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1.
2-Methylhistidine (2MH) and 1,2,4-triazole-3-alanine (TRA) inhibited the growth of Serratia marcescens. These inhibitory effects were counteracted by L-histidine. Enzymatic studies showed that 2MH acts as a false feedback inhibitor and TRA acts as both a false feedback inhibitor and a repressor. Mutants resistant to each analog were isolated from a histidase-less mutant, because the wild-type strain possesses a potent histidase activity. 2MH-resistant mutants had a feedback-insensitive phosphoribosyltransferase, but they produced only small amounts of L-histidine. TRA-resistant mutants were divided into two types according to their histidine productivity. A mutant of one type produced about 8 mg of L-histidine per ml and had about a 10-fold increase in the enzyme levels of histidine biosynthesis. Moreover, this mutant had a partially feedback-insensitive phosphoribosyltransferase. A mutant of the second type produced only a small amount of L-histidine and had only derepressed enzyme levels. Accordingly, strains possessing the genetic alterations in both 2MH- and TRA-resistant mutants were constructed by PS20-mediated transduction. They had both feedback-insensitive phosphoribosyltransferase and derepressed enzyme levels. The representative strain HT-2604 produced about 17 mg of L-histidine per ml.  相似文献   

2.
In Serratia marcescens, the mutation responsible for triazolealanine (TRA) resistance was transferred from a TRA-resistant mutant to a urocanase-less mutant by PS20-mediated transduction. The two crosses were performed using as donors two TRA-resistant mutants, whose phenotypes included increased levels of histidine-biosynthetic enzymes and feedback-insensitive phosphoribosyltransferase. In one cross, TRA-resistant transductants were urocanase-less mutants having only increased levels of the enzymes and barely detectable levels of urocanic acid. In the other cross, the transductants were urocanase-less mutants having both phenotypes of the donor, and most produced high concentrations (10.5 mg/ml) of urocanic acid.  相似文献   

3.
In Serratia marcescens, the mutation responsible for triazolealanine (TRA) resistance was transferred from a TRA-resistant mutant to a urocanase-less mutant by PS20-mediated transduction. The two crosses were performed using as donors two TRA-resistant mutants, whose phenotypes included increased levels of histidine-biosynthetic enzymes and feedback-insensitive phosphoribosyltransferase. In one cross, TRA-resistant transductants were urocanase-less mutants having only increased levels of the enzymes and barely detectable levels of urocanic acid. In the other cross, the transductants were urocanase-less mutants having both phenotypes of the donor, and most produced high concentrations (10.5 mg/ml) of urocanic acid.  相似文献   

4.
Thiazolealanine, a false feedback inhibitor, causes transient repression of the his operon previously derepressed by a severe histidine limitation in strains with a wild-type or feedback-hypersensitive first enzyme but not in feedback-resistant mutants. Since experiments reported here clearly demonstrate that thiazolealanine is not transferred to tRNAHis, it is proposed that this "transient repression" is effected through the interaction of thiazolealanine with the feedback site of the enzyme. Experiments in the presence of rifampin indicate that this thiazolealanine-mediated effect is exerted at the level of translation. We conclude that histidine (free), in addition to forming co-repressor, also represses the operon at the level of translation through feedback interaction with the first enzyme of the pathway (adenosine 5'-triphosphate phosphoribosyltransferase). Rates of derepression in feedback-resistant strains are roughly half of those observed in controls, suggesting a positive role played by a first enzyme with a normal but unoccupied feedback site. Some feedback-resistant mutants, in contrast to the wild type, were unable to exhibit derepression under histidine limitation caused by aminotriazole.  相似文献   

5.
Corynebacterium glutamicum mutants carrying both auxotrophy and histidine analog-resistance were derived by a mutagenic treatment, and their histidine productivity was compared with that of a triazolealanine (TRA)-resistant histidine producer, C. glutamicum KY-10260. As a result, a leucine auxotrophic TRA-resistant mutant, Rα-88 was selected out of 164 auxotrophic derivatives of KY-10260. It produced histidine at a distinctly higher concentration than the parent strain under every condition tested. The concentration reached 11 mg/ml or 5.8% (w/w) of the initial sugar. Addition of an excessive amount of leucine to the medium inhibited the histidine production together with the by-production of valine by this mutant. Thiazolealanine-resistant mutants derived from a tyrosine auxotroph, a phenylalanine auxotroph and a tryptophan auxotroph gave the same or lower production in comparison with KY-10260.  相似文献   

