Preferred Nitrogen Source Research Articles

Ammonium-induced internalisation of UapC, the general purine permease from Aspergillus nidulans

The Aspergillus nidulans UapC protein is a high-affinity, moderate-capacity, uric acid–xanthine transporter, which also displays a low transport capacity for hypoxanthine, adenine, and guanine. It has been previously shown that a functional UapC–GFP fusion protein localises at the plasma membrane [Fungal Genet. Biol. 30 (2000) 105]. Here, we demonstrate that ammonium, a preferred nitrogen source, dramatically changes the subcellular distribution of UapC. After addition of ammonium, UapC–GFP is removed from the plasma membrane and is concentrated into the vacuolar compartment. A chimeric gene construct in which an inducible promoter, insensitive to nitrogen repression, drives the expression of UapC–GFP, allowed us to demonstrate that the ammonium-dependent redistribution of UapC can be dissociated from the transcriptional repression of the gene. These results provide further support for the occurrence of endocytosis and the lysosomal–endosomal function of the vacuolar compartment in A. nidulans.

Nitrogen metabolism and virulence of Candida albicans require the GATA-type transcriptional activator encoded by GAT1.

Nitrogen acquisition and metabolism is central to microbial growth. A conserved family of zinc-finger containing transcriptional regulators known as GATA-factors ensures efficient utilization of available nitrogen sources by fungi. GATA factors activate expression of nitrogen catabolic pathways when preferred nitrogen sources are absent or limiting, a phenomenon known as nitrogen catabolite repression. GAT1 of Candida albicans encodes a GATA-factor homologous to the AREA protein of Aspergillus nidulans and related transcription factors involved in nitrogen regulation. Two observations implicated GAT1 in nitrogen regulation. The growth of mutants lacking GAT1 was reduced when isoleucine, tyrosine or tryptophan were the sole source of nitrogen. Secondly, when cultured on a secondary nitrogen source, gat1Delta mutants were unable to activate expression of GAP1, UGA4 or DAL5, which were shown to be nitrogen regulated in C. albicans. This regulatory defect did not prevent filamentation of gat1Delta mutants in nitrogen repressing or non-repressing conditions, demonstrating that nitrogen catabolite repression does not influence dimorphism. The mutants were, however, highly attenuated in a murine model of disseminated candidiasis. Attenuation was not associated with any diminution of growth in serum or ability to utilize serum amino acids. The results indicate an important role for nitrogen regulation in the virulence of C. albicans.

Nitrogen-source regulation of yeast gamma-glutamyl transpeptidase synthesis involves the regulatory network including the GATA zinc-finger factors Gln3, Nil1/Gat1 and Gzf3.

In Saccharomyces cerevisiae, the CIS2 gene encodes gamma-glutamyl transpeptidase (gamma-GT; EC 2.3.2.2), the main GSH-degrading enzyme. The promoter region of CIS2 contains one stress-response element (CCCCT) and eight GAT(T/A)A core sequences, probably involved in nitrogen-regulated transcription. We show in the present study that expression of CIS2 is indeed regulated according to the nature of the nitrogen source. Expression is highest in cells growing on a poor nitrogen source such as urea. Under these conditions, the GATA zinc-finger transcription factors Nil1 and Gln3 are both required for CIS2 expression, Nil1 appearing as the more important factor. We further show that Gzf3, another GATA zinc-finger protein, acts as a negative regulator in nitrogen-source control of CIS2 expression. During growth on a preferred nitrogen source like NH(4)(+), CIS2 expression is repressed through a mechanism involving (at least) the Gln3-binding protein Ure2/GdhCR. Induction of CIS2 expression during nitrogen starvation is dependent on Gln3 and Nil1. Furthermore, rapamycin causes similar CIS2 activation, indicating that the target of rapamycin signalling pathway controls CIS2 expression via Gln3 and Nil1 in nitrogen-starved cells. Finally, our results show that CIS2 expression is induced mainly by nitrogen starvation but apparently not by other types of stress.

