Changes in gene expression elicited by amino acid limitation in Neurospora crassa strains having normal or mutant cross-pathway amino acid control

Summary The effects of amino acid limitation on gene expression have been investigated in Neurospora crassa strains carrying normal (cpc-1 ⁺) or mutant (cpc-1) alleles at a locus implicated in cross-pathway amino acid control. Electrophoresis and fluorography were used to reveal the patterns of label incorporation into polypeptides in vivo, or after in vitro translation of extracted mRNAs. In a cpc-1 ⁺ strain at least 20% of detectable in vitro translation products showed relative increases in incorporation when RNA was obtained from mycelium grown under conditions of arginine limitation, by comparison with conditions of arginine sufficiency. A cpc-1 mutation, which impairs derepression of a variety of amino acid synthetic enzymes following amino acid limitation, had little detectable effect on in vivo polypeptide synthesis during amino acid sufficient growth or following pyrimidine limitation. However the mutation substantially altered the response to arginine or histidine limitation. The majority of in vitro translation products that showed increased expression in arginine limited cpc-1 ⁺ failed to increase in cpc-1 strains, but arginine limitation of cpc-1 also resulted in increases that did not occur in cpc-1 ⁺ strains. This may reflect both direct and indirect consequences of the impairment of cross-pathway control.     

A lethal allele at the putative regulatory locus, cpc-1, of cross-pathway control in Neurospora crassa

Summary A lethal allele at the putative regulatory locus, cpc-1, of cross-pathway control in Neurospora crassa was discovered by genetic analysis. cpc-1 ^j-5 is viable only in the presence of a second mutation, slo, causing slow growth. The detection of a lethal allele at a regulatory locus is a rare event and points to the physiological importance of the regulatory circuit concerned, namely the cross-pathway or general control of amino acid biosynthetic enzymes in lower eukaryotes.     

A <Emphasis Type="Italic">Sordaria macrospora</Emphasis> mutant lacking the <Emphasis Type="Italic">leu1</Emphasis> gene shows a developmental arrest during fruiting body formation

Developmental mutants with defects in fruiting body formation are excellent resources for the identification of genetic components that control cellular differentiation processes in filamentous fungi. The mutant pro4 of the ascomycete Sordaria macrospora is characterized by a developmental arrest during the sexual life cycle. This mutant generates only pre-fruiting bodies (protoperithecia), and is unable to form ascospores. Besides being sterile, pro4 is auxotrophic for leucine. Ascospore analysis revealed that the two phenotypes are genetically linked. After isolation of the wild-type leu1 gene from S. macrospora, complementation experiments demonstrated that the gene was able to restore both prototrophy and fertility in pro4. To investigate the control of leu1 expression, other genes involved in leucine biosynthesis specifically and in the general control of amino acid biosynthesis (“cross-pathway control”) have been analysed using Northern hybridization and quantitative RT-PCR. These analyses demonstrated that genes of leucine biosynthesis are transcribed at higher levels under conditions of amino acid starvation. In addition, the expression data for the cpc1 and cpc2 genes indicate that cross-pathway control is superimposed on leucine-specific regulation of fruiting body development in the leu1 mutant. This was further substantiated by growth experiments in which the wild-type strain was found to show a sterile phenotype when grown on a medium containing the amino acid analogue 5-methyl-tryptophan. Taken together, these data show that pro4 represents a novel mutant type in S. macrospora, in which amino acid starvation acts as a signal that interrupts the development of the fruiting body. Electronic Supplementary Material Supplementary material is available for this article at http://dx.doi.org/10.1007/s00438-005-0021-8     

Biosynthesis of aromatic amino acids in Nocardia sp. 239: effects of amino acid analogues on growth and regulatory enzymes

Summary Further steps required for overproduction of aromatic amino acids by a mutant strain of Nocardia sp. 239 (Noc 87-13), unable to grow on l-phenylalanine as a sole carbon and energy source, were investigated. A number of analogues of the aromatic amino acids displayed severe inhibitory effects on the activities of regulatory enzymes in the biosynthetic pathway and growth of the organism in glucose mineral medium. l-Tryptophane analogues strongly inhibited 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase activity. l-Tyrosine analogues especially inhibited DAHP synthase and chorismate mutase, whereas l-phenylalanine analogues strongly inhibited chorismate mutase and prephenate dehydratase activity. Addition of the aromatic amino acids and their precursors chorismate, 4-hydroxyphenylpyruvate, phenylpyruvate and anthranilate, to the medium counteracted the growth inhibitory effect of specific analogues. The data indicate that ortho- (OFP) and para-fluoro-d,l-phenylalanine (PFP), and l-phenylalanine amide, are the most suitable analogues for the isolation of feedback-inhibition-insensitive prephenate dehydratase mutants. Attempts to isolate l-tyrosine and l-trytophane auxotrophic mutants were only successful in the latter case, resulting in the selection of a stable anthranilate synthase-negative mutant (Noc 87-13-14). Uptake of aromatic amino acids in Nocardia sp. 239 most likely involves a common transport system. This necessitates the use of anthranilate, rather than l-trytophane, as a supplement during the isolation of l-tyrosine auxotrophic and OFP- and/or PFP-resistant mutant derivative strains of Noc 87-13-14. Offprint requests to: L. Dijkhuizen     

