Studies on the metabolic role of peptidyl-tRNA hydrolase

Summary A mutant strain of Eschrichia coli that is temperature-sensitive for growth stopped protein biosynthesis at 43° C after a brief lag (Fig. 1). Cell-free extracts from the strain showed no specific defect in aminoacyl-tRNA synthetases, binding initiator tRNA to ribosomes (Table 1), protein chain elongation (Tables 2, 5) or protein chain termination (Tables 3, 4) at high temperature.The partially purified enzyme peptidyl-tRNA hydrolase, however, was temperature-sensitive (Table 6); the mutant hydrolase was inactivated rapidly at 43° C (Table 7). Mixing experiments ruled out the presence, in the mutant enzyme preparation, of an inhibitor and also demonstrated, on the mutant enzyme, a protective effect by wild type enzyme that was not shown by general coli proteins (Tables 8, 9).Interrupted mating allowed the temperature-sensitive growth phenotype to be mapped near to and before trp (Figs. 4, 5). Co-transsduction, mediated by bacteriophage P1, with trp ⁺ (frequency 7.5%) located the marker at 24 min on the coli map. All transductants for temperature-sensitive growth also had temperature-sensitive peptidyl-tRNA hydrolase activity in crude sonicates (Table 10). We provisionally conclude that the temperature-sensitive protein synthesis and growth are caused by a single genetic change in the structural gene (pth) for peptidyl-tRNA hydrolase.After shift to 43° C the polysomes of the mutant cells broke down into 70S particles (Figs. 2, 3). A defect in protein biosynthesis thus appeared to be located after termination and before reformation of new polysomes.The metabolic role of peptidyl-tRNA hydrolase is discussed in the light of these experiments.Journal paper No. J-7465 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, project no. 1747.     

S-adenosylmethionine synthetase in methionine regulatory mutants of Salmonella typhimurium

Summary In wild-type bacteria, S-adenosylmethionine (SAM) synthetase activity was repressed by growth in methionine. MetJ regulatory mutants had elevated activities which did not show this repression. Two metK mutants with normal regulation of the methionine biosynthetic enzymes had elevated Km's (methionine) for SAM synthetase while five metK mutants with constitutive methionine enzymes showed no measurable SAM synthetase activity. One mutant, metK ^X 721, similar in phenotype to these five had a wild-type level of SAM synthetase in conditions where SAM decarboxylase activity was blocked. By using an F-factor carrying the metK region of the genome, this mutant was shown to complement six other metK mutants.These results indicate that SAM or a derivative of it, rather than methionine itself, is the co-repressor of the methionine system, regulatory abnormalities resulting from the absence or reduction of the amount of SAM formed by the SAM synthetase reaction. As SAM is essential for bacteria it is likely that there is some alternative biosynthetic route for SAM.     

Mechanism of gene conversion in Ascobolus immersus. II. The relationships between the genetic alterations in b 1 or b 2 mutants and their conversion spectrum

Summary In order to establish a relationship between the type of genetic alteration occurring in the mutant and the conversion spectrum which is associated with it, an attempt was made to characterize the genetic alterations in a sample of five spore colour mutants by specific reversion.All the mutants revert by back-mutation. Reversion of one of them is possible by mutation in external suppressor gene: at least two of the external suppressors behave like super-suppressors. Reversion of two other mutants by intragenic second-site mutation and study of the intragenic suppressors indicate that they are of the frameshift type. One of the frameshift mutants (ICR₁₇₀ induced) reverts by two alkylating agents suggesting that it originates from base(s) addition. The inhability of ICR₁₇₀ to induce reversion of this mutant suggests the preferential occurrence of base addition in ICR₁₇₀ mutagenesis. Conversly the other frameshift mutant (induced by ethyl methanesulfonate) reverts strongly by ICR₁₇₀. Thus it is concluded that it originates from deletion.These two frameshift mutants and all the ICR₁₇₀ induced mutants give no or very few asci with postmeiotic segregation and either an excess of conversion towards the mutant type allele (addition type mutant) or towards the wild type allele (deletion type mutant).Evidence supports the hypothesis that mutants which give many asci with postmeiotic segregation originate from substitution.These data imply differential recognition of non-pairing and mispairing of bases and in the case of non-pairing, that the direction of conversion is determined by the non-pairing itself.     

