Regulation of a transport operon promoter in Salmonella typhimurium: identification of sites essential for nitrogen regulation

Summary The promoter of nitrogen-regulated transport, argTr, has been mutationally altered in order to determine the features that are essential for its response to nitrogen availability. Deletions of all sequences upstream of position-44 or downstream of position +2 had no effect no nitrogen regulation of argTr. These deletions define a small region of 44 bp where all necessary features for nitrogen regulation are located. This region includes for nitrogen regulation are located. This region includes sequences highly homologous to the nif consensus promoter. Alteration of this particular sequence caused drastic changes in the response to changes of nitrogen availability, thus indicating that they are directly involved in regulation. This implies that the NtrC protein must also act within this small region of the promoter. The data are discussed in terms of current-hypotheses concerning nitrogen regulation. In addition, we have shown 1. that carbon regulation at this promoter must occur at a site upstream from the nitrogen promoter; 2. that nifA can replace ntrC in the regulation of argTr.     

The General Amino Acid Permease FfGap1 of Fusarium fujikuroi Is Sorted to the Vacuole in a Nitrogen-Dependent,but Npr1 Kinase-Independent Manner

The rice pathogenic fungus Fusarium fujikuroi is well known for the production of a broad spectrum of secondary metabolites (SMs) such as gibberellic acids (GAs), mycotoxins and pigments. The biosynthesis of most of these SMs strictly depends on nitrogen availability and of the activity of permeases of nitrogen sources, e.g. the ammonium and amino acid permeases. One of the three ammonium permeases, MepB, was recently shown to act not only as a transporter but also as a nitrogen sensor affecting the production of nitrogen-repressed SMs. Here we describe the identification of a general amino acid permease, FfGap1, among the 99 putative amino acid permeases (AAPs) in the genome of F. fujikuroi. FfGap1 is able to fully restore growth of the yeast gap1∆ mutant on several amino acids including citrulline and tryptophane. In S. cerevisiae, Gap1 activity is regulated by shuttling between the plasma membrane (nitrogen limiting conditions) and the vacuole (nitrogen sufficiency), which we also show for FfGap1. In yeast, the Npr1 serine/threonine kinase stabilizes the Gap1 position at the plasma membrane. Here, we identified and characterized three NPR1-homologous genes, encoding the putative protein kinases FfNpr1-1, FfNpr1-2 and FfNpr1-3 with significant similarity to yeast Npr1. Complementation of the yeast npr1Δ mutant with each of the three F. fujikuroi NPR1 homologues, resulted in partial restoration of ammonium, arginine and proline uptake by FfNPR1-1 while none of the three kinases affect growth on different nitrogen sources and nitrogen-dependent sorting of FfGap1 in F. fujikuroi. However, exchange of the putative ubiquitin-target lysine 9 (K9A) and 15 (K15A) residues of FfGap1 resulted in extended localization to the plasma membrane and increased protein stability independently of nitrogen availability. These data suggest a similar regulation of FfGap1 by nitrogen-dependent ubiquitination, but differences regarding the role of Fusarium Npr1 homologues compared to yeast.     

Lysine transporters in human trypanosomatid pathogens

In previous studies we characterized arginine transporter genes from Trypanosoma cruzi and Leishmania donovani, the etiological agents of chagas disease and kala azar, respectively, both fatal diseases in humans. Unlike arginine transporters in higher eukaryotes that transport also lysine, these parasite transporters translocate only arginine. This phenomenon prompted us to identify and characterize parasite lysine transporters. Here we demonstrate that LdAAP7 and TcAAP7 encode lysine-specific permeases in L. donovani and T. cruzi, respectively. These two lysine permeases are both members of the large amino acid/auxin permease family and share certain biochemical properties, such as specificity and Km. However, we evidence that LdAAP7 and TcAAP7 differ in their regulation and localization, such differences are likely a reflection of the dissimilar L. donovani and T. cruzi life cycles. Failed attempts to delete both alleles of LdAAP7 support the premise that this is an essential gene that encodes the only lysine permeases expressed in L. donovani promastigotes and T. cruzi epimastigotes, respectively.     