Cytoplasmic Compartmentation of Gln3 during Nitrogen Catabolite Repression and the Mechanism of Its Nuclear Localization during Carbon Starvation in Saccharomyces cerevisiae

Regulated intracellular localization of Gln3, the transcriptional activator responsible for nitrogen catabolite repression (NCR)-sensitive transcription, permits Saccharomyces cerevisiae to utilize good nitrogen sources (e.g. glutamine and ammonia) in preference to poor ones (e.g. proline). During nitrogen starvation or growth in medium containing a poor nitrogen source, Gln3 is nuclear and NCR-sensitive transcription is high. However, when cells are grown in excess nitrogen, Gln3 is localized to the cytoplasm with a concomitant decrease in gene expression. Treating cells with the Tor protein inhibitor, rapamycin, mimics nitrogen starvation. Recently, carbon starvation has been reported to cause nuclear localization of Gln3 and increased NCR-sensitive transcription. Here we show that nuclear localization of Gln3 during carbon starvation derives from its indirect effects on nitrogen metabolism, i.e. Gln3 does not move into the nucleus of carbon-starved cells if glutamine rather than ammonia is provided as the nitrogen source. In addition, these studies have clearly shown Gln3 is not uniformly distributed in the cytoplasm, but rather localizes to punctate or tubular structures. Analysis of these images by deconvolution microscopy suggests that Gln3 is concentrated in or associated with a highly structured system in the cytosol, one that is possibly vesicular in nature. This finding may impact significantly on how we view (i) the mechanism by which Tor regulates the intracellular localization of Gln3 and (ii) how proteins move into and out of the nucleus.

Nitrogen-assimilating enzymes in land plants and algae: phylogenic and physiological perspectives.

An important biochemical feature of autotrophs, land plants and algae, is their incorporation of inorganic nitrogen, nitrate and ammonium, into the carbon skeleton. Nitrate and ammonium are converted into glutamine and glutamate to produce organic nitrogen compounds, for example proteins and nucleic acids. Ammonium is not only a preferred nitrogen source but also a key metabolite, situated at the junction between carbon metabolism and nitrogen assimilation, because nitrogen compounds can choose an alternative pathway according to the stages of their growth and environmental conditions. The enzymes involved in the reactions are nitrate reductase (EC 1.6.6.1-2), nitrite reductase (EC 1.7.7.1), glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 1.4.1.13-14, 1.4.7.1), glutamate dehydrogenase (EC 1.4.1.2-4), aspartate aminotransferase (EC 2.6.1.1), asparagine synthase (EC 6.3.5.4), and phosphoenolpyruvate carboxylase (EC 4.1.1.31). Many of these enzymes exist in multiple forms in different subcellular compartments within different organs and tissues, and play sometimes overlapping and sometimes distinctive roles. Here, we summarize the biochemical characteristics and the physiological roles of these enzymes. We also analyse the molecular evolution of glutamine synthetase, glutamate synthase and glutamate dehydrogenase, and discuss the evolutionary relationships of these three enzymes.

The TOR-controlled transcription activators GLN3, RTG1, and RTG3 are regulated in response to intracellular levels of glutamine.

The essential, rapamycin-sensitive TOR kinases regulate a diverse set of cell growth-related readouts in response to nutrients. Thus, the yeast TOR proteins function as nutrient sensors, in particular as sensors of nitrogen and possibly carbon. However, the nutrient metabolite(s) that acts upstream of TOR is unknown. We investigated the role of glutamine, a preferred nitrogen source and a key intermediate in yeast nitrogen metabolism, as a possible regulator of TOR. We show that the glutamine synthetase inhibitor L-methionine sulfoximine (MSX) specifically provokes glutamine depletion in yeast cells. MSX-induced glutamine starvation caused nuclear localization and activation of the TOR-inhibited transcription factors GLN3, RTG1, and RTG3, all of which mediate glutamine synthesis. The MSX-induced nuclear localization of GLN3 required the TOR-controlled, type 2A-related phosphatase SIT4. Other TOR-controlled transcription factors, GAT1/NIL1, MSN2, MSN4, and an unknown factor involved in the expression of ribosomal protein genes, were not affected by glutamine starvation. These findings suggest that the TOR pathway senses glutamine. Furthermore, as glutamine starvation affects only a subset of TOR-controlled transcription factors, TOR appears to discriminate between different nutrient conditions to elicit a response appropriate to a given condition.

