The Klebsiella pneumoniae PII protein (glnB gene product) is not absolutely required for nitrogen regulation and is not involved in NifL-mediated nif gene regulation

Summary The role of theKlebsiella pneumoniae P_II protein (encoded byglnB) in nitrogen regulation has been studied using two classes ofglnB mutants. In Class I mutants P_II appears not to be uridylylated in nitrogen-limiting conditions and in Class II mutants P_II is not synthesised. The effects of these mutations on expression from nitrogen-regulated promoters indicate that P_II is not absolutely required for nitrogen control. Furthermore the uridylylated form of P_II(P_II-UMP) plays a significant role in the response to changes in nitrogen status by counteracting the effect of P_II on NtrB-mediated dephosphorylation of NtrC. P_II is not involved in thenif-specific response to changes in nitrogen status mediated by NifL.     

Nucleotide sequence of the Vibrio alginolyticus glnA region

The nucleotide sequence of a 4 kb fragment containing the Vibrio alginolyticus glnA, ntrB and ntrC genes was determined. The upstream region of the glnA gene contained tandem promoters. The upstream promoter resembled the consensus sequence for Escherichia coli ⁷⁰ promoters whereas the presumptive downstream promoter showed homology with nitrogen regulated promoters. Four putative NR_I binding sites were located between the tandem promoters. The ntrB gene was preceded by a single presumptive NR_I binding site. The ntrC gene was located 45 base pairs downstream from the ntrB gene. The V. alginolyticus ntrB and ntrC genes were able to complement ntrB, ntrC deletions in E. coli.Abbreviations bp base pair(s) - CAP catabolite-activating protein - GS glutamine synthetase - kb kilobase(s) - ORF open reading frame - SD Shine-Dalgarno     

Regulation of nif gene expression in Enterobacter agglomerans: nucleotide sequence of the nifLA operon and influence of temperature and ammonium on its transcription

The nucleotide sequence of a plasmid-borne 3.9 kb XhoI-SmaI fragment comprising the 3-region of the nifM gene, the nifL and nifA genes and the 5-region of nifB gene of Enterobacter agglomerans was determined. The genes were identified by their homology to the corresponding nif genes of Klebsiella pneumoniae. A typical ⁵⁴-dependent promoter and a consensus NtrC-binding motif were identified upstream of nifL. The predicted amino acid sequence of NifL showed close similarities to NifL of K. pneumoniae and Azotobacter vinelandii. However, no histidine residue was found to correspond to histidine-304 of A. vinelandii NifL, which had been proposed to be required for the repressor activity of NifL. The NifA sequence with a putative DNA binding motif (Q(X₃) A (X₃) G (X₅)I) and an ATP binding site in the C-terminal and central domains, respectively, resembles that of other known NifA proteins. The function of the nifL and nifA genes was demonstrated in vivo using a binary plasmid system by their ability to activate a nifH promoter-lacZ fusion at different temperatures and concentrations of NH ₄ ⁺ . Maximal promoter activity occurred at 25°C, and it appears that the sensitivity of NifA to elevated temperatures is independent of NifL. The expression of nifL inhibited promoter activity in the presence of NifA when the initial NH ₄ ⁺ concentration in the medium exceeded 4 mM.Communicated by H. Böhme     

Nitrogen metabolism inRhodopseudomonas globiformis

Rhodopseudomonas globiformis strain 7950 grew with a variety of amino acids, urea, or N₂ as sole nitrogen sources. Cultures grown on N₂ reduced acetylene to ethylene; this activity was absent from cells grown on nonlimiting NH ₄ ⁺ . Glutamate dehydrogenase could not be detected in extracts of cells of strain 7950, although low levels of an alanine dehydrogenase were present. Growth ofR. globiformis on NH ₄ ⁺ was severely inhibited by the glutamate analogue and glutamine synthetase inhibitor, methionine sulfoximine. High levels of glutamine synthetase (as measured in the -glutamyl transferase assay) were observed in cell extracts of strain 7950 regardless of the nitrogen source, although N₂ and amino acid grown cells contained somewhat higher glutamine synthetase contents than cells grown on excess NH ₄ ⁺ . Levels of glutamate synthase inR. globiformis were consistent with that reported from other phototrophic bacteria. Both glutamate synthase and alanine dehydrogenase were linked to NADH as coenzyme. We conclude thatR. globiformis is capable of fixing N₂, and assimilates NH ₄ ⁺ primarily via the glutamine synthetase/glutamate synthase pathway.Abbreviations GS glutamine synthetase - GOGAT Glutamineoxoglutarate aminotransferase - GDH Glutamate dehydrogenase - ADH Alanine dehydrogenase - MSO Methionine sulfoximine     

