Nitrogen metabolite repression in Aspergillus nidulans

Summary In Aspergillus nidulans, mutations, designated areA^r, can result in the inability to utilise a wide variety of nitrogen sources including amino acids, purines, amides, nitrate, and nitrite, whilst not affecting growth on ammonium. Other allelic areA mutations, designated areA^d, lead to derepression of one or more activities which are ammonium repressible in wild type (areA⁺) strains, whilst not affecting their inducibility. Various areA mutations exhibit a wide variety of phenotypes: areA^r alleles can be temperature sensitive on some nitrogen sources while not on others, and different alleles can be temperature sensitive for utilisation of different nitrogen sources. areA^d alleles can be derepressed for one ammonium-repressible activity, be normally repressible for another, and lead to abnormally low levels for a third. Once again each areA^d allele has its own highly specific phenotype. The inability of areA^r strains to utilise most nitrogen sources is paralleled by low activities of certain ammonium-repressible enzymes. areA^r mutations appear to be epistatic to some but not all regulatory mutations leading to constitutive synthesis of inducible enzymes and also epistatic to gdhA mutations which lead both to loss of NADP-linked glutamate dehydrogenase and to derepression of ammonium-repressible activities. areA^r mutations do not interfere with repair of a large number of auxotrophies in double mutants. Furthermore, although areA^r mutations prevent utilisation of L-arginine, L-ornithine, and L--amino-n-butyrate as nitrogen sources, they do not prevent the metabolism of these compounds necessary for repairing auxotrophies for proline and isoleucine in the appropriate double mutants. Utilisation of acetamide and most amino acids as carbon or carbon and nitrogen sources is unaffected by areA^r mutations, and areA^r strains are able to utilise acetamide and L-proline (but not other amino acids) as nitrogen sources in the presence of non-catabolite-repressing carbon sources such as L-arabinose, glycerol, melibiose, and lactose. Suppressor mutations, designated creA^d, probably leading to loss of carbon catabolite repression, allow utilisation of acetamide and proline as nitrogen sources in areA^r double mutants in the presence of carbon catabolite-repressing carbon sources. creA^d mutations allow ethanol to serve as a source of acetate for pyruvate dehydrogenaseless (pdhA) strains in the presence of carbon catabolite-repressing carbon sources, whereas pdhA single mutants respond to ethanol as sole carbon source only in the presence of non-carbon catabolite-repressing carbon sources. Specific suppressor mutations, designated amd ^d and prn ^d, allow utilisation of acetamide or proline, respectively, in areA^r double mutants.The areA locus can be interpreted as specifying a protein which is capable of (and in most cases essential for) allowing the synthesis of a number of enzymes of nitrogen metabolism but which cannot function in the presence of ammonium (i.e., as specifying a positive regulatory element which mediates ammonium repression) although the possibility that the areA product also plays a negative regulatory role cannot at present be ruled out.     

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.     

A mutation in Aspergillus nidulans which affects the regulation of nitrite reductase and is tightly linked to its structural gene.

Summary The selection of nis-5, a mutation which is tightly linked to the structural genes for nitrate reductase (niaD) and nitrite reductase (niiA) but which only affects nitrite reductase activities, is described. nis-5 single mutants have only 40% of the wild type activity of nitrite reductase after induction by nitrate and, for this reason, grow poorly on nitrate and nitrite. Nitrate reductase activity is not affected, and nis-5 is shown to complement with a niaD^- mutation but not with a niiA^- mutation.When grown without inducer, nis-5 strains have higher than the non-induced wild type activity of nitrite reductase. This low, constitutive activity is insensitive to repression by ammonium. These facts explain why the nis-5 mutation weakly suppresses many nirA^- and areA^r mutations for utilization of nitrite.Three of the possible explanations of this unusual phenotype are considered. Studies of nitrite reductase in cell-free extracts provided no evidence for the already unlikely possibility that nis-5 is a structural gene mutation resulting in the observed phenotype because of alteration in the catalytic activity and/or stability of the nitrite reductase.A more plausible explanation is that it defines a receptor site for either the nirA gene product and/or the areA gene product. However, no evidence for this has yet been obtained from a study of double mutants carrying nis-5 and areA or nirA mutations.A third possibility is that nis-5 creates a new, but inefficient promoter or initiator, which is not subject to the normal control systems (and therefore causes constitutive, deprepressed synthesis) but whose physical presence reduces maximal enzyme synthesis. The presence of a translocation in nis-5 strains suggests a means by which niiA could come to be under the control of another promoter/initiator.     