Permeability of methylamine across the membrane of a cyanobacterial cell

Summary Ammonia (NH3) is the preferred nitrogen source for many eukaryotic algae and the cyanobacteria, Synechococcus R‐2 (PCC 7942) and Synechocystis (PCC 6803). Ammonia in solution is a weak base with a pKa of 9.25; hence, under environmental conditions it is present in solution as NH3 and NH4+. The uncharged form is a readily diffusible small molecule but NH4+ has an ionic radius similar to K+ and behaves like an alkali metal cation. Accumulation and retention of NH3 + NH4+ is therefore a complicated function of the permeabilities of two very different chemical species. NH3 is usually thought to be highly permeable across cell membranes although it is difficult to find actual measurements primarily because there is no convenient radioactive tracer for N. Many studies have therefore used 14C‐methylamine (14CH3‐NH2) as a convenient analogue tracer. Permeability of 14CH3‐NH2 was measured in Synechocystis over a range of pH values; permeability of uncharged amines was shown to be governed by pH. The permeability of CH3NH2 is c. 50 µm s−1 at pHo 10 but increases very rapidly to c. 300 µm s−1 at pHo 7. This finding has serious implications for attempts to develop stochastic models of amine uptake and retention by cells.

The N-Terminal Domain of the Yeast Permease Bap2p Plays a Role in Its Degradation

The amino acid permease Bap2p in Saccharomyces cerevisiae mediates a major part of the uptake of leucine, isoleucine, and valine from media containing a preferred nitrogen source. Although the transcriptional controls of BAP2 have been well studied, the posttranslational down-regulation mechanisms for Bap2p have not been established. Here we show that Bap2p is subject to a starvation-induced degradation upon rapamycin treatment or cultivation with proline as the sole nitrogen source. The starvation-induced degradation of Bap2p was dependent on the cellular functions of ubiquitination and endocytosis. Down-regulation of the permease required the most probable ubiquitination sites, the lysine residues situated in the N-terminal 49 residues, as well as the C-terminal domain. Furthermore, when the N-terminal domain of Bap2p was fused to the general amino acid permease Gap1p, the resultant chimeric permease became susceptible to the starvation-induced degradation, indicating that the Bap2p N-terminus contains a determinant responsive to the starvation signals.

Functional analysis of 14 genes that constitute the purine catabolic pathway in Bacillus subtilis and evidence for a novel regulon controlled by the PucR transcription activator.

The soil bacterium Bacillus subtilis has developed a highly controlled system for the utilization of a diverse array of low-molecular-weight compounds as a nitrogen source when the preferred nitrogen sources, e.g., glutamate plus ammonia, are exhausted. We have identified such a system for the utilization of purines as nitrogen source in B. subtilis. Based on growth studies of strains with knockout mutations in genes, complemented with enzyme analysis, we could ascribe functions to 14 genes encoding enzymes or proteins of the purine degradation pathway. A functional xanthine dehydrogenase requires expression of five genes (pucA, pucB, pucC, pucD, and pucE). Uricase activity is encoded by the pucL and pucM genes, and a uric acid transport system is encoded by pucJ and pucK. Allantoinase is encoded by the pucH gene, and allantoin permease is encoded by the pucI gene. Allantoate amidohydrolase is encoded by pucF. In a pucR mutant, the level of expression was low for all genes tested, indicating that PucR is a positive regulator of puc gene expression. All 14 genes except pucI are located in a gene cluster at 284 to 285 degrees on the chromosome and are contained in six transcription units, which are expressed when cells are grown with glutamate as the nitrogen source (limiting conditions), but not when grown on glutamate plus ammonia (excess conditions). Our data suggest that the 14 genes and the gde gene, encoding guanine deaminase, constitute a regulon controlled by the pucR gene product. Allantoic acid, allantoin, and uric acid were all found to function as effector molecules for PucR-dependent regulation of puc gene expression. When cells were grown in the presence of glutamate plus allantoin, a 3- to 10-fold increase in expression was seen for most of the genes. However, expression of the pucABCDE unit was decreased 16-fold, while expression of pucR was decreased 4-fold in the presence of allantoin. We have identified genes of the purine degradation pathway in B. subtilis and showed that their expression is subject to both general nitrogen catabolite control and pathway-specific control.