Physiological effects of nitrogen deficiency and recovery on the macroalga Gracilariopsis lemaneiformis (Rhodophyta)

Algal metabolites are the most promising feedstocks for bio‐energy production. Gracilariopsis lemaneiformis seems to be a good candidate red alga for polysaccharide production, especially relating to the agar production industry. Nitrogen deficiency is an efficient environmental pressure used to increase the accumulation of metabolites in algae. However, there are no studies on the physiological effects of G. lemaneiformis in response to nitrogen deficiency and its subsequent recovery. Here we integrated physiological data with molecular studies to explore the response strategy of G. lemaneiformis under nitrogen deficiency and recovery. Physiological measurements indicated that amino acids and protein biosynthesis were decreased, while endogenous NH₄⁺ and soluble polysaccharides levels were increased under nitrogen stress. The expression of key genes involved in these pathways further suggested that G. lemaneiformis responded to nitrogen stress through up‐regulation or down‐regulation of genes related to nitrogen metabolism, and increased levels of endogenous NH₄⁺ to complement the deficiency of exogenous nitrogen. Consistent with the highest accumulation of soluble polysaccharides, the gene encoding UDP‐glucose pyrophosphorylase, a molecular marker used to evaluate agar content, was dramatically up‐regulated more than 4‐fold compared to the relative expression of actin after 4 d of nitrogen recovery. The present data provide important information on the mechanisms of nutrient balance in macroalgae.     

Temperature and oxygen regulated expression of a glutamine synthetase gene fromVibrio alginolyticus cloned inEscherichia coli

Glutamine synthetase (GS) synthesis inVibrio alginolyticus was regulated by temperature, oxygen and nitrogen levels. A GS gene,glnA fromV. alginolyticus was cloned on a 5.67 kb insert in the recombinant plasmid pRM210, which enabledEscherichia coli glnA, ntrB, ntrC deletion mutants to utilize (NH₄)₂SO₄ as a sole source of nitrogen. TheV. alginolyticus glnA gene was expressed from a regulatory region contained within the cloned fragment.V. alginolyticus glnA expression from pRM210 was subject to regulation by temperature, oxygen and nitrogen levels. GS specific activity in anE. coli wild-type strain was not affected by temperature or oxygen. pRM211 was a deletion derivative of pRM210 and GS production by pRM211 was not regulated by temperature, oxygen or nitrogen levels inE. coli.Abbreviation GS glutamine synthetase     

Nitrogen regulation of the xyl genes of Pseudomonas putida mt‐2 propagates into a significant effect of nitrate on m‐xylene mineralization in soil

The nitrogen species available in the growth medium are key factors determining expression of xyl genes for biodegradation of aromatic compounds by Pseudomonas putida. Nitrogen compounds are frequently amended to promote degradation at polluted sites, but it remains unknown how regulation observed in the test tube is propagated into actual catabolism of, e.g. m‐xylene in soil, the natural habitat of this bacterium. To address this issue, we have developed a test‐tube‐to‐soil model system that exposes the end‐effects of remediation practices influencing gene expression of P. putida mt‐2. We found that NO₃^? compared with NH₄⁺ had a stimulating effect on xyl gene expression in pure culture as well as in soil, and that this stimulation was translated into increased m‐xylene mineralization in soil. Furthermore, expression analysis of the nitrogen‐regulated genes amtB and gdhA allowed us to monitor nitrogen sensing status in both experimental systems. Hence, for nitrogen sources, regulatory patterns that emerge in soil reflect those observed in liquid cultures. The current study shows how distinct regulatory traits can lead to discrete environmental consequences; and it underpins that attempts to improve bioremediation by nitrogen amendment should integrate knowledge on their effects on growth and on catabolic gene regulation under natural conditions.     

Ammonia regulation of amino acid permeases in Saccharomyces cerevisiae.