Peculiarities of amino acid transport inSchizosaccharomyces pombe: Effects of growth medium

Transport systems for amino acids in the wild-type strain ofSchizosaccharomyces pombe are not constitutive. During growth on different media no transport of acidic, neutral and basic amino acids is detectable. To acquire the ability to transport amino acids, cells must be preincubated with a metabolic source of energy, such as glucose. The appearance of transport activity is associated with protein synthesis (suppression by cycloheximide) at all phases of culture growth. After such preincubation the initial rate of amino acid uptake depends on the phase of growth of the culture and on the amount of glucose in the growth medium but not on the nitrogen source used.l-Proline and 2-aminoisobutyric acid are practically not transported under any of the conditions tested.     

Construction of an arginine-producing strain of Serratia marcescens

Summary In Serratia marcescens Sr41, l-canavanine was demonstrated to be a weak cell growth inhibitor in minimal medium containing glucose as the sole carbon source. The inhibition of cell growth was enhanced by changing the carbon source from glucose to l-glutamic acid. An arginine regulatory mutant (i.e., argR mutant) in which formation of l-arginine biosynthetic enzymes was genetically derepressed was isolated by selecting for l-canavanine resistance on the glutamate medium. Furthermore, an l-arginine-producing strain was constructed by introducing the mutation leading to feedback-resistant N-acetylglutamate synthase into the argR mutant. The resulting transductant produced about 40 g/l of l-arginine, whereas the wild strain produced no l-arginine and the argR mutant only 3 g/l.     

Silencing of Vlaro2 for chorismate synthase revealed that the phytopathogen Verticillium longisporum induces the cross-pathway control in the xylem

The first leaky auxotrophic mutant for aromatic amino acids of the near-diploid fungal plant pathogen Verticillium longisporum (VL) has been generated. VL enters its host Brassica napus through the roots and colonizes the xylem vessels. The xylem contains little nutrients including low concentrations of amino acids. We isolated the gene Vlaro2 encoding chorismate synthase by complementation of the corresponding yeast mutant strain. Chorismate synthase produces the first branch point intermediate of aromatic amino acid biosynthesis. A novel RNA-mediated gene silencing method reduced gene expression of both isogenes by 80% and resulted in a bradytrophic mutant, which is a leaky auxotroph due to impaired expression of chorismate synthase. In contrast to the wild type, silencing resulted in increased expression of the cross-pathway regulatory gene VlcpcA (similar to cpcA/GCN4) during saprotrophic life. The mutant fungus is still able to infect the host plant B. napus and the model Arabidopsis thaliana with reduced efficiency. VlcpcA expression is increased in planta in the mutant and the wild-type fungus. We assume that xylem colonization requires induction of the cross-pathway control, presumably because the fungus has to overcome imbalanced amino acid supply in the xylem.     

Condensed phosphate deposition,sulfur amino acid use,and unidirectional transsulfuration in Synechococcus leopoliensis

Cells of the cyanobacterium, Synechococcus leopoliensis, have previously been shown to exhibit diminished growth, increased condensed phosphate accumulation, enlarged polyphosphate bodies, and severe chlorosis when cultured under conditions of sulfur deficiency. These characteristics were used to identify which of several sulfur amino acids and a tripeptide served as a sole sulfur source for this unicellular microorganism. Completely serving sulfur compounds were l-cystine, dl-lanthionine, l-djenkolic acid, and glutathione. Sulfur amino acids serving poorly or not at all were l-cystathionine, dl-homocystine, l-methionine, l-cysteic acid, and taurine. This pattern of use suggests that the unidirectional transsulfuration pathway demonstrated in enteric bacteria and green plants, i.e. l-cysteine to l-homocysteine, operates as well in cyanobacteria of the Synechococcus type.     