Conformational changes play a role in regulating the activity of the proline utilization pathway-specific regulator in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the ability to use proline as a nitrogen source requires the Put3p transcriptional regulator, which turns on the expression of the proline utilization genes, PUT1 and PUT2, in the presence of the inducer proline and in the absence of preferred nitrogen sources. Changes in target gene expression occur through an alteration in activity of the DNA-bound Put3p, a member of the Zn(II)2Cys6 binuclear cluster family of proteins. Here, we report that the 'on' conformation can be mimicked in the absence of proline by the insertion of an epitope tag in several different places in the protein, as well as by specific amino acid changes that suppress a put3 mutation leading to non-inducibility of the pathway. In addition, the presence of proline causes a conformational change in the Put3 protein detected by increased sensitivity to thrombin or V8 protease. These findings suggest that Put3p shifts from an inactive to an activate state via conformational changes.

Production of Indole Acetic Acid in Culture by a Rhizobium Species from the Root Nodules of a Monocotyledonous Tree, Roystonea regia

The study of the rhizobial root nodules of the monocotyledonous tree Roystonea regia revealed that the Rhizobium sp. isolated from the root nodules produced high amounts (45.6 μg/ml) of indole acetic acid (IAA) from L-tryptophan supplemented basal medium. The IAA production reached its optimum using 3 mg/ml of L-tryptophan. The preferred carbon and nitrogen sources were glucose and KNO 3 and the optimum concentrations 1% and 0.02%, respectively. FeSO 4 x 7 H 2 O was found to be the only metal ion that increased IAA production. An optimum IAA production was also achieved when the basal medium was supplemented with glucose (1%), FeSO 4 x 7 H 2 O (10 μg/ml), KNO 3 (0.02%) as well as EDTA (5 μg/ml) and L-tryptophan (3 mg/ml). The possible role of IAA production in the monocotyledonous tree-Rhizobium symbiosis is discussed. Hormone production is shown to be the beneficial aspect of this symbiosis as shown earlier in dicotyledonous plants.

Production of Indole Acetic Acid in Culture by a Rhizobium Species from the Root Nodules of a Monocotyledonous Tree, Roystonea regia

The study of the rhizobial root nodules of the monocotyledonous tree Roystonea regia revealed that the Rhizobium sp. isolated from the root nodules produced high amounts (45.6 μg/ml) of indole acetic acid (IAA) from L-tryptophan supplemented basal medium. The IAA production reached its optimum using 3 mg/ml of L-tryptophan. The preferred carbon and nitrogen sources were glucose and KNO3 and the optimum concentrations 1% and 0.02%, respectively. FeSO4 × 7 H2O was found to be the only metal ion that increased IAA production. An optimum IAA production was also achieved when the basal medium was supplemented with glucose (1%), FeSO4 × 7 H2O (10 μg/ml), KNO3 (0.02%) as well as EDTA (5 μg/ml) and L-tryptophan (3 mg/ml). The possible role of IAA production in the monocotyledonous tree-Rhizobium symbiosis is discussed. Hormone production is shown to be the beneficial aspect of this symbiosis as shown earlier in dicotyledonous plants.

Nitrogen control in cyanobacteria.