The activities of the proline-specific permease (PUT4) and the general amino acid permease (GAP1) of Saccharomyces cerevisiae vary 70- to 140-fold in response to the nitrogen source of the growth medium. The PUT4 and GAP1 permease activities are regulated by control of synthesis and control of activity. These permeases are irreversibly inactivated by addition of ammonia or glutamine, lowering the activity to that found during steady-state growth on these nitrogen sources. Mutants altered in the regulation of the PUT4 permease (Per-) have been isolated. The mutations in these strains are pleiotropic and affect many other permeases, but have no direct effect on various cytoplasmic enzymes involved in nitrogen assimilation. In strains having one class of mutations (per1), ammonia inactivation of the PUT4 and GAP1 permeases did not occur, whereas glutamate and glutamine inactivation did. Thus, there appear to be two independent inactivation systems, one responding to ammonia and one responding to glutamate (or a metabolite of glutamate). The mutations were found to be nuclear and recessive. The inactivation systems are constitutive and do not require transport of the effector molecules per se, apparently operating on the inside of the cytoplasmic membrane. The ammonia inactivation was found not to require a functional glutamate dehydrogenase (NADP). These mutants were used to show that ammonia exerts control of arginase synthesis largely by inducer exclusion. This may be the primary mode of nitrogen regulation for most nitrogen-regulated enzymes of S. cerevisiae.     

Effect of low-temperature fermentation on yeast nitrogen metabolism

The aim of this study was to analyse the influence of low-temperature wine fermentation on nitrogen consumption and nitrogen regulation. Synthetic grape must was fermented at 25 and 13°C. Low-temperature decreased both the fermentation and the growth rates. Yeast cells growing at low-temperature consumed less nitrogen than at 25°C. Specifically, cells at 13°C consumed less ammonium and glutamine, and more tryptophan. Low-temperature seemed to relax the nitrogen catabolite repression (NCR) as deduced from the gene expression of ammonium and amino acid permeases (MEP2 and GAP1) and the uptake of some amino acids subjected to NCR (i.e. arginine and glutamine). Low-temperature influences the quantity and the quality of yeast nitrogen requirements. Nitrogen-deficient grape musts and low temperature are two of the main prevalent causes of sluggish fermentations and, therefore, the effects of both growth conditions on yeast metabolism are of considerable interest for wine making.     

Altered expression of the PTR/NRT1 homologue OsPTR9 affects nitrogen utilization efficiency,growth and grain yield in rice

The plant PTR/NRT1 (peptide transporter/nitrate transporter 1) gene family comprises di/tripeptide and low‐affinity nitrate transporters; some members also recognize other substrates such as carboxylates, phytohormones (auxin and abscisic acid), or defence compounds (glucosinolates). Little is known about the members of this gene family in rice (Oryza sativa L.). Here, we report the influence of altered OsPTR9 expression on nitrogen utilization efficiency, growth, and grain yield. OsPTR9 expression is regulated by exogenous nitrogen and by the day‐night cycle. Elevated expression of OsPTR9 in transgenic rice plants resulted in enhanced ammonium uptake, promotion of lateral root formation and increased grain yield. On the other hand, down‐regulation of OsPTR9 in a T‐DNA insertion line (osptr9) and in OsPTR9‐RNAi rice plants had the opposite effect. These results suggest that OsPTR9 might hold potential for improving nitrogen utilization efficiency and grain yield in rice breeding.     

Reciprocal regulation of distinct asparagine synthetase genes by light and metabolites in Arabidopsis thaliana

In plants, the amino acid asparagine serves as an important nitrogen transport compound whose levels are dramatically regulated by light in many plant species, including Arabidopsis thaliana . To elucidate the mechanisms regulating the flux of assimilated nitrogen into asparagine, we examined the regulation of the gene family for asparagine synthetase in Arabidopsis. In addition to the previously identified ASN1 gene, we identified a novel class of asparagine synthetase genes in Arabidopsis ( ASN2 and ASN3 ) by functional complementation of a yeast asparagine auxotroph. The proteins encoded by the ASN2/3 cDNAs contain a Pur-F type glutamine-binding triad suggesting that they, like ASN1 , encode glutamine-dependent asparagine synthetase isoenzymes. However, the ASN2/3 isoenyzmes form a novel dendritic group with monocot AS genes which is distinct from all other dicot AS genes including Arabidopsis ASN1 . In addition to these distinctions in sequence, the ASN1 and ASN2 genes are reciprocally regulated by light and metabolites. Time-course experiments reveal that light induces levels of ASN2 mRNA while it represses levels of ASN1 mRNA in a kinetically reciprocal fashion. Moreover, the levels of ASN2 and ASN1 mRNA are also reciprocally regulated by carbon and nitrogen metabolites. The distinct regulation of ASN1 and ASN2 genes combined with their distinct encoded isoenzymes suggest that they may play different roles in nitrogen metabolism, as discussed in this paper.     