Symbiotically defective histidine auxotrophs of Bradyrhizobium japonicum

Four histidine auxotrophs of Bradyrhizobium japonicum strain USDA 122 were isolated by random transposon Tn5 mutagenesis. These mutants arose from different, single transposition events as shown by the comparison of EcoRI and XhoI-generated Tn5 flanking sequences of genomic DNA. The mutants grew on minimal medium supplemented with l-histidine or l-histidinol but failed to grow with l-histidinol phosphate. While two of the muants were symbiotically defective and did not form nodules on Glycine max cvs. Lee and Peking and on Glycine soja, the other two mutants were symbiotically competent. Reversion to prototrophy occurred at a frequency of about 10^-7 on growth medium without added antibiotics, but prototrophs could not be isolated from growth medium containing 200 g/ml kanamycin and streptomycin. The prototrophic revertants formed nodules on all the soybean cultivars examined. When histidine was supplied to the plant growth medium, both nodulation deficient mutants formed effective symbioses. On histidine unamended plants, nodules were observed infrequently. Three classes of bacterial colonies were isolated from such infrequent nodules: class 1 were kanamycin resistant-auxotrophs; class 2 were kanamycin sensitive-prototrophs; and class 3 were kanamycin-sensitive auxotrophs. Our results suggest that two Tn5 insertion mutations in B. japonicum leading to histidine auxotrophy, affect nodulation in some way. These mutations are in regions that show no homology to the Rhizobium meliloti common nodulation genes.     

Nucleotide sequence of the pyrimidine specific carbamoyl phosphate synthetase, a part of the yeast multifunctional protein encoded by the URA2 gene

Summary Yeast URA2 encodes a multifunctional carbamoyl phosphate synthetase-aspartate transcarbamylase of 220,000 molecular weight. We determined the nucleotide sequence of the 5 proximal part of the gene which is responsible for the glutamine amide transfer function of the carbamoyl phosphate synthetase activity. Alignment of the enzyme sequence derived from URA2 with sequences from Escherichia coli carA carB and yeast arginine-specific CPA1 CPA2 indicates that monofunctional and bifunctional carbamoyl phosphate synthetases are probably homologous. The URA2-derived enzyme organization is NH₂-carbamoyl phosphate synthetase-aspartate transcarbamylase-CO₂H.     

Nitrogen limitation induces expression of the avirulence gene avr9 in the tomato pathogen Cladosporium fulvum

The avirulence gene avr9 of the fungal tomato pathogen Cladosporium fulvum encodes a race-specific peptide elicitor that induces the hypersensitive response in tomato plants carrying the complementary resistance gene Cf9. The avr9 gene is not expressed under optimal growth conditions in vitro, but is highly expressed when the fungus grows inside the tomato leaf. In this paper we present evidence for the induction of avr9 gene expression in C. fulvum grown in vitro under conditions of nitrogen limitation. Only growth medium with very low amounts of nitrogen (nitrate, ammonium, glutamate or glutamine) induced the expression of avr9. Limitation of other macronutrients or the addition of plant factors did not induce the expression of avr9. The induced expression of avr9 is possibly mediated by a positive-acting nitrogen regulatory protein, homologous to the Neurospora crassa NIT2 protein, which induces the expression of many genes under conditions of nitrogen limitation. The avr9 promoter contains several putative NIT2 binding sites. The expression of avr9 during the infection process was explored cytologically using transformants of C. fulvum carrying an avr9 promoter--glucuronidase reporter gene fusion. The possibility that expression of avr9 in C. fulvum growing in planta is caused by nitrogen limitation in the apoplast of the tomato leaf is discussed.     