Single gene alteration of plasma and mitochondrial membrane function in Saccharomyces cerevisiae.

Summary Some physiological properties of a multiple-drug-resistant mutant with a permeability barrier to chloramphenicol and its isogenic parental strain were compared. The ATPase specific activity of plasma and mitochondrial membranes isolated from the mutant strain was approximately 20% lower (P(0.001, Tables 1 and 2) than that of membranes isolated from the isogenic parental strain. Additional evidence of altered mitochondrial function was: (i) the enhanced growth of the parental strain was eliminated by the [rho-] state (Table 3); (ii) the mutant strain had a greater resistance to petite induction by ethidium bromide (Table 4); (iii) the mutant strain was unable to use a nonfermentable energy source for respiratory adaptation (Table 5). It is proposed that a single gene mutation has resulted in an alteration of some physiological properties of the plasma and mitochondrial membranes.     

Mechanism of repression of methionine biosynthesis in Escherichia coli. II. The effect of metJ mutations on the free amino acid pool

Summary Ethionine-resistant mutants (metJ mutants) were isolated and characterized as constitutive in the biosynthesis of methionine. Such mutations resulted in marked differences or alterations in the free amino acid pool. In some strains the levels of threonine and histidine were elevated by as much as 13 and 22 times that of the wild type level. The possibility that structural modifications of methionyl-tRNA were giving rise to constitutive methionine biosynthesis and the apparent aberrations in the free amino acid pool, was in large part ruled out by a comparison of the mobilities of wild type and mutant methionyl-tRNA on benzoylated DEAE-cellulose columns. The results obtained are consistent with the view that the product of the metJ locus is a repressor protein which is directly involved in the repression of the methionine genes.     

Effects of Regulatory Mutations upon Methionine Biosynthesis in Saccharomyces cerevisiae: Loci eth2-eth3-eth10

The effects of mutations occurring at three independent loci, eth2, eth3, and eth10, were studied on the basis of several criteria: level of resistance towards two methionine analogues (ethionine and selenomethionine), pool sizes of free methionine and S-adenosyl methionine (SAM) under different growth conditions, and susceptibility towards methionine-mediated repression and SAM-mediated repression of some enzymes involved in methionine biosynthesis (met group I enzymes). It was shown that: (i) the level of resistance towards both methionine analogues roughly correlates with the amount of methionine accumulated in the pool; (ii) the repressibility of met group I enzymes by exogenous methionine is either abolished or greatly lowered, depending upon the mutation studied; (iii) the repressibility of the same enzymes by exogenous SAM remains, in at least three mutants studied, close to that observed in a wild-type strain; (iv) the accumulation of SAM does not occur in the most extreme mutants either from endogenously overproduced or from exogenously supplied methionine: (v) the two methionine-activating enzymes, methionyl-transfer ribonucleic acid (tRNA) synthetase and methionine adenosyl transferase, do not seem modified in any of the mutants presented here; and (vi) the amount of tRNA^met and its level of charging are alike in all strains. Thus, the three recessive mutations presented here affect methionine-mediated repression, both at the level of overall methionine biosynthesis which results in its accumulation in the pool, and at the level of the synthesis of met group I enzymes. The implications of these findings are discussed.     

