the ADP/ATP carrier isoforms in Saccharomyces cerevisiae

AAC2 is one of three paralogs encoding mitochondrial ADP/ATP carriers in the yeast Saccharomyces cerevisiae, and because it is required for respiratory growth it has been the most extensively studied. To comparatively examine the relative functionality of Aac1, Aac2, and Aac3 in vivo, the gene encoding each isoform was expressed from the native AAC2 locus in aac1Delta aac3Delta yeast. Compared to Aac2, Aac1 exhibited reduced capacity to support growth of yeast lacking mitochondrial DNA or of yeast lacking the ATP/Mg-P(i) carrier, both conditions requiring ATP import into the mitochondrial matrix through the ADP/ATP carrier. Sixteen AAC1/AAC2 chimeric genes were constructed and analyzed to determine the key differences between residues or sections of Aac1 and Aac2. On the basis of the growth rate differences of yeast expressing different chimeras, the C1 and M2 loops of the ADP/ATP carriers contain divergent residues that are responsible for the difference(s) between Aac1 and Aac2. One chimeric gene construct supported growth on nonfermentable carbon sources but failed to support growth of yeast lacking mitochondrial DNA. We identified nine independent intragenic mutations in this chimeric gene that suppressed the growth phenotype of yeast lacking mitochondrial DNA, identifying regions of the carrier important for nucleotide exchange activities.     

Characterization of genes encoding dimethyl sulfoxide reductase of Rhodobacter sphaeroides 2.4.1T: an essential metabolic gene function encoded on chromosome II.

Rhodobacter sphaeroides 2.4.1T is a purple nonsulfur facultative phototrophic bacterium which exhibits remarkable metabolic diversity as well as genomic complexity. Under anoxic conditions, in the absence of light and the presence of dimethyl sulfoxide (DMSO) or trimethylamine N-oxide (TMAO), R. sphaeroides 2.4.1T utilizes DMSO or TMAO as the terminal electron acceptor for anaerobic respiration, which is mediated by the molybdoenzyme DMSO reductase. Sequencing of a 13-kb region of chromosome II revealed the presence of 10 putative open reading frames, of which 5 possess homology to genes encoding the TMAO reductase (the tor system) of Escherichia coli. The dorS and dorR genes encode a sensor-regulator pair of the two-component sensory transduction protein family, homologous to the torS and torR gene products. The dorC gene was shown to encode a 44-kDa DMSO-inducible c-type cytochrome. The dorB gene encodes a membrane protein of unknown function homologous to the torD gene product. The dorA gene encodes DMSO reductase, containing the molybdopterin active site. Mutations were constructed in each of these dor genes, and the resulting mutants were shown to be impaired for DMSO-dependent anaerobic growth in the dark. The mutant strains exhibited negligible levels of DMSO reductase activity compared to the wild-type strain under similar growth conditions. Further, no DorA protein was detected in DorS and DorR mutant strains with anti-DorA antisera, suggesting that the products of these genes are required for the positive regulation of dor expression in response to DMSO. This characterization of the dor gene cluster is the first evidence that genes of chromosome CII encode metabolic functions which are essential under particular growth conditions.     

Characterization of a novel tetracycline resistance that functions only in aerobically grown Escherichia coli.

A tetracycline resistance (Tcr) gene that was found originally on two Bacteroides plasmids (pBF4 and pCP1) confers tetracycline resistance on Escherichia coli, but only when it is grown aerobically. Using maxicells, we have identified a 44-kilodalton protein which is encoded by the region that carries the Tcr gene and which may be the Tcr gene product. Localization experiments indicate that this 44-kilodalton protein is cytoplasmic. To determine whether the tetracycline resistance gene is expressed under anaerobic conditions, we have constructed a protein fusion between the Tcr gene and lacZ. In strains of E. coli carrying the fusion, beta-galactosidase activity was the same when the cells were grown under anaerobic conditions as when the cells were grown under aerobic conditions. This indicates that the tetracycline resistance gene product is made under anaerobic conditions but does not work. The failure of the Tcr protein to function under anaerobic conditions was not due to a requirement for function of the anaerobic electron transport system, because neither nitrate nor fumarate added to anaerobic media restored tetracycline resistance. Inhibition of the aerobic electron transport system with potassium cyanide did not prevent growth on tetracycline of cells containing the Tcr gene. A heme-deficient mutant, E. coli SHSP19, which carries the Tcr gene, was still resistant to tetracycline even when grown in heme-free medium. These results indicate that functioning of the Tcr gene product is not dependent on the aerobic electron transport system. Thus the requirement for aerobic conditions appears to reflect a requirement for oxygen. Spent medium from an E. coli strain carrying the Tcr gene, which was grown in medium containing tetracycline (50 micrograms/ml), did not inhibit growth of a tetracycline-susceptible strain of E. coli. Thus, the Tcr gene product may be detoxifying tetracycline.     