Ammonium sensing in nitrogen fixing bacteria: Functions of theglnB andglnD gene products

A plentiful supply of fixed nitrogen as ammonium (or other compounds such as nitrate or amino acids) inhibits nitrogen fixation in free-living bacteria by preventing nitrogenase synthesis and/or activity. Ammonium and nitrate have variable effects on the ability ofRhizobiaceae (Rhizobium, Bradyrhizobium andAzorhizobium) species to nodulate legume hosts and on nitrogen fixation capacity in bacteroid cells contained in nodules or in plant-free bacterial cultures. In addition to effects on nitrogen fixation, excess ammonium can inhibit activity or expression of other pathways for utilization of nitrogenous compounds such as nitrate (through nitrate and nitrite reductase), or glutamine synthetase (GS) for assimilation of ammonium. This paper describes the roles of two key genesglnB andglnD, whose gene products sense levels of fixed nitrogen and initiate a cascade of reactions in response to nitrogen status. While work onEscherichia coli and other enteric bacteria provides the model system,glnB and, to a lesser extent,glnD have been studied in several nitrogen fixing bacteria. Such reports will be reviewed here. Recent results on the identity and function of theglnB andglnD gene products inAzotobacter vinelandii (a free-living soil diazotroph) and inRhizobium leguminosarum biovarviciae, hereinafter designatedR.l. viciae will be presented. New data suggests thatAzotobacter vinelandii probably contains aglnB-like gene and this organism may have twoglnD-like genes (one of which was recently identified and namednfrX). In addition, evidence for uridylylation of theglnB gene product (the PII protein) ofR. l. viciae in response to fixed nitrogen deficiency is presented. Also, aglnB mutant ofR. l. viciae has been isolated; its characteristics with respect to expression of nitrogen regulated genes is described.     

Cloning and nucleotide sequence of the ptsG gene of Bacillus subtilis

Summary The ptsG gene of Bacillus subtilis encodes Enzyme II^G1c of the phosphoenolpyruvate: glucose phosphotransferase system. The 3 end of the gene was previously cloned and the encoded polypeptide found to resemble the Enzymes III^Glc of Escherichia coli and Salmonella typhimurium. We report here cloning of the complete ptsG gene of B. subtilis and determination of the nucleotide sequence of the 5 end. These results, combined with the sequence of the 3 end of the gene, revealed that ptsG encodes a protein consisting of 699 amino acids and which is similar to other Enzymes II. The N-terminal domain contains two small additional fragments, which share no similarities with the closely related Enzymes II^Glc and II^Nag of E. coli but which are present in the II^G1c-like protein encoded by the E. coli malX gene.     

Copper-containing nitrite reductase from Pseudomonas aureofaciens is functional in a mutationally cytochrome cd 1-free background (NirS−) of Pseudomonas stutzeri

The structural gene, nirK, for the respiratory Cu-containing nitrite reductase from denitrifying Pseudomonas aureofaciens was isolated and sequenced. It encodes a polypeptide of 363 amino acids including a signal peptide of 24 amino acids for protein export. The sequence showed 63.8% positional identity with the amino acid sequence of Achromobacter cycloclastes nitrite reductase. Ligands for the blue, type I Cu-binding site and for a putative type-II site were identified. The nirK gene was transferred to the mutant MK202 of P. stutzeri which lacks cytochrome cd ₁ nitrite reductase due to a transposon Tn5 insertion in its structural gene, nirS. The heterologous enzyme was active in vitro and in vivo in this background and restored the mutationally interrupted denitrification pathway. Transfer of nirK to Escherichia coli resulted in an active nitrite reductase in vitro. Expression of the nirS gene from P. stutzeri in P. aureofaciens and E. coli led to nonfunctional gene products. Nitrite reductase activity of cell extract from either bacterium could be reconstituted by addition of heme d ₁, indicating that both heterologous hosts synthesized a cytochrome cd ₁ without the d ₁-group.Abbreviations Cu-NIR Cu-containing nitrite reductase - DDC diethyldithiocarbamate - EPR electron paramagnetic resonance - IPTG isopropyl--D-galactoside - SDS sodium dodecyl sulfate - LB medium Luria-Bertani medium     

A newly discovered tRNA(1Asp) gene (aspV) of Escherichia coli K12

Summary We report a new tRNA ₁ ^Asp gene near the dnaQ gene, which is located at 5 min on the Escherichia coli linkage map. We named it aspV. The sequence corresponding to the mature tRNA is identical with that of the two previously identified tRNA ₁ ^Asp genes (aspT and aspU), but there is no homology in the sequences of their 3-and 5-flanking regions.Abbreviations kb kilo base pair(s) - rrn ribosomal RNA     

Cloning and expression of the genes for xylose isomerase and xylulokinase from Klebsiella pneumoniae 1033 in Escherichia coli K12