6.
Phosphoribosyl-ATP pyrophosphorylase of two l-histidine-producers of Corynebacterium glutamicum, each selected as a 2-thiazolealanine (TA)-resistant and a 1,2,4-triazole-3-alanine (TRA)-resistant, was found to be 100-fold resistant to l-histidine-inhibition in comparison with wild-type enzyme. It was also resistant to the inhibition by TA, but still as sensitive as the wild-type enzyme to the inhibition by α-methylhistidine. Formation of the pyrophosphorylase in these mutants was not significantly derepressed. However, two-fold derepression was noted with a further improved l-histidine producer KY-10522, a derivative of the above TRA-resistant. KY-10522 is an improved strain in l-histidine-productivity through the additions of resistance markers including increased resistance to TRA. Phosphoribosyl-ATP pyrophosphorylase of KY-10522 was found to be resistant to the feedback inhibition, like it’s parent strain.  相似文献   

7.
Nodules produced by ineffective mutants of R. meliloti strain L5-30 requiring arginine+uracil (arg-55) and cysteine requiring mutants (cys-243, cys-244, cys-246) studied under light microscopy were found to be occupied by bacteria. This indicates on defect in transformation of these mutants into N2 fixing bacteroids. These defects were not associated with auxotrophy. In the nodules induced by histidine requiring mutant (his-240) only few host plant cells were occupied by bacteria. This indicate that his-240 mutant is defective in liberation from the infection thread and its multiplication since supplementation of the plant growth medium with 50 microgram/ml of L-histidine enabled establishment of fully effective association. Prototrophic transductants and revertants were fully effective.  相似文献   

8.
This study describes the isolation and characterization of a mutant (strain GP122) of Salmonella typhimurium with a partial deficiency of phosphoribosylpyrophosphate (PRPP) synthetase activity. This strain was isolated in a purE deoD gpt purin auxotroph by a procedure designed to select guanosine-utilizing mutants. Strain GP122 had roughly 15% of the PRPP synthetase activity and 25% of the PRPP pool of its parent strain. The mutant exhibited many of the predicted consequences of a decreased PRPP pool and a defective PRPP synthetase enzyme, including: poor growth on purine bases; decreased accumulation of 5-aminoimidazole ribonucleotide (the substrate of the blocked purE reaction) under conditions of purine starvation; excretion of anthranilic acid when grown in medium lacking tryptophan; increased resistance to inhibition by 5-fluorouracil; derepressed levels of aspartate transcarbamylase and orotate phosphoribosyltransferase, enzymes involved in the pyrimidine de novo biosynthetic pathway; growth stimulation by PRPP-sparing compounds (e.g. guanosine, histidine); poor growth in low phosphate medium; and increased heat lability of the defective enzyme. This mutant strain also had increased levels of guanosine 5'-monophosphate reductase. This genetic lesion, designated prs, was mapped by conjugation and phage P22-mediated transduction at 35 units on the Salmonella linkage map.  相似文献   

9.
An effort to find growth conditions leading to conditional regulation of the histidine operon of Salmonella typhimurium by the allosteric first enzyme of the pathway, adenosine triphosphate phosphoribosyltransferase (EC 2.4.2.17), is reported. A strain deleting the enzyme, TR3343, behaved simply and predictably under all growth conditions, whereas histidine auxotrophs containing active enzyme behaved in complicated ways dependent upon the location of the histidine pathway lesion. hisE strains derepressed the operon only one-half as much as TR3343 when grown on limiting histidine and a poor carbon source, but they also grew more slowly, probably as a result of high N1-(5-phospho-beta-D-ribosyl)-adenosine triphosphate levels in the cell. hisC strains exhibited oscillatory growth behavior and oscillatory histidine operon expression when grown on intermediate concentrations of the histidine precursor histidinol. This behavior probably was caused by synergistic in-phase variations in the histidine, purine nucleotide, and ppGpp pools of the cell. All of the growth and histidine operon expression effects associated with the presence of adenosine triphosphate phosphoribosyltransferase could be assigned to metabolic perturbation of the cell caused by unregulated enzymatic activity.  相似文献   