Transport of basic amino acids in Riccia fluitans: Evidence for a second binding site

The transport of several amino acids with different side-chain characteristics has been investigated in the aquatic liverwort Riccia fluitans. i) The saturation of system I (neutral amino acids) by addition of excess -aminoisobutyric acid to the external medium completely eliminated the electrical effects which are usually set off by neutral amino acids. Under these conditions arginine and lysine significantly depolarized the plasmalemma. ii) L- and D-lysine/arginine were discriminated against in favour of the L-isomers. iii) Increasing the external proton concentration in the interval pH 9 to 4.5 stimulated plasmalemma depolarization, electrical net current, and uptake of [¹⁴C]-basic amino acids. iv) Uptake of [¹⁴C]-glutamic acid took place only at acidic pHs. v) [¹⁴C]-histidine uptake had an optimum between pH 6 and 5.5. vi) Overlapping of the transport of basic, neutral, and acidic amino acids was common. It is suggested that besides system I, a second system (II), specific for basic amino acids, exists in the plasmalemma of Riccia fluitans. It is concluded that the amino-acid molecule with an uncharged side chain is the substrate for system I, which also binds and transports the neutral species of acidic amino acids, whereas system II is specific for amino acids with a positively charged side chain. The possibility of system II being a proton cotransport is discussed.Abbreviation AiB -aminoisobutyric acid     

Temperature sensitivity of the cdc9-1 allele of Saccharomyces cerevisiae DNA ligase is dependent on specific combinations of amino acids in the primary structure of the expressed protein.

Summary In this study we present the characterization of the temperature-sensitive mutant allele cdc9-1 encoding DNA ligase, of Saccharomyces cerevisiae strain A364A by DNA sequencing. Comparison with the published wild-type sequence from strain SKI revealed 13 nucleotide exchanges between these two sequences, which are derived from non-isogenic genetic backgrounds. Only four of these changes, distributed over the whole coding region, lead to amino acid exchanges in the protein chain. Our analysis of the sequence of the wild-type CDC9 allele from strain A364A revealed differences from the isogenic cdc9-1 allele in only two nucleotides: one silent change and one leading to a single amino acid exchange. The latter is therefore responsible for the temperature-sensitive phenotype. A mosaic protein, in which a region carrying this amino acid exchange has been inserted in place of the corresponding part of CDC9 from the non-isogenic strain SKI, is not temperature sensitive. The exchange of a longer stretch of DNA leading to atteration of three amino acids of the protein compared with the original sequence of SKI is required to obtain a temperature-sensitive DNA ligase in this strain, while in strain A364A a single amino acid change is sufficient for expression of a temperature-sensitive protein.     

Effect of amino acids on growth ofSphaerotilus discophorus

The effect of casein hydrolysate, of mixtures of amino acids and of individual amino acids on the growth of 4 strains ofSphaerotilus discophorus was determined. Growth was virtually completely inhibited by 1.0% Bacto Casamino Acids, 0.54% simulated casein hydrolysate and 0.2% of a uniform mixture of 18 amino acids. The latter were prepared withl amino acids except thatdl-serine,dl-valine anddl-threonine were present in the uniform amino acid mixture.Experiments designed to test the toxicity of the 18 individual amino acids at 0.018 – 0.36% concentration indicated that arginine, glutamic acid, leucine, lysine and proline were non-toxic. However, aspartic acid and methionine were moderately toxic; growth was greatly repressed at a concentration of 0.36%. The remaining 11 amino acids which included alanine, cystine, glycine, tyrosine, histidine, isoleucine, phenylalanine, serine, threonine, tryptophane and valine were the most toxic of the group. They prevented growth partially or completely, at a concentration of 0.18% or 0.36%.dl-Serine anddl-valine were especially toxic and prevented growth at a concentration of 0.018%. The toxicity of the individuall-amino acids can account for the toxicity of Casamino Acids and simulated casein hydrolysate. l-Methionine or cyanocobalamin (vitamin B₁₂) is required for the growth ofS. discophorus. Alsod- anddl-methionine can replace cyanocobalamin although they completely repress growth when used at the relatively high concentration of 200 µg per ml of medium.     

Regulation of biosynthesis of aspartate family amino acids in the photosynthetic bacterium Rhodopseudomonas palustris

The four amino acids of the aspartate family (l-lysine, l-methionine, l-threonine, and l-isoleucine) are produced in bacteria by a branched biosynthetic pathway. Regulation of synthesis of early common intermediates and of carbon flow through distal branches of the pathway requires operation of a number of subtle feedback controls, which are integrated so as to ensure balanced synthesis of the several end products. Earlier studies with nonsulfur purple photosynthetic bacteria were instrumental in revealing the existence of alternative regulatory schemes, and in this communication we report on the control pattern of a representative of this physiological group not previously investigated, Rhodopseudomonas palustris. The results obtained from study of the properties of four key regulatory enzymes of the aspartate family pathway (-aspartokinase, homoserine dehydrogenase, homoserine kinase, and threonine deaminase) and of the effects of exogenous amino acids (i. e., the end products) on growth of the bacterium indicate that the control schema in Rps. palustris differs substantially from the schemes described for other Rhodopseudomonas species, but resembles the regulatory pattern observed in Rhodospirillum rubrum.Abbreviations A absorbancy - AK -aspartokinase - ASA aspartate -semialdehyde - DTT dithiothreitol - HS l-homoserine - HSDH homoserine dehydrogenase - HSK homoserine kinase - I l-isoleucine - KU Klett-Summerson photometer units - L l-lysine - M l-isoleucine - KU Klett-Summerson photometer units - L l-lysine - M l-methionine - ME -mercaptoethanol - PABA p-aminobenzoic acid - T l-threonine - TD threonine deaminase - RCV synthetic growth medium (see text) - YP agar medium containing 0.3% yeast extract, 0.3% peptone, and 1.5% agar - Y2T synthetic growth medium (see text)     