Bradyrhizobium japonicum mutants defective in root-nodule bacteroid development and nitrogen fixation

The genome of the slow-growing Bradyrhizobium japonicum (strain 110) was mutagenized with transposon Tn5. A total of 1623 kanamycin/streptomycin resistant derivatives were screened in soybean infection tests for nodulation (Nod) and symbiotic nitrogen fixation (Fix). In this report we describe 14 strains possessing a stable, reproducible Nod⁺Fix^- phenotype. These strains were also grown under microaerobic culture conditions to test them for free-living nitrogen fixation activity (Nif). In addition to strains having reduced Fix and Nif activities, there were also strains that had reduced symbiotic Fix activity but were Nif⁺ ex planta.Analysis of the genomic structure revealed that the majority of the strains had a single Tn5 insertion without any further apparent physical alteration. A few strains had additional insertions (by Tn5 or IS50), or a deletion, or had cointegrated part of the vector used for Tn5 mutagenesis. One of the insertions was found in a known nif gene (nifD) whereas all other mutations seem to affect different, hitherto unknown genes or operons.Several mutant strains had an altered nodulation phenotype, inducing numerous, small, widely distributed nodules. Light and electron microscopy revealed that most of these mutants were defective in different stages of bacteroid development and/or bacteroid persistence. The protein patterns of the mutants were inspected by two-dimensional gel electrophoresis after labelling microaerobic cultures with l-(³⁵S)methionine. Of particular interest were mutants lacking a group of proteins the synthesis of which was known to be under oxygen control. Such strains can be regarded as potential regulatory mutants.     

长双歧杆菌N-乙酰氨基己糖1-位激酶的活性位点

【目的】研究长双歧杆菌(Bifidobacterium longum)JCM1217的N-乙酰氨基己糖1-位激酶(Nacetylhexosamine 1-kinase,Nah K)中对催化活性有影响的位点。【方法】利用点突变试剂盒,获得Nah K的4个位点的共10种单点突变体表达菌株。诱导表达并纯化野生型和突变体酶,用DNS法和NADH偶联的微孔板分光光度法检测野生型及突变体酶的最适p H和最适Mg~(2+)浓度,并测定酶促反应动力学参数。【结果】D208A、D208N、D208E和I24A四种突变体的催化活性几乎丧失。突变体H31A、H31V、F247A和I24V的最适p H由野生型的7.5变为7.0,突变体H31A和F247A的最适Mg~(2+)浓度由野生型的5 mmol/L变为10 mmol/L。反应动力学参数测定结果表明,突变体F247Y对底物Glc NAc/Gal NAc及ATP的催化活性均高于野生型。【结论】通过定点突变,确定了对Nah K催化活性有影响的4个位点,并且获得了一个催化效率提高的突变体(F247Y),为进一步对Nah K进行分子改造奠定了一定基础。     

Effect of septum-initiation mutations on sporulation and competent cell formation in Bacillus subtilis

Summary Sporulation and competent cell formation have been studied in four Bacillus subtilis strains, carrying septum-initiation mutations of different loci, div-31, div-341, div-12 and div-355 which exhibit filamentous growth at 45° C. The div-31 mutant was found to be defective in competence development at 30°–40°C whereas the div-12 mutant was affected only slightly. The div-341 and div-355 mutants showed lower competence, particularly at the higher temperatures. The four div mutant strains all showed poor sporulation at higher temperatures compared to the wild-type strain. We propose that some of the initial steps of septation are involved both in sporulation (possibly in forespore septum formation) and in competent cell formation and that these two processes share certain common features distinct from those in vegetative cell division.     

Characterization of the Staphylococcus aureus chromosomal gene pcrA,identified by mutations affecting plasmid pT181 replication

Mutations in four genes: sconA (formerly suA25meth, mapA25), sconB (formerly mapBl), sconC and sconD, the last two identified in this work, relieve a group of sulphur amino acid biosynthetic enzymes from methionine-mediated sulphur metabolite repression. Exogenous methionine has no effect on sulphate assimilation in the mutant strains, whereas in the wild type it causes almost complete elimination of sulphate incorporation. In both mutant and wild-type strains methionine is efficiently taken up and metabolized to S-adenosylmethionine, homocysteine and other compounds. scon mutants also show elevated levels of folate-metabolizing enzymes which results from the large pool of homocysteine found in these strains. The folate enzymes apear to be inducible by homocysteine and repressible by methionine (or Sadenosylmethionine).     