Global gene expression profiles of Bacillus subtilis grown under anaerobic conditions

Bacillus subtilis can grow under anaerobic conditions, either with nitrate or nitrite as the electron acceptor or by fermentation. A DNA microarray containing 4,020 genes from this organism was constructed to explore anaerobic gene expression patterns on a genomic scale. When mRNA levels of aerobic and anaerobic cultures during exponential growth were compared, several hundred genes were observed to be induced or repressed under anaerobic conditions. These genes are involved in a variety of cell functions, including carbon metabolism, electron transport, iron uptake, antibiotic production, and stress response. Among the highly induced genes are not only those responsible for nitrate respiration and fermentation but also those of unknown function. Certain groups of genes were specifically regulated during anaerobic growth on nitrite, while others were primarily affected during fermentative growth, indicating a complex regulatory circuitry of anaerobic metabolism.     

Engineering of the metabolism of Saccharomyces cerevisiae for anaerobic production of mannitol

Under anaerobic conditions, Saccharomyces cerevisiae uses NADH-dependent glycerol-3-phosphate dehydrogenase (Gpd1p and Gpd2p) to re-oxidize excess NADH, yielding substantial amounts of glycerol. In a Deltagpd1 Deltagpd2 double-null mutant, the necessary NAD+ regeneration through glycerol production is no longer possible, and this mutant does not grow under anaerobic conditions. The excess NADH formed can potentially be used to drive other NADH-dependent reactions or pathways. To investigate this possibility, a double-null mutant was transformed with a heterologous gene (mtlD) from Escherichia coli, coding for NADH-dependent mannitol-1-phosphate dehydrogenase. Expression of this gene in S. cerevisiae should result in NADH oxidation by the NADH-requiring formation of mannitol-1-phosphate from fructose-6-phosphate. The strain was characterized using step-change experiments, in which, during the exponential growth phase, the inlet gas was changed from air to nitrogen. It was found that the mutant produced mannitol only under anaerobic conditions. However, anaerobic growth was not regained, which was probably due to the excessive accumulation of mannitol in the cells.     

Yeast ADP/ATP carrier (AAC) proteins exhibit similar enzymatic properties but their deletion produces different phenotypes.

Saccharomyces cerevisiae strains expressing a single type of ADP/ATP carrier (AAC) protein were prepared from a mutant in which all AAC genes were disrupted, by transformation with plasmids containing a chosen AAC gene. As demonstrated by measurements of [14C]ADP specific binding and transport, all three translocator proteins, AAC1, AAC2 and AAC3 when present in the mitochondrial membrane, exhibited similar translocation properties. The disruption of some AAC genes, however, resulted in phenotypes indicating that the function of these proteins in whole cells can be quite different. Specifically, we found that the disruption of AAC1 gene, but not AAC2 and AAC3, resulted in a change in colony phenotype.     

Requirement for terminal cytochromes in generation of the aerobic signal for the arc regulatory system in Escherichia coli: study utilizing deletions and lac fusions of cyo and cyd.