Catabolite repression of the lac operon: effect of operator-constitutive mutations

Summary We have constructed and tested three lac diploid strains in an attempt to show whether operator-constitutive mutations relieve catabolite repression of the lac operon. Each of these carries a different operator mutation on the chromosome, and all three have the genotype I⁺P⁺O^cZ⁺Y^-polar/Flac I⁺P⁺O⁺Z^delY⁺A⁺. When these strains were grown in medium containing glucose plus gluconate, synthesis of -galactosidase (directed by a gene cis to a mutant operator) and of thiogalactoside transacetylase (directed by a gene cis to an intact operator) suffered equal catabolite repression. We conclude that the operator-constitutive mutations have no effect on catabolite repression. Since it has been shown in analogous experiments that all promoter mutations tested do alleviate catabolite repression, these results are consistent with the view that the operator and promoter are functionally distinct.     

Release of glucose-mediated catabolite repression due to a defect in the membrane fraction of phosphoenolpyruvate: mannose phosphotransferase system in Pediococcus halophilus

A spontaneous mutant 9R-4 resistant to 2-deoxyglucose (2DG) was derived from a wild-type strain Pediococcus halophilus I-13. Phosphoenolpyruvate (PEP)-dependent glucose-6-phosphate formation by the permeabilized 9R-4 cells was < 5% of that observed with the parent I-13. In vitro complementation of PEP-dependent 2DG-6-phosphate formation was assayed with combination of the cytoplasmic and membrane fractions prepared from the I-13 and the mutants (9R-4, and X-160 isolated from nature), which were defective in PEP: mannose phosphotransferase system (man:PTS). The defects in man:PTS of both the strain 9R-4 and X-160 were restricted to the membrane fraction (e.g. EII^man), not to the cytoplasmic one. Kinetic studies on the glucose transport with intact cells and iodoacetate-treated cells also supported the presence of two distinct transport systems in this bacterium as follows: (i) The wild-type I-13 possessed a high-affinity man:PTS (K _m=11 M) and a low-affinity proton motive force driven glucose permease (GP) (K _m=170 M). (ii) Both 9R-4 and X-160 had only the low-affinity system (K _m=181 M for 9R-4, 278 M for X-160). In conclusion, a 2DG-induced selective defect in the membrane component (EII^man) of the man:PTS could partially release glucose-mediated catabolite repression but not frutose-mediated catabolite repression in soy pediococci.Abbreviations GCR glucose-mediated catabolite repression - FCR fructose-mediated catabolite repression - PEP phosphoenolpyruvate - man:PTS phosphoenolpyruvate:mannose phosphotransferase system - glc:PTS phosphoenolpyruvate:glucose phosphotransferase system - GP glucose permease - CCCP carbonylcyanide mchlorophenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - P proton motive force - G-6-P glucose-6-phosphate - 2DG 2-deoxyglucose - IAA iodoacetic acid - EII^man enzyme II component of man:PTS - EIII^man enzyme III component of man:PTS - EII^glc enzyme II component of glc:PTS - EIII^glc enzyme III component of glc:PTS     

A possible rôle for acid phosphatase in γ-amino-n-butyrate uptake in Aspergillus nidulans

Previously published work from another laboratory has shown that the mutation pacC-5 in the ascomycete Aspergillus nidulans leads to loss of an acid phosphatase (EC 3.1.3.2) activity and is probably located in the structural gene for this enzyme. Here, we show that, pleiotropically, pacC-5 considerably reduces -amino-n-butyrate transport levels as shown both by direct uptake measurements and two kinds of growth tests. A reduction in expression of the permease specified by the gabA gene is almost certainly reponsible for the -amino-n-butyrate uptake defect in pacC-5 strains. pacC-5 does not reduce l-proline uptake, mainly mediated by the prnB permease, or -alanine uptake. This work and our previously published results suggest that, although it does not uniquely reduce -amino-n-butyrate uptake, pacC-5 is highly selective in its effects on transport processes. It is therefore probable that the acid phosphatase specified by the pacC gene plays some rôle in the synthesis, membrane integration or functioning of a particular class of permeases. A rôle for acid phosphatases in membrane processes casts an intriguing new light on the fact that these enzymes are periplasmic and extracellular in many micro-organisms including A. nidulans.Non-Standard Abbreviations GABA -amino-n-butyrate - P5C l-¹pyrroline-5-carboxylate     