Summary The genes xy1A and xy1B were cloned together with their promoter region from the chromosome of Klehsiella pneumoniae var. aerogenes 1033 and the DNA sequence (3225 bp) was determined. The gene xy1A encodes the enzyme xylose isomerase (XI or XylA) consisting of 440 amino acids (calculated M_r of 49 793). The gene xy1B encodes the enzyme xylulokinase (XK or Xy1B) with a calculated M, of 51 783 (483 amino acids). The two genes successfully complemented xy1 mutants of Escherichia coli K12, but no gene dosage effect was detected. E. coli wild-type cells which harbored plasmids with the intact xylA _Kp 5 upstream region in high copy number (but lacking an active xy1B gene on the plasmids) were phenotypically xylose-negative and xylose isomerase and xylulokinase activities were drastically diminished. Deletion of 5 upstream regions of xy1A on these plasmids and their substitution by a lac promoter resulted in a xylose-positive phenotype. This also resulted in overproduction of plasmid-encoded xylose isomerase and xylulokinase activities in recombinant E. coli cells.     

Molecular cloning of a cysteine synthase cDNA from Citrullus vulgaris (watermelon) by genetic complementation in an Escherichia coli Cys? auxotroph

We have isolated cDNA clones encoding cysteine synthase (CSase, EC 4.2.99.8), which catalyzes the terminal step in cysteine biosynthesis, by direct genetic complementation of a Cys^– mutation in Escherichia coli with an expression library of Citrullus vulgaris (watermelon) cDNA. The library was constructed from 8-day-old etiolated seedlings of C. vulgaris in the ZAPII vector, converted to a plasmid library by in vivo excision, and then used for transformation of cysteine auxotroph E. coli NK3, which lacks the cysK and cysM loci. The complementing cDNA containing a 560 by 5-untranslated region encodes a polypeptide of 325 amino acids of M_r 34342. The translational product reacted with an antibody raised against CSase A of Spinacia oleracea. CSase and -pyrazolealanine synthase activities were demonstrated in vitro in extracts from E. coli cells expressing the cDNA. Genomic DNA blot analysis indicated the presence of a single copy of the gene, designated cysA, in the C. vulgaris genome. RNA blot hybridization indicated constitutive expression of cysA in cotyledons, hypocotyls and radicles of green and etiolated seedlings. These data suggested that this cDNA clone encodes CSase A the homolog of which in spinach is localized in the cytoplasm. The molecular phylogenetic tree of the amino acid sequences of CSaes from plants and bacteria suggested that there are three families in the CSase superfamily; the plant CSase A family, the plant CSase B family and the bacterial CSase family. The proteins in the plant CSase A family are the most conserved relative to the ancestral CSase protein.     

Detection of developmentally regulated genes of the myxobacterium Stigmatella aurantiaca with the transposon Tn5lacZ

Stigmatella aurantiaca is a prokaryotic organism that undergoes a multicellular cycle of development resulting in the formation of a fruiting body. Insertional mutations were introduced at random sites into the Stigmatella aurantiaca genome with the promotor probe Tn5lacZ derived from Tn5lac by deleting non-essential sequences. 638 transconjugants were obtained with a frequency of 1×10^-7. In 260 of the transconjugants isolated the -glactosidase gene of Tn5lacZ is fused to vegetative promotors of Stigmatella aurantiaca. In 65 of the strains -galactosidase is induced by starvation; in 14 of the transconjugants -galactosidase activity is observed after chemical induction of sporulation by 3-methyl-indole. Thirtytwo of the mutants are affected in fruiting body formation and morphology.     

Molecular organization of the Escherichia coli gab cluster: nucleotide sequence of the structural genes gabD and gabP and expression of the GABA permease gene