Nitrogen is a quantitatively important bioelement which is incorporated into the biosphere through assimilatory processes carried out by microorganisms and plants. Numerous nitrogencontaining compounds can be used by different organisms as sources of nitrogen. These include, for instance, inorganic ions like nitrate or ammonium and simple organic compounds like urea, amino acids, and some nitrogen-containing bases. Additionally, many bacteria are capable of fixing N 2. Nitrogen control is a phenomenon that occurs widely among microorganisms and consists of repression of the pathways of assimilation of some nitrogen sources when some other, more easily assimilated source of nitrogen is available to the cells. Ammonium is the preferred nitrogen source for most bacteria, but glutamine is also a very good source of nitrogen for many microorganisms. Two thoroughly investigated nitrogen control systems are the NtrB-NtrC two-component regulatory system found in enterics and some other proteobacteria (80) and the GATA family global nitrogen control transcription factors of yeast and some fungi (75). Novel nitrogen control systems have, however, been identified in bacteria other than the proteobacteria, like Bacillus subtilis (26), Corynebacterium glutamicum (52), and the cyanobacteria. The cyanobacterial system is the subject of this review. The cyanobacteria are prokaryotes that belong to the Bacteria domain and are characterized by the ability to perform oxygenic photosynthesis. Cyanobacteria have a wide ecological distribution, and they occupy a range of habitats, which includes vast oceanic areas, temperate soils, and freshwater lakes, and even extreme habitats like arid deserts, frigid lakes, or hot springs. Photoautotrophy, fixing CO 2 through the Calvin cycle, is the dominant mode of growth of these organisms (109). A salient feature of the intermediary metabolism of cyanobacteria is their lack of 2-oxoglutarate dehydrogenase (109). As a consequence, they use 2-oxoglutarate mainly as a substrate for the incorporation of nitrogen, a metabolic arrangement that may have regulatory consequences. Notwithstanding their rather homogeneous metabolism, cyanobacteria exhibit remarkable morphological diversity, being found as either unicellular or filamentous forms and exhibiting a number of cell differentiation processes, some of which take place in response to defined environmental cues, as is the case for the differentiation of N 2-fixing heterocysts (109). Nitrogen control in cyanobacteria is mediated by NtcA, a transcriptional regulator which belongs to the CAP (the catabolite gene activator or cyclic AMP [cAMP] receptor protein) family and is therefore different from the well-characterized Ntr system. Interestingly, however, the signal transduction P II protein, which plays a key role in Ntr regulation, is found in cyanobacteria but with characteristics which differentiate it from proteobacterial P II. In the following paragraphs, we shall first briefly summarize our current knowledge of the cyanobacterial nitrogen assimilation pathways and of what is known about their regulation at the protein level. This description will introduce most of the known cyanobacterial nitrogen assimilation genes. We shall then describe the ntcA gene and the NtcA protein themselves to finally discuss NtcA function through a survey of the NtcA-regulated genes which participate in simple nitrogen assimilation pathways or in heterocyst differentiation and function.

Transcriptional regulation of the Saccharomyces cerevisiae amino acid permease gene BAP2.

Uptake of branched-chain amino acids by Saccharomyces cerevisiae from media containing a preferred nitrogen source is mediated by the permeases encoded by BAP2, BAP3, and VAP1/TAT1. The transcriptional activity of the BAP2 promoter is affected by a number of genes, including SSY1, which encodes an amino acid permease homologue that is necessary for transcription of BAP2. Other genes that control BAP2 encode known (Leu3p, Tup1p) and putative (Stp1p, Stp2p) transcription factors. We present evidence that the zinc-finger proteins Stp1p and Stp2p bind directly to the BAP2 promoter. Binding of Stplp to the BAP2 promoter in vivo and in vitro indicates that the STP gene family indeed encodes transcription factors. The presence of a Leu3p binding site in the BAP2 promoter is required for full promoter activity on synthetic complete medium. The capacity of Leu3p to activate BAP2 transcription correlates with conditions that affect the level of alpha-isopropyl malate. The effect of a tup1 deletion on BAP2 transcription depends on SSY1. In an ssy1 strain, the phenotype of tup1 conforms to the well-established role of Tup1p as part of a repressor complex, but in the SSY1 strain deletion of TUP1 causes a decrease in transcription, indicating that Tup1p may also have an activating role at the BAP2 promoter. Our results thus suggest a complex interplay between several transcription factors in the expression of BAP2.