ScoC Mediates Catabolite Repression of Sporulation in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Sporulation in Bacillus subtilis can be triggered by carbon catabolite limitation. Conversely, carbon source excess can repress the production of extracellular enzymes, motility, and sporulation. Recent studies have implicated a pH-sensing mechanism, involving AbrB, the TCA cycle, Spo0K, and Ï^H in controlling the catabolite repression of sporulation gene expression. In an accompanying paper, we demonstrate that the AbrB-dependent pH-sensing mechanism may not be the only means by which carbon catabolites affect sporulation. In the studies reported here, we have examined the molecular basis underlying the catabolite repression phenotype of mutations in the hpr (scoC), rpoD (crsA47), and spo0A (rvtA11) loci. Loss of function mutations in hpr (scoC) restored sporulation gene expression and sporulation in the presence of excess catabolite(s), suggesting that Hpr (ScoC) has a pivotal role in mediating catabolite repression. Moreover, hpr gene expression increased substantially in the presence of excess catabolite(s), further supporting the involvement of Hpr (ScoC) in the carbon catabolite response system. We suggest that alterations in the phosphorelay response to catabolites may be one mechanism by which catabolite-resistant mutants such as crsA and rvtA are able to sporulate in the presence of excess glucoseReceived: 12 November 2002 / Accepted: 13 December 2002     

Lactam utilisation in Aspergillus nidulans: evidence for a fourth gene under the control of the integrator gene intA.

Summary Mutations in the lamA gene of Aspergillus nidulans prevent the conversion of exogenous 2-pyrrolidone (-butyrolactam) to -amino-n-butyrate (GABA) and also prevent the (probably analogous) utilisation of 2-piperidone (-valerolactam). The lamA gene, in linkage group VIII, probably specifies a lactamase but a rôle in lactam uptake cannot be ruled out. lamA is probably under the control of the positive acting regulatory gene intA, which can integrate its expression with the expression of three other genes, specifying acetamidase, GABA transaminase, and GABA permease, respectively.     

The role fo glutamine synthetase and glutamine metabolism in nitrogen metabolite repression, a regulatory phenomenon in the lower eukaryote Neurospora crassa

Summary Growth of Neurospora crassa on media containing NH ₄ ⁺ leads to the repression of a variety of permeases and alternative pathways which would generate NH ₄ ⁺ , so called ammonium repression. The mutant am ₂ which lacks NADP-GDH is not subject to ammonium repression of nitrate reductase or urea permease, but like the wild type has repressed levels of these systems when grown in the presence of proline, glutamate or glutamine. The glutamine synthetase (GS) mutant gln-la has derepressed levels of the aforementioned systems unless grown with glutamine.The oligomeric state of GS depends upon the nitrogen sufficiency of the cell, a tetrameric form predominates under conditions of nitrogen limitation and an octameric form under conditions of nitrogen sufficiency. We have found that the tetrameric form GS predominates in the mutants am ₂ and gln-la when they are ammonium derepressed.The mechanism of NH ₄ ⁺ repression in N. crassa is thought to entail a cessation of positive gene action by the product of the nit-2 regulatory gene. We propose that under conditions of NH ₄ ⁺ sufficiency, and hence glutamine sufficiency, the octameric form of GS represses nit-2 gene expression and thereby achieves ammonium repression.     

Mutations affecting the regulation of γ-aminobutyrate utilization inEscherichia coli K-12

Four genes,gabCPDT, are involved in the utilization of γ-aminobutyrate (GABA) byEscherichia coli K-12. Thegab gene cluster maps nearrecA andsrl, at 57.5 min.gabP, gabD andgabT specify the synthesis of GABA transport carrier, succinic semialdehyde dehydrogenase (SSDH), and glutamate-succinic semialdehyde transaminase (GSST), respectively;gabC controls the synthesis of all three proteins. GABA-nonutilizing mutants carrying deletions insrl extended into thegab cluster have been isolated. The mutants completely lost the capacity for GABA transport, while preserving full activity of GSST and SSDH, suggesting thatgabC is not a promoter-operator locus or a gene coding for an activator protein. A mutation ingabD (M-16) that abolished SSDH activity had the following additional properties: It exerted a bipolar effect on the neighboring genes, greatly reducing the activities of GSST and SSDH; the polar effect ongabP but not ongabT was fully suppressed by the knownrho mutation suA78; at least three classes of GABA-utilizing revertants of M-16 were obtained: (i) revertants with allgab activities restored to the parental levels; (ii) revertants with SSDH activity still missing, but with the other activities fully repaired; (iii) revertants with no SSDH activity, with GSST partly recovered, but with transport fully repaired. It is suggested that thegab cluster is transcribed bidirectionally from a promoter in thegabD region and that the mutation in strain M-16 may be due to DNA insertion in that region.     