10.
Previous studies showed that when triazolalanine was added to a derepressed culture of a histidine auxotroph, repression of the histidine operon occurred as though histidine had been added (6). However, when triazolalanine was added to a derepressed culture of a strain with a mutation in the first gene of the histidine operon which rendered the first enzyme for histidine biosynthesis resistant to inhibition by histidine, repression did not occur. The studies reported here represent a cis/trans test of this effect of mutations to feedback resistance. Using specially constructed merodiploid strains, we were able to show that the wild-type allele is dominant to the mutant (feedback resistant) allele and that the effect operates in trans. We conclude that the enzyme encoded by the first gene of the histidine operon exerts its regulatory effect on the operon not by acting locally at its site of synthesis, but by acting as a freely diffusible protein.  相似文献   

11.
A general search has been made for mutants defective in their ability to derepress the histidine operon. The procedure was to select for mutants with an increased sensitivity to the false feedback inhibitor, 2-thiazolealanine. Five mutant strains defective in derepression have been isolated. All five strains are unable to derepress normally because of mutations located in the operator-promoter region of the histidine operon.  相似文献   

12.
alpha-Aminobutyric acid, norvaline, and norleucine, which are analogues of branched-chain amino acids, inhibited the growth of Serratia marcescens. The inhibitory effect of these three analogues was counteracted by branched-chain amino acids. A number of mutants resistant to these analogues were isolated. alpha-Aminobutyric acid-resistant (abu-r) mutants markedly accumulated l-valine in the culture medium, but the other analogue-resistant mutants did not. Acetohydroxy acid synthetase, which seems to be rate-limiting for the biosynthesis of l-valine, was derepressed in abu-r mutants. One of the abu-r mutants, no. 140, which accumulated over 8 mg of l-valine per ml, had about a 20-fold increase in the enzyme level. Most of the abu-r mutants had acetohydroxy acid synthetase activity which was sensitive to feedback inhibition by l-valine to the same extent as in the parent strain. However, the enzyme of two of abu-r mutants was less sensitive to l-valine, and one of the two was the best valine accumulator.  相似文献   

13.
Previous studies suggested that phosphoribosyltransferase, which catalyzes the first step of the pathway for histidine biosynthesis in Salmonella typhimurium and which is sensitive to inhibition by histidine, plays a role in repression of the histidine operon. Recently, we showed that the enzyme has a high affinity for histidyl transfer ribonucleic acid (His-tRNA), which is known to participate in the repression process. In the present study, we have investigated further the interaction between the enzyme and His-tRNA. We found that His-tRNA binds at a site on phosphoribosyltransferase distinct from the catalytic site and the histidine-sensitive site; that the substrates of the enzyme inhibit the binding of His-tRNA, whereas histidine does not do so; that, once a complex has been formed between phosphoribosyltransferase and His-tRNA, the substrates of the enzyme decrease the stability of the complex, whereas histidine is without effect; and that purified phosphoribosyltransferase which has a defect in its inhibition by histidine (produced by mutation) displays an altered ability to bind His-tRNA, a finding which may be a reflection of the fact that mutants producing such a defective enzyme display an alteration of the repression process.  相似文献   