Production of γ-Linolenic Acid by Rhizopus nigricans SSSD-8

A gamma linolenic acid (GLA, cis-6,9,12-Octadecatrienoic acid) producing fungus was isolated and identified for the first time as Rhizopus nigricans, labeled as SSSD-8. The alteration of carbon and nitrogen sources on the production of GLA by Rhizopus nigricans SSSD-8 was examined in flask cultures for two separate media. The cultivation conditions were optimised for GLA production by appropriate selection of carbon and nitrogen sources and their concentrations, temperature, time of incubation, and pH. On an identical weight basis, soluble starch is the best carbon source and urea the most preferred nitrogen source for GLA yield irrespective of the media composition. A maximum GLA yield of about 1640 mg/L was obtained for Rhizopus nigricans SSSD-8 when incubated at 30°C and at pH 5.5 in the medium containing 15% (w/v) potato extract, 15% (w/v) soluble starch, 0.5% (w/v) yeast extract, and 0.2% (w/v) of urea.

Global regulation of gene expression.

Microorganisms such as Escherichia coli live in environments subject to rapid changes in the availability of the carbon and nitrogen compounds necessary to provide energy and building blocks for the synthesis of cell material. Their survival depends on their ability to regulate the expression of genes coding for the enzymes and transport proteins required for growth in the altered environment. The availability of microarrays of the entire genome of E. coli has made it possible to measure the expression of all genes by determining the levels of the corresponding species of RNA. As reported in this issue of PNAS, Sydney Kustu, Robert A. Bender, and their coworkers (1) have used this method to identify all of the genes whose expression is activated in response to the replacement of the preferred nitrogen source, ammonia, by a non-preferred source of nitrogen (nitrogen regulation) (2). Their study revealed that the products of many genes that had previously not been known to be subject to nitrogen regulation are responsible for the scavenging of amino acids and peptides, among them the dipeptide d-alanyl-d-alanine. Under conditions of stress, probably including nitrogen limitation, E. coli switches the mechanism for crosslinking the peptidoglycan layer of the cell wall to one that releases d-alanyl-d-alanine into the growth medium. The activation of the expression …

Mechanism of metabolic control. Target of rapamycin signaling links nitrogen quality to the activity of the Rtg1 and Rtg3 transcription factors.

De novo biosynthesis of amino acids uses intermediates provided by the TCA cycle that must be replenished by anaplerotic reactions to maintain the respiratory competency of the cell. Genome-wide expression analyses in Saccharomyces cerevisiae reveal that many of the genes involved in these reactions are repressed in the presence of the preferred nitrogen sources glutamine or glutamate. Expression of these genes in media containing urea or ammonia as a sole nitrogen source requires the heterodimeric bZip transcription factors Rtg1 and Rtg3 and correlates with a redistribution of the Rtg1p/Rtg3 complex from a predominantly cytoplasmic to a predominantly nuclear location. Nuclear import of the complex requires the cytoplasmic protein Rtg2, a previously identified upstream regulator of Rtg1 and Rtg3, whereas export requires the importin-beta-family member Msn5. Remarkably, nuclear accumulation of Rtg1/Rtg3, as well as expression of their target genes, is induced by addition of rapamycin, a specific inhibitor of the target of rapamycin (TOR) kinases. We demonstrate further that Rtg3 is a phosphoprotein and that its phosphorylation state changes after rapamycin treatment. Taken together, these results demonstrate that target of rapamycin signaling regulates specific anaplerotic reactions by coupling nitrogen quality to the activity and subcellular localization of distinct transcription factors.