Stimulation in trans of synthesis of E. coli gal operon enzymes by lambdoid phages during low catabolite repression

Summary The infection of E. coli cells with different lambdoïd prophages triggers a stimulation of galactokinase synthesis when cells are grown in a medium giving rise to a mild catabolite repression (tryptone broth) with an inducer of the gal operon (fucose). These results show that during phage infection (or induction) some factor acting in trans is produced which is able to overcome efficiently catabolite repression of the kinase cistron. Using different strains of pbio252 (P^am, Q^am, H1), pbio256H1 and NNS7 we have concluded that the factor is the N gene product which is known for its anti- (rho) action.Studies of the whole gal operon in the same conditions show that epimerase unlike transferase and galactokinase is practically insensitive to catabolite repression by tryptone broth and that viral development has a low effect on it. This indicates that there is an internal modulation of gal operon expression. A mRNA termination site sensitive to the factor is known in the gal operon between galE and galT. Another site weaker than this one might exist between galE and operator-promoter region.     

Localization of the upstream regulatory sites of yeast iso2-cytochrome c gene

Summary In order to study the regulation of expression of the iso2-cytochrome c gene, we have constructed a fused gene between the 5 flanking region of the gene coding for the yeast iso2-cytochrome c and the coding region of the E. coli beta-galactosidase lacZ gene. When introduced in yeast cells this hybrid gene is expressed and regulated like the production of iso2-cytochrome c: it is under the control of the general catabolic repression and of the unlinked trans-acting CYP1 gene whose CYP1-18 allele causes an overproduction of iso2-cytochrome c. The expression of hybrid genes whose upstream region has been progressively shortened or altered by internal deletions was studied either in wild-type CYP1 ⁺ cells or in cells carrying the CYP1-18 allele grown either on glucose or on glycerol.It appears that the expression and the regulation of the iso2-cytochrome c gene is controlled by an upstream regulatory site composed of a positive and a negative element. This site is the target of regulation by the CYP1 gene product and, directly or through this gene, of the control by the general catabolic repression.     

The mglB sequence of Salmonella typhimurium LT2; promoter analysis by gene fusions and evidence for a divergently oriented gene coding for the mgl repressor

Summary The mglB gene of Salmonella typhimurium LT2 coding for the galactose-binding protein (GBP) was sequenced. We compared the deduced amino acid sequence with the GBP sequence of Escherichia coli K12. The mature proteins differ in only 19 of 309 amino acid residues, corresponding to 94% homology. Analysis of the mglB control region by promoter-probe vectors revealed that two promoters, P1 and P2, constitute the mgl control region (P _mgl ). P1 and P2 function in a synergistic way. P1 is the main promoter of the operon; its activity is 20 times the activity of P2. Both promoters are activated by the cyclic adenosine monophosphate catabolite activator protein (cAMP/CAP) complex. While P1 is inactive in the absence of the cAMP/CAP complex, there is residual activity of P2 under these conditions. Studies on the inducibility of the mglBAEC operon using multicopy plasmid promoter-probe vectors were hampered by the titration of the mgl repressor resulting in a partially constitutive expression of the mgl operon. The results indicate that only P1 is responding to induction by D-fucose. A weak promoter, P _D , within the P1 region but divergent to it was found. P _D is neither stimulated by the cAMP/CAP complex nor by D-fucose. We cloned the gene located downstream to P _D and found it to strongly repress the expression of the mgl operon. We termed this gene mglD. The presence of D-fucose abolished the repression caused by the plasmid-encoded mglD gene product.Abbreviations IPTG isopropyl-1-thic--D-galatopyranoside - ONPG 2-nitrophenyl--D-galatopyranoside - XG 5-bromo-4-chloro-3-indolyl--D-galatopyranoside - Kan^r Kanamycin resistance     