We have determined the nucleotide sequences of two structural genes of the Escherichia coli gab cluster, which encodes the enzymes of the 4-aminobutyrate degradation pathway: gabD, coding for succinic semialdehyde dehydrogenase (SSDH, EC 1.2.1.16) and gabP, coding for the 4-aminobutyrate (GABA) transport carrier (GABA permease). We have previously reported the nucleotide sequence of the third structural gene of the cluster, gabT, coding for glutamate: succinic semialdehyde transaminase (EC 2.6.1.19). All three gab genes are transribed unidirectionally and their orientation within the cluster is 5-gabD-gabT-gabP-3. gabT and gabP are separated by an intergenic region of 234-bp, which contains three repetetive extragenic palindromic (REP) sequences. The gabD gene consists of 1,449 nucleotides specifying a protein of 482 amino acids with a molecular mass of 51.7 kDa. The protein shows significant homologies to the NAD⁺-dependent aldehyde dehydrogenase (EC 1.2.1.3) from Aspergillus nidulans and several mammals, and to the tumor associated NADP⁺-dependent aldehyde dehydrogenase (EC 1.2.1.4) from rat. The permease gene gabP comprises 1,401 nucleotides coding a highly hydrophobic protein of 466 amino acids with a molecular mass of 51.1 kDa. The GABA permease shows features typical for an integral membrane protein and is highly homologous to the aromatic acid carrier from E. coli, the proline, arginine and histidine permeases from Saccharomyces cerevisiae and the proline transport protein from A. nidulans. Uptake of GABA was increased ca. 5-fold in transformants of E. coli containing gabP plasmids. Strong overexpression of the gabP gene under control of the isopropyl-2-d-thiogalactoside (IPTG) inducible tac promoter, however, resulted in a severe growth inhibition of the transformed strains. The GABA carrier was characterized using moderately overexpressing transformants. The K _m of GABA uptake was found to be 11.8 M and the V_max 0.33 nmol/min · mg cells. Uptake of GABA was stimulated by ammonium sulfate and abolished by 2,4-dinitrophenol. Aspartate competed with GABA for uptake.     

Genetic interactions indicate a role for Mdg1p and the SH3 domain protein Bem1p in linking the G-protein mediated yeast pheromone signalling pathway to regulators of cell polarity

The pheromone signal in the yeastSaccharomyces cerevisiae is transmitted by the and subunits of the mating response G-protein. TheSTE20 gene, encoding a protein kinase required for pheromone signal transduction, has recently been identified in a genetic screen for high-gene-dosage suppressors of a partly defective G mutation. The same genetic screen identifiedBEM1, which encodes an SH3 domain protein required for polarized morphogenesis in response to pheromone, and a novel gene, designatedMDG1 (multicopy suppressor ofdefectiveG-protein). TheMDG1 gene was independently isolated in a search for multicopy suppressors of abem1 mutation. TheMDG1 gene encodes a predicted hydrophilic protein of 364 amino acids with a molecular weight of 41 kDa that has no homology with known proteins. A fusion of Mdg1p with the green fluorescent protein fromAequorea victoria localizes to the plasma membrane, suggesting that Mdg1p is an extrinsically bound membrane protein. Deletion ofMDG1 causes sterility in cells in which the wild-type G has been replaced by partly defective G derivatives but does not cause any other obvious phenotypes. The mating defect of cells deleted forSTE20 is partially suppressed by multiple copies ofBEM1 andCDC42, which encodes a small GTP-binding protein that binds to Ste20p and is necessary for the development of cell polarity. Elevated levels ofSTE20 andBEM1 are capable of suppressing a temperature-sensitive mutation inCDC42. This complex network of genetic interactions points to a role for Bem1p and Mdg1p in G-protein mediated signal transduction and indicates a functional linkage between components of the pheromone signalling pathway and regulators of cell polarity during yeast mating.     

Patterns of [13N]ammonium uptake and assimilation by Frankia HFPArl3

Nitrogen-starved cells of Frankia strain HFPArl3 incorporated [¹³N]-labeled ammonium into glutamine serine (glutamate, alanine, aspartate), after five-minute radioisotope exposures. High initial endogenous pools of glutamate were reduced, while total glutamine increased, during short term NH _inf4 ^sup+ incubation. Preincubation of cells in methionine sulfoximine (MSX) resulted in [¹³N]glutamine reduced by more than 80%, while [¹³N]glutamate and [¹³N]alanine levels increased. The results suggest that glutamine synthetase is the primary enzyme of ammonium assimilation, and that glutamate dehydrogenase and alanine dehydrogenase may also function in ammonium assimilation at low levels. Efflux of [¹³N]serine and lesser amounts of [¹³N]glutamine was detected from the Frankia cells. The identity of both Ser and Gln in the extracellular compartment was confirmed with gas chromatography/mass spectrometry. Serine efflux may be of significance in nitrogen transfer in Frankia.Abbreviations Pthr phosphothreonine - Aad -amino-adipate - MSX methionine sulfoximine