Escherichia coli has two terminal oxidases for its respiratory chain: cytochrome o (low O2 affinity) and cytochrome d (high O2 affinity). Expression of the cyo operon, encoding cytochrome o, is decreased by anaerobic growth, whereas expression of the cyd operon, encoding cytochrome d, is increased by anaerobic growth. We show by the use of lac gene fusion that the expressions of cyo and cyd are under the control of the two-component arc system. In a cyo+ cyd+ background, expression of phi(cyo-lac) is higher when the organism is grown aerobically than when it is grown anaerobically. A mutation in either the sensor gene arcB or the pleiotropic regulator gene arcA almost abolishes the anaerobic repression. In the same background, expression of phi(cyd-lac) is higher under anaerobic growth conditions than under aerobic growth conditions. A mutation in arcA or arcB lowers both the aerobic and anaerobic expressions, suggesting that ArcA plays an activating role instead of the typical repressing role. Under aerobic growth conditions, double deletions of cyo and cyd lower phi(cyo-lac) expression but enhance phi(cyd-lac) expression. The double deletions also prevent elevated aerobic induction of the lct operon (encoding L-lactate dehydrogenase), another target operon of the arc system. In contrast, these deletions do not circumvent aerobic repression of the nar operon (encoding the anaerobic respiratory enzyme nitrate reductase) under the control of the pleiotropic fnr gene product. It thus appears that ArcB senses the presence of O2 by level of an electron transport component in reduced form or that of an nonautoxidizable compound linked to the process by a redox reaction, whereas Fnr senses O2 by a different mechanism.     

Soluble fumarate reductase isoenzymes from Saccharomyces cerevisiae are required for anaerobic growth

In Saccharomyces cerevisiae, the cytosolic and promitochondrial isoenzymes of fumarate reductase are encoded by the FRDS and OSM1 genes, respectively. The product of the OSM1 gene is reported to be required for growth in hypertonic medium. Simultaneous disruption of the FRDS and OSM1 genes resulted in the inability of the yeasts to grow anaerobically on glucose as a carbon source, and disruption of the OSM1 gene caused poor growth under anaerobic conditions. However, the disruption of both the FRDS and/or OSM1 genes had no effect on aerobic growth or growth under hypertonic conditions. These results suggest that the fumarate reductase isoenzymes in Saccharomyces cerevisiae are essential for anaerobic growth but not for growth under hypertonic conditions.     

Regulation of the citrate synthase (gltA) gene of Escherichia coli in response to anaerobiosis and carbon supply: role of the arcA gene product.

As an enzyme of the tricarboxylic acid cycle pathway, citrate synthase participates in the generation of a variety of cellular biosynthetic intermediates and in that of reduced purine nucleotides that are used in energy generation via electron transport-linked phosphorylation reactions. It catalyzes the condensation of oxaloacetate and acetyl coenzyme A to produce citrate plus coenzyme A. In Escherichia coli this enzyme is encoded by the gltA gene. To investigate how gltA expression is regulated, a gltA-lacZ operon fusion was constructed and analyzed following aerobic and anaerobic cell growth on various types of culture media. Under aerobic culture conditions, expression was elevated to a level twofold higher than that reached under anaerobic culture conditions. ArcA functions as a repressor of gltA expression under each set of conditions: in a delta arcA strain, gltA-lacZ expression was elevated to levels two- and eightfold higher than those seen in a wild-type strain under aerobic and anaerobic conditions, respectively. This control is independent of the fnr gene product, an alternative anaerobic gene regulator in E. coli. When the richness or type of carbon compound used for cell growth was varied, gltA-lacZ expression varied by 10- to 14-fold during aerobic and anaerobic growth. This regulation was independent of both the crp and fruR gene products, suggesting that another regulatory element in E. coli is responsible for the observed control. Finally, gltA-lacZ expression was shown to be inversely proportional to the cell growth rate. These findings indicate that the regulation of gltA gene expression is complex in meeting the differential needs of the cell for biosynthesis and energy generation under various cell culture conditions.     

Identification of a methyl-accepting chemotaxis protein in Rhodobacter sphaeroides

Analysis of the DNA sequence directly upstream of the chemotaxis operon of Rhodobacter sphaeroides identified a single gene whose product has strong similarity to the methyl-accepting chemotaxis proteins (MCPs) found in enteric bacteria. The deduced protein had a highly conserved signalling sequence and only one very hydrophobic region at the N-terminus, in contrast to enteric MCPs. A possible cytoplasmic location of the majority of the protein was supported by Western blotting. The mcpA gene was insertionally inactivated and the resulting phenotype examined using swarm plate assays. The mutant lacking McpA lost chemotaxis to a wide range of attractant stimuli but only under aerobic conditions; it retained almost normal chemotaxis under anaerobic/photosynthetic conditions. The identification of a sensory protein which is active only under one set of growth conditions suggests that R. sphaeroides probably has several MCPs, which co-ordinately respond to changes in environmental conditions. Southern hybridization at relaxed stringency to the conserved sequence of the R. sphaeroides and Caulobacter crescentus mcp genes identified three possible additional mcp genes.     