Effect of total ammonia nitrogen concentration and pH on growth rates of duckweed ( Spirodela polyrrhiza)

The use of duckweed in domestic wastewater treatment is receiving growing attention over the last few years. Duckweed-based ponds in combination with anaerobic pre-treatment may be a feasible option for organic matter and nutrient removal. The main form of nitrogen in anaerobic effluent is ammonium. This is the preferred nitrogen source of duckweed but at certain levels it may become inhibitory to the plant. Renewal fed batch experiments at laboratory scale were performed to assess the effect of total ammonia (NH 3+NH + 4) nitrogen and pH on the growth rate of the duckweed Spirodela polyrrhiza. The experiments were performed at different total ammonia nitrogen concentrations, different pH ranges and in three different growth media. The inhibition of duckweed growth by ammonium was found to be due to a combined effect of ammonium ions (NH + 4) and ammonia (NH 3), the importance of each one depending on the pH.

Saccharomyces cerevisiae GATA Sequences Function as TATA Elements during Nitrogen Catabolite Repression and When Gln3p Is Excluded from the Nucleus by Overproduction of Ure2p

Saccharomyces cerevisiae selectively uses good nitrogen sources (glutamine) in preference to poor ones (proline) by repressing GATA factor-dependent transcription of the genes needed to transport and catabolize poor nitrogen sources, a physiological process designated nitrogen catabolite repression (NCR). We show that some NCR-sensitive genes (CAN1, DAL5, DUR1,2, and DUR3) produce two transcripts of slightly different sizes. Synthesis of the shorter transcript is NCR-sensitive and that of the longer transcript is not. The longer transcript also predominates in gln3Delta mutants irrespective of the nitrogen source provided. We demonstrate that the longer mRNA species arises through the use of an alternative transcription start site generated by Gln3p-binding sites (GATAAs) being able to act as surrogate TATA elements. The ability of GATAAs to serve as surrogate TATAs, i.e. when synthesis of the shorter, NCR-sensitive transcripts are inhibited, correlates with sequestration of enhanced green fluorescent protein (EGFP)-Gln3p in the cytoplasm in a way that is indistinguishable from that seen with EGFP-Ure2p. However, when the shorter, NCR-sensitive DAL5 transcript predominates, EGFP-Gln3p is nuclear. These data suggest that the mechanism underlying NCR involves the cytoplasmic association of Ure2p with Gln3p, an interaction that prevents Gln3p from reaching it is binding sites upstream of NCR-sensitive genes.

Modestobacter multiseptatus gen. nov., sp. nov., a budding actinomycete from soils of the Asgard Range (Transantarctic Mountains).

Oligotrophic PYGV medium, inoculated with soils from Linnaeus Terrace (1600 m, Antarctica), yielded four aerobic actinomycetes with short rods, multiple and irregular septa and often motile buds. Cells were 1.0-2.8 x 1.0-3.0 microm and colonies were beige to pink. The isolates were nearly identical in physiological and biochemical tests. Three strains grew from 0 degrees C to 25-28 degrees C, but one was psychrophilic with a maximum growth temperature of 20 degrees C. Carbon sources utilized were D-glucose, D-galactose, lactose, sucrose or mannitol; malate, succinate, fumarate, pyruvate or glutarate were decarboxylated aerobically. Peptone and yeast extract were the preferred nitrogen sources. Nitrate was reduced aerobically or anaerobically. Cell walls contained meso-diaminopimelic acid, glutamate, alanine, glycine, galactose, glucose and ribose. Major fatty acids of strains AA-802, -824, -825 and -826T were n18:1, i16:0 and ai17:0. Major respiratory quinones were MK-9(H4) and MK-8(H4). Polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol. Phosphatidylglycerol was found in most strains. The DNA G+C contents were 68-70 mol%. In 16S rDNA analyses, similarity values obtained for 500 nucleotides from the 5' terminus were > 99.5%. Almost complete sequences from AA-826T and -825 were 99.9% similar. Strain AA-826T belonged to a novel cluster of desert soil and rock isolates within the Geodermatophilaceae and was equidistantly related to members of Geodermatophilus and to a Blastococcus lineage. The four isolates represent a new genus, Modestobacter gen. nov., with Modestobacter multiseptatus sp. nov. as the type species. The type strain, Modestobacter multiseptatus AA-826T, was deposited in the DSMZ as DSM 44406T.