The Rhizobium meliloti early nodulation genes (nodABC) are nitrogen-regulated: Isolation of a mutant strain with efficient nodulation capacity on alfalfa in the presence of ammonium

Summary The presence of combined nitrogen in the soil suppresses the formation of nitrogen-fixing root nodules by Rhizobium. We demonstrate that bacterial genes determining early nodulation functions (nodABC) as well as the regulatory gene nodD3 are under nitrogen (NH ₄ ⁺ ) control. Our results suggest that the gene product of nodD3 has a role in mediating the ammonia regulation of early nod genes. The general nitrogen regulatory (ntr) system as well as a chromosomal locus mutated in Rhizobium meliloti were also found to be involved in the regulation of nod gene expression. A R. meliloti mutant with altered sensitivity to ammonia regulation was isolated, capable of more efficient nodulation of alfalfa than the wild-type strain in the presence of 2 mM ammonium sulfate.     

Qualitative network models and genome-wide expression data define carbon/nitrogen-responsive molecular machines in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Background  

Carbon (C) and nitrogen (N) metabolites can regulate gene expression in Arabidopsis thaliana. Here, we use multinetwork analysis of microarray data to identify molecular networks regulated by C and N in the Arabidopsis root system.     

Differential regulation of ssb genes in the nitrogen‐fixing cyanobacterium,Anabaena sp. strain PCC71201

Anabaena sp. PCC7120 possesses three genes coding for single‐stranded DNA‐binding (SSB) protein, of which ssb1 was a single gene, and ssb2 and ssb3 are the first genes of their corresponding operons. Regulation of the truncated ssb genes, ssb1 (alr0088) and ssb2 (alr7559), was unaffected by N‐status of growth. They were negatively regulated by the SOS‐response regulatory protein LexA, as indicated by the (i) binding of Anabaena LexA to the LexA box of regulatory regions of ssb1 and ssb2, and (ii) decreased expression of the downstream gfp reporter gene in Escherichia coli upon co‐expression of LexA. However, the full‐length ssb gene, ssb3 (all4779), was regulated by the availability of Fe²⁺ and combined nitrogen, as indicated by (i) increase in the levels of SSB3 protein on Fe²⁺‐depletion and decrease under Fe²⁺‐excess conditions, and (ii) 1.5‐ to 1.6‐fold decrease in activity under nitrogen‐fixing conditions compared to nitrogen‐supplemented conditions. The requirement of Fe²⁺ as a co‐factor for repression by FurA and the increase in levels of FurA under nitrogen‐deficient conditions in Anabaena (Lopez‐Gomollon et al. 2007) indicated a possible regulation of ssb3 by FurA. This was substantiated by (i) the binding of FurA to the regulatory region of ssb3, (ii) repression of the expression of the downstream gfp reporter gene in E. coli upon co‐expression of FurA, and (iii) negative regulation of ssb3 promoter activity by the upstream AT‐rich region in Anabaena. This is the first report on possible role of FurA, an important protein for iron homeostasis, in DNA repair of cyanobacteria.     

Differential regulation of two myo-inositol transporter genes of Saccharomyces cerevisiae

We previously isolated two distinct Saccharomyces cerevisiae myo-inositol transporter genes, ITR1 and ITR2 (Nikawa et al., 1991). Here, we studied the regulation of their expression by measuring steady-state mRNA levels and β-galactosidase activities of lacZ fusion genes under different conditions. The results show that the expression of the two ITR genes is differently regulated: ITR1 was repressed by inositol and choline whereas ITR2 was constitutive. Deletion analysis of the ITR1 upstream region and comparison with the upstream regions of other genes involved in phospholipid synthesis indicate that the octamer sequence 5′-TTCACATG-3′ is important for the expression and inositol/choline regulation of the ITR1 gene.