Studies on utilization of 2-ketoglutarate,glutamate and other amino acids by the unicellular alga Cyanidium caldarium

Two strains of Cyanidium caldarium which possess different biochemical and nutritional characteristics were examined with respect to their ability to utilize amino acids or 2-ketoglutarate as substrates.One strain utilizes alanine, glutamate or aspartate as nitrogen sources, and glutamate, alanine or 2-ketoglutarate as carbon and energy sources for growth in the dark. The growth rate in the dark on 2-ketoglutarate is almost twice as high or higher than that on glutamate or alanine. During growth or incubation of this alga on amino acids, large amounts of ammonia are formed; however, ammonia formation is strongly inhibited by 2-ketoglutarate. The capacity of the alga to form ammonia from amino acids is inducible and develops fully only when the cells are grown or incubated in the presence of glutamate.By contrast, the other strain of Cyanidium caldarium cannot utilize alanine or aspartate as nitrogen sources. It utilizes glutamate only very poorly and does not excrete ammonia into the external medium. This strain is unable to utilize amino acids or 2-ketoglutarate as carbon and energy sources for heterotrophic growth.Cell-free extracts were tested for the occurrence of enzymes which could account for amino acid metabolism and ammonia formation.     

Zinc affects siderophore-mediated high affinity iron uptake systems in the rhizosphere Pseudomonas aeruginosa 7NSK2

Zinc concentrations ranging between 0.1 and 1 mm only slightly reduced maximal growth of wild-type Pseudomonas aeruginosa 7NSK2 in iron-limiting casamino acid medium, but had a clear negative effect on the growth of mutant MPFM1 (pyoverdin negative) and especially mutant KMPCH (pyoverdin and pyochelin negative). Production of pyoverdin by wild-type strain 7NSK2 was significantly increased in the presence of 0.5 mm zinc and could not be repressed by iron even at a concentration of 100 m. Siderophore detection via isoelectrofocusing revealed that mutant KMPCH did not produce any siderophores, while mutant MPFM1 overproduced a siderophore with an acidic isoelectric point, most likely pyochelin. Pyochelin production by MPFM1 was stimulated by the presence of zinc in a similar way as pyoverdin for the wild-type. Analysis of outer membrane proteins revealed that three iron regulated outer membrane proteins (IROMPs) (90, 85 and 75 kDa) were induced by iron deficiency in the wild-type, while mutants were found to have altered IROMP profiles. Zinc specifically enhanced the production of a 85 kDa IROMP in 7NSK2, a 75 kDa IROMP in MPFM1 and a 90 kDa IROMP in KMPCH.     

Resistance to l-methionine-S-sulfoximine in Chlamydomonas reinhardtii is due to an alteration in a general amino acid transport system

It was suggested that the mutant ARF1 of Chlamydomonas reinhardtii is resistant to l-methionine-S-sulfoximine (MSX, an irreversible inhibitor of glutamine synthetase, EC 6.3.1.2) because this strain degraded and utilized this compound as a nitrogen source for growth (A.R. Franco et al., 1996, Plant Physiol 110: 1215–1222). Resistance to MSX has now been characterized in a double mutant of this alga, called MPA1, which is resistant to MSX and lacks l-amino acid oxidase (LAO activity, EC 1.4.3.2). Biochemical and genetic evidence indicate that the mutant MPA1 is altered in the same MSX-resistance locus as mutant ARF1. However, mutant MPA1 neither degraded nor utilized MSX as a nitrogen source. This led us to conclude that (i) resistance to MSX is not linked to its utilization, and (ii) that LAO activity accounts for the degradation of MSX in mutant ARF1. Data indicate that C. reinhardtii possesses a broad-specificity carrier system responsible for the transport of arginine and other amino acids, including MSX. We propose that the alteration of this carrier confers resistance to MSX in mutants ARF1 and MPA1. Received: 6 April 1998 / Accepted: 8 June 1998