Relative Heterothallism and Production of Hybrid Perithecia by Auxotrophic Mutants of Glomerella cingulata from Apple

Glomerella cmgulata is a homothallic species but produces a ridge of fertile perithecia at a frontier between certain wild-type strains on agar. To account for the presence or absence of perithecia earlier workers suggested that alleles at A and B loci control the formation of perithecia at mycelial frontiers in + and – strains. We propose that G. cingulata actually demonstrates “relative heterothallism”. Of 7 induced nutritionally deficient mutants (auxotrophs) in 2 wild-type strains from apple, only one methionine (met-1) and one arginine (arg) mutant in only one wild-type strain gave a heavy ridge of perithecia at their junctures. Neither the met-l nor arg mutations have been identified as those in the A or B locus. The perithecia were either homozygous (selfs) for met-1 or arg, or heterozygous (hybrids). Paired met-1 and arg segregants from hybrid perirhecia as well as diauxotrophic strains from met-l or arg mutants also gave hybrids of selfs. Specific nutritional deficiencies in certain wild-type strains which can direct sexuality are not yet known. Genetic studies are now feasible in G. cingulata to define enzymatic factors responsible for pathogenicity.     

A new gene (madI) involved in the phototropic response of Phycomyces

Summary Only eight genes are known to be involved in the phototropic response of Phycomyces (madA-H). Mutants affected in these genes have played a major role in the analysis of photosensory transduction processes in this system. A set of new mutants isolated by Alvarez et al. (1989) that are unable to bend towards dim unilateral blue light were studied by complementation and recombination. Two of these mutants have mutations in madE, one has a mutation in madF and one is a double madE madF mutant. The three remaining mutants tested did not complement each other and showed positive complementation with strains carrying mutations in the genes madA, madB, and madC, indicating that they carried mutations in a new gene designated madI. Recombination analysis showed that madI is unlinked to madA, madB and madC.     

Analysis of pathogenicity mutants of a bacteriocin producing Xanthomonas perforans

Since the initial discovery of Xanthomonas perforans on tomato in 1991, it has completely displaced Xanthomonas euvesicatoria as the bacterial spot of tomato pathogen in Florida. Previous research has shown that X. perforans produces at least three different bacteriocin-like compounds (BcnA, BcnB, BcnC) antagonistic toward X. euvesicatoria strains. In this study pathogenicity-attenuated, bacteriocin-producing mutants of X. perforans were created to determine their potential as biological control agents for control of X. euvesicatoria. Several candidate genes were chosen based on previous studies in which mutant phenotypes exhibited reduced virulence in either X. perforans (OpgH_Xcv) or the closely related X. euvesicatoria strain 85-10 (hpaB, hpaC, xopA, xopD, avrBs2 and gumD). Each candidate gene in X. perforans was amplified and PCR-assisted deletion mutagenesis was performed in the wild-type (wt) X. perforans strain to create potential attenuation mutants. Each mutant was tested for growth rate, disease severity and antagonism toward X. euvesicatoria strains. Three mutants, XopA, opgH, and gumD were significantly less pathogenic than the wild-type strain with the opgH mutant reaching significantly lower internal populations than all other mutants except hpaC. The opgH-strain was the most affected in its ability to grow internally in plant tissue while inhibiting X. euvesicatoria populations equal to or more than the other mutant strains. This mutant strain could potentially be used as part of an effective biological control strategy.     