14.
Phosphoribosylpyrophosphate (PRPP) synthetase participates in the biosynthesis in bacteria of purine nucleotides, pyrimidine nucleotides, tryptophan, and histidine. The regulation of the synthesis of PRPP synthetase in Salmonella typhimurium was studied. Addition of end products to the growth medium, singly or in combination, resulted in small decreases in the specific activity of PRPP synthetase, but levels of the enzyme were never decreased to less than half of those found when the bacteria were grown on minimal medium. Growth of the bacteria on several different carbon sources or starvation for phosphate had little effect on the specific activity of PRPP synthetase. Over-production of histidine in a histidine regulatory mutant, which would be expected to result in a depletion of intracellular PRPP pools, did not alter PRPP synthetase specific activity. PRPP synthetase levels were examined in auxotrophic strains of S. typhimurium that had been starved for the end products of PRPP. In each case derepression of an enzyme in the biosynthetic pathway for the limiting end product was demonstrated. However, only alterations in the levels of pyrimidine bases in the culture medium brought about derepression and repression of PRPP synthetase. Excess pyrimidines do not completely repress the enzyme. Deprivation of exponentially growing cells for pyrimidines by growth of an auxotrophic mutant on media containing orotic acid, which enters the cells slowly, resulted in a 10-fold derepression of PRPP synthetase. Derepression of PRPP synthetase during uracil starvation was prevented by chloramphenicol. The PRPP synthetase activities of extracts from repressed and derepressed cells responded in identical fashion to heat inactivation, cellulose acetate electrophoresis at several pH values, and in kinetic experiments.  相似文献   

15.
In the Hupc mutants of Bradyrhizobium japonicum SR, regulation of expression of hydrogenase is altered; the mutants synthesize hydrogenase constitutively in the presence of atmospheric levels of oxygen. The DNA gyrase inhibitors nalidixic acid, novobiocin, and coumermycin were used to inhibit growth of wild-type and mutant cells. For each inhibitor tested, growth of mutant and wild-type strains was equally sensitive. However, in contrast to the wild type, the Hupc mutants synthesized hydrogenase in the presence of high levels of any inhibitor. Cells were incubated with the drugs and simultaneously labeled with 14C-labeled amino acids, and hydrogenase was immunoprecipitated with antibody to the large subunit of the enzyme. Fluorograms of antibody blots then were scanned to determine the relative amount of hydrogenase (large subunit) synthesized in the presence or absence of the gyrase inhibitors. The amount of hydrogenase synthesized by the Hupc mutants in the presence of 300 micrograms of nalidixic acid per ml was near the level of enzyme synthesized in the absence of the inhibitor. No hydrogenase was detected in antibody blots of wild-type cultures which were derepressed for hydrogenase in the presence of 100 micrograms of coumermycin or novobiocin per ml. In contrast, hydrogenase was synthesized by the Hupc mutants in the presence of 100 micrograms of either drug per ml. The amount synthesized ranged from 5 to 32% and 20 to 49%, respectively, of that in the absence of those inhibitors, but nevertheless, hydrogenase synthesis was detected in all of the mutants examined.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

16.
In the Hupc mutants of Bradyrhizobium japonicum SR, regulation of expression of hydrogenase is altered; the mutants synthesize hydrogenase constitutively in the presence of atmospheric levels of oxygen. The DNA gyrase inhibitors nalidixic acid, novobiocin, and coumermycin were used to inhibit growth of wild-type and mutant cells. For each inhibitor tested, growth of mutant and wild-type strains was equally sensitive. However, in contrast to the wild type, the Hupc mutants synthesized hydrogenase in the presence of high levels of any inhibitor. Cells were incubated with the drugs and simultaneously labeled with 14C-labeled amino acids, and hydrogenase was immunoprecipitated with antibody to the large subunit of the enzyme. Fluorograms of antibody blots then were scanned to determine the relative amount of hydrogenase (large subunit) synthesized in the presence or absence of the gyrase inhibitors. The amount of hydrogenase synthesized by the Hupc mutants in the presence of 300 micrograms of nalidixic acid per ml was near the level of enzyme synthesized in the absence of the inhibitor. No hydrogenase was detected in antibody blots of wild-type cultures which were derepressed for hydrogenase in the presence of 100 micrograms of coumermycin or novobiocin per ml. In contrast, hydrogenase was synthesized by the Hupc mutants in the presence of 100 micrograms of either drug per ml. The amount synthesized ranged from 5 to 32% and 20 to 49%, respectively, of that in the absence of those inhibitors, but nevertheless, hydrogenase synthesis was detected in all of the mutants examined.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