cobA function is required for both de novo cobalamin biosynthesis and assimilation of exogenous corrinoids in Salmonella typhimurium

Salmonella typhimurium is able to synthesize cobalamin (B12) under anaerobic growth conditions. The previously described cobalamin biosynthetic mutations (phenotypic classes CobI, CobII, and CobIII) map in three operons located near the his locus (minute 41). A new class of mutant (CobIV) defective in B12 biosynthesis was isolated and characterized. These mutations map between the cysB and trp loci (minute 34) and define a new genetic locus, cobA. The anaerobic phenotype of cobA mutants suggests an early block in corrin ring formation; mutants failed to synthesize cobalamin de novo but did so when the corrin ring is provided as cobyric acid dicyanide or as cobinamide dicyanide. Under aerobic conditions, cobA mutants were unable to convert either cobyric acid dicyanide or cobinamide dicyanide to cobalamin but could use adenosylcobyric acid or adenosylcobinamide as a precursor; this suggests that the mutants are unable to adenosylate exogenous corrinoids. To explain the anaerobic CobI phenotype of a cobA mutant, we propose that the cobA gene product catalyzes adenosylation of an early intermediate in the de novo B12 pathway and also adenosylates exogenous corrinoids. Under anaerobic conditions, a substitute function, known to be encoded in the main Cob operons, is induced; this substitute function can adenosylate exogenous cobyric acid and cobinamide but not the early biosynthetic intermediate. The cobA gene of S. typhimurium appears to be functionally equivalent to the btuR gene of Escherichia coli.     

Functional Divergence in a Multi-gene Family Is a Key Evolutionary Innovation for Anaerobic Growth in Saccharomyces cerevisiae

The amplification and diversification of genes into large multi-gene families often mark key evolutionary innovations, but this process often creates genetic redundancy that hinders functional investigations. When the model budding yeast Saccharomyces cerevisiae transitions to anaerobic growth conditions, the cell massively induces the expression of seven serine/threonine-rich anaerobically-induced cell wall mannoproteins (anCWMPs): TIP1, TIR1, TIR2, TIR3, TIR4, DAN1, and DAN4. Here, we show that these genes likely derive evolutionarily from a single ancestral anCWMP locus, which was duplicated and translocated to new genomic contexts several times both prior to and following the budding yeast whole genome duplication (WGD) event. Based on synteny and their phylogeny, we separate the anCWMPs into four gene subfamilies. To resolve prior inconclusive genetic investigations of these genes, we constructed a set of combinatorial deletion mutants to determine their contributions toward anaerobic growth in S. cerevisiae. We found that two genes, TIR1 and TIR3, were together necessary and sufficient for the anCWMP contribution to anaerobic growth. Overexpressing either gene alone was insufficient for anaerobic growth, implying that they encode non-overlapping functional roles in the cell during anaerobic growth. We infer from the phylogeny of the anCWMP genes that these two important genes derive from an ancient duplication that predates the WGD event, whereas the TIR1 subfamily experienced gene family amplification after the WGD event. Taken together, the genetic and molecular evidence suggests that one key anCWMP gene duplication event, several auxiliary gene duplication events, and functional divergence underpin the evolution of anaerobic growth in budding yeasts.     

Physiological role of soluble fumarate reductase in redox balancing during anaerobiosis in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, there are two isoenzymes of fumarate reductase (FRDS1 and FRDS2), encoded by the FRDS and OSM1 genes, respectively. Simultaneous disruption of these two genes results in a growth defect of the yeast under anaerobic conditions, while disruption of the OSM1 gene causes slow growth. However, the metabolic role of these isoenzymes has been unclear until now. In the present study, we found that the anaerobic growth of the strain disrupted for both the FRDS and OSM1 genes was fully restored by adding the oxidized form of methylene blue or phenazine methosulfate, which non-enzymatically oxidize cellular NADH to NAD(+). When methylene blue was added at growth-limiting concentrations, growth was completely arrested after exhaustion of oxidized methylene blue. In the double-disrupted strain, the accumulation of succinate in the supernatant was markedly decreased during anaerobic growth in the presence of methylene blue. These results suggest that fumarate reductase isoenzymes are required for the reoxidation of intracellular NADH under anaerobic conditions, but not aerobic conditions.