Gene-directed mutagenesis on the chromosome of Bacillus subtilis 168

Summary We have devised a method whereby any mutagenized cloned DNA from Bacillus subtilis can be reinserted at the original site on the B. subtilis chromosome. The procedure depends on the accuracy and high frequency of homologous recombination between the B. subtilis chromosome and the DNA taken up by the cell. The method makes use of two drug resistance selection markers (the chloramphenicol resistance gene and the neomycin resistance gene) and a marker gene which functions as a catalyst. The utility of the method has been demonstrated using leuB and pro of B. subtilis as target gene and catalyst, respectively, and mutations such as leuB: : cat, leuB ^–, and pro: : neo constructed in vitro on the cloned DNA fragments. Transformation in sequential steps as (leuB ⁺ pro⁺)(leuB: : cat pro ⁺) (leuB ^– pro: : neo)(leuB ^– pro ⁺) resulted in a leuB ^– single mutant without affecting other regions of the B. subtilis chromosome (gene-directed mutagenesis). We also demonstrate that other single mutations such as metD ^– and pro ^–, as well as the double mutation leuB ^– pro ^– can be introduced by the same procedure. In principle, true isogenies with multiple mutations can be constructed by the method described in this paper. Furthermore, the procedure should be generally applicable to any organisms in which homologous recombination is proficient.     

A temperature-sensitive Escherichia coli mutant defective in DNA replication: dnaA, a new gene adjacent to the dnA, gene

Summary An Escherichia coli mutant defective in replication of the chromosome has been isolated from temperature-sensitive mutants that cannot support colicin E1 plasmid DNA synthesis in the presence of chloramphenicol. Cellular DNA synthesis of the mutant ceases almost immediately after transfer to the nonpermissive temperature. The defect is due to a single mutation, dna-59, which is located close to the sites of dnaA mutations and a cou ^R mutation conferring DNA gyrase with resistance to coumermycin. The dna-59 mutant is not able to support DNA synthesis of phage at the high temperature. The mutant also restricts growth of X174 phage at the high temperature, but permits formation of supercoiled closedcircular duplex replicative intermediates. T7 phage can grow on the mutant even at the high temperature.A specialized transducing phage imm ²¹[tna dnaA]#2 (Miki et al., 1978) supports growth of dna-59, dnaA46 and dna-167 cells at the high temperature. Some of the EDTA-resistant derivatives of the phage have lost part or all of the dnaA gene, but carry gene function complementing the defect of dna-59 cells, as judged by conversion of the above dna strains to wild type cells by phage infection, and by suppression of the loss of viability of dna-59 cells at the high temperature by phage infection. The gene containing the dna-59 mutation site is thus distinct from the dnaA gene. Since the dna-59 mutation does not affect expression of the cou ^r gene of DNA gyrase, which is another known gene involved in DNA synthesis near the dnaA gene, this mutation is probably in a new gene, dnaN. From analysis of the suppression activities of imm ²¹[tna dnaA]#2 phage and its deletion derivatives against dnaN59 cells, it is suggested that the expression of the dnaN gene function is reduced by deletion in the dnaA region.     

Ribosome activity and degradation in meiotic cells of Saccharomyces cerevisiae.

Summary The acetamidase of Aspergillus nidulans is induced by sources of acetyl CoA, benzoate and benzamide and by -alanine and other -amino acids. The effects of these groups of inducers are approximately additive. The cis-acting control site mutant, amdI9, affects induction by sources of acetyl-CoA specifically. Lesions in the amdR and gatA genes affect induction by -amino acids specifically. Mutations in the amdA gene can lead to elevated acetamidase levels which still respond to the various inducers. The induction controls act independently of repression control by nitrogen metabolites and are not altered by the areA102 mutation. The properties of double mutants with lesions affecting the different control mechanisms also indicate their independence of each other. It is suggested that the acetamidase is subject to complex control by multiple regulatory circuits and that functionally independent control sites adjacent to the structural gene occur.     

RifR mutations in the beginning of the Escherichia coli rpoB gene

In Escherichia coli, mutations conferring rifampicin (Rif) resistance map to the rpoB gene, which encodes the 1342-amino acid subunit of RNA polymerase. Almost all sequenced RifR mutations occur within the Rif region, encompassing rpoB codons 500–575. A strong Rif^R mutation lying outside the Rif region, which changed Val¹⁴⁶ to Phe was previously reported, but was not recovered in subsequent studies. Here, we used site-directed mutagenesis followed by selection on Rif to search for Rif^R mutations in the evolutionarily conserved segment of rpoB around codon 146. Strong Rif^R mutations were obtained when Val¹⁴⁶ was mutated, and several weak Rif^R mutations were also isolated near position 146. The results define a new, N-terminal cluster of Rif^R mutations, in addition to the classical central Rif region.