17.
A colony screening procedure was devised to detect Bacillus subtilis mutants containing temperature-sensitive trypsin-like intracellular protease activity. The enzyme was characterized as a non-sulfhydryl serine protease on the basis of inhibitor studies. It was also inhibited by D- or L-histidine but not by any other amino acid tested. The long-term survival at 45 degrees C of these mutants in a minimal salts medium was decreased, with rapid lysis occurring within 24 h. A D-histidine function in long-term survival and inhibition accounted for the presence of additional protease mutants among survivors of histidine auxotrophs selected for their ability to utilize D-histidine. In addition to being lysed when incubated at 45 degrees C under nongrowth conditions, all of the protease mutants had a decreased rate of protein turnover and produced spores deficient in a major low-molecular-weight spore coat polypeptide. The morphology of the undercoat layers was altered, but there was no effect on spore heat resistance or on germination. The missing spore coat polypeptide appeared to be processed from a larger precursor by cleavage to produce N-terminal histidine. A defect in this protease could account for the lack of processing and thus the absence of this polypeptide in spore coats.  相似文献   

18.
Histidine production by a regulatory mutant of Streptomyces coelicolor   总被引:1,自引:0,他引:1  
Streptomyces coelicolor mutant RF-59, isolated as a revertant of a histidine auxotroph after mutagenic treatment with N-methylN'-nitro-N-nitrosoguanidine, was found to accumulate L-histidine. The mutant was sensitive to 2-thiazo-lealanine and L-2,4-diaminobutyric acid and partially sensitive to alpha-methylhistidine but resistant to 1,2,4-triazolealanine, indicating that repression of the histidine operon was modified in the mutant. Culture conditions were investigated, and optimal media for L-histidine production were developed, resulting in L-histidine accumulation of 2.1 to 3.5 g/liter.  相似文献   

19.
Streptomyces coelicolor mutant RF-59, isolated as a revertant of a histidine auxotroph after mutagenic treatment with N-methylN'-nitro-N-nitrosoguanidine, was found to accumulate L-histidine. The mutant was sensitive to 2-thiazo-lealanine and L-2,4-diaminobutyric acid and partially sensitive to alpha-methylhistidine but resistant to 1,2,4-triazolealanine, indicating that repression of the histidine operon was modified in the mutant. Culture conditions were investigated, and optimal media for L-histidine production were developed, resulting in L-histidine accumulation of 2.1 to 3.5 g/liter.  相似文献   

20.
M R Atkinson  L V Wray  Jr    S H Fisher 《Journal of bacteriology》1993,175(14):4282-4289
During growth of Bacillus subtilis in nutrient sporulation medium containing histidine (DSM-His medium), the expression of histidase, the first enzyme in the histidine-degradative pathway (hut), is derepressed 40- to 200-fold at the onset of stationary phase. To identify the gene products responsible for this regulation, histidase expression was examined in various hut regulatory mutants as well as in mutants defective in stationary-phase gene regulation. Histidase expression during growth in DSM-His medium was significantly altered only in a strain containing the hutC1 mutation. The hutC1 mutation allows the hut operon to be expressed in the absence of its inducer, histidine. During logarithmic growth in DSM-His medium, histidase levels were 25-fold higher in the HutC mutant than in wild-type cells. Moreover, histidase expression in the HutC mutant increased only four- to eightfold after the end of exponential growth in DSM-His medium. This suggests that histidine transport is reduced in wild-type cells during exponential growth in DSM-His medium and that this reduction is largely responsible for the repression of hut expression in cells growing logarithmically in this medium. Indeed, the rate of histidine uptake in DSM-His medium was fourfold lower in exponentially growing cells than in stationary-phase cells. The observation that the degradation of histidine is inhibited when B. subtilis is growing rapidly in medium containing a mixture of amino acids suggests that a hierarchy of amino acid utilization may be present in this bacterium.  相似文献   

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