Adaptation to phenylacetamide as a growth substrate by an acetanilide-utilizing mutant of Pseudomonas aeruginosa

Mutants able to utilize phenylacetamide as sole nitrogen source were isolated from the acetanilide (N-phenylacetamide)-utilizing Pseudomonas aeruginosa mutant strain AI3 and from its parent strain L10. Growth properties of the mutants (Ph strains) on amide media and the physicochemical properties of their amidases in cell free extracts indicated that their phenylacetamidase activities were attributable to alterations in their amidases. Differences in amide hydrolase specificities between the AI3-and the L10-Ph mutants were observed. The AI3 group had a high level of activity towards 4-nitrophenylacetamide, activity towards phenylacetyl-4-nitroaniline but, unlike strain AI3, no activity towards acetyl-4-nitroaniline; the L10 group had a low activity towards 4-nitrophenylacetamide, no activity towards phenylacetyl-4-nitroaniline but retained the low level of activity towards acetyl-4-nitroaniline exhibited by strain L10. Confirmation of the association between these altered specificition of alterations in amidases was obtained from analysis of the properties of phenylacetamidases purified from an AI3-Ph mutant (pH5) and an L 10-Ph mutant (Ph14). The original mutation in the amidase gene of strain AI3 appeared responsible for the differences between the two groups of Ph mutants and the binding interactions with acetanilide that it determined were eliminated in AI3-Ph mutants.     

Allelspezifische suppressormutationen vonSchizosaccharomyces pombe

By comparing the intragenic distribution of suppressor sensitive mutants in fine structure maps, 13 allele specific suppressor mutations (isolated from revertants in adenine dependent mutants of constitutionad ₇) have been analyzed for their allele specific patterns of action in three different groups of mutants blocked in adenine biosynthesis. The 13 suppressor mutations, which have resulted from mutations at seven different suppressor loci, are characterized by four different suppression patterns. Three of these patterns, which partially overlap, are not locus specific since they include sensitive mutants at each of the three lociad ₇, ad₆ andad ₁ studied. The relative frequency of mutants sensitive to one or the other of the suppressors of this type, the absence of osmotic-remedial strains among the suppressor sensitive mutants, and the polarized complementation behaviour of one suppressiblead ₆ mutant and two suppressiblead ₁ mutants capable of interallelic complementation, suggest that the suppression mechanism involves misreading of a mutant triplet of the nonsense type.     

Isolation of l-methionine-enriched mutant of a methylotrophic yeast,Candida biodinii No. 2201

Six strains of methylotrophic yeast were examined for production of l-methionine-enriched cells. Candida biodinii (Kloeckera sp.) No. 2201, which accumulated 0.54 mg/g-dry cell weight (DCW) of free l-methionine (pool methionine), was selected as the parental strain for breeding l-methionine-rich mutants. Ethionine-resistant mutants were derived from the strain by UV irradation. A mutant strain, E500-78, which was resistant to 500 μg/ml of dl-ethionine, accumated 6.02 mg/g-DCW of pool methionine. The culture conditions for mutant strain E500-78 to increase pool methionine accumulation were o-ptimized. As a result, the mutant strain accumulated 8.80 mg/g-DCW of pool methionine and contained 16.02 mg/g-DCW total methionine.     

Dihydrofolate reductase and aminopterin resistance in Pneumococcus

Summary Wild-type pneumococci derived from Avery's strain R36A are sensitive to extracellular concentrations of the folate antimetabolite aminopterin exceeding 1.0x10^-6 M. Three classes of resistant strains are phenotypically distinguishable: amiB-r, amiA-r and amiD-r strains are resistant to low (1.5x10^-6 M), intermediate (0.5–4.0×10^-5 M) and high (4.5x10^-4 M) aminopterin levels respectively. The amiA and amiB regions are weakly linked, but linkage has not been established between either of these loci and the amiD region.Consistent with the maximum resistance conferred by mutations in the amiA locus, dihydrofolate (FH₂) reductase in cell-free extracts (CFE) of amiA-r strains has a two- to six-fold greater affinity for the substrate than dose the enzyme in wild-type CFE (Table 1); FH₂ reductase from amiA-r strains may also have reduced affinity for aminopterin. Specific activity of the enzyme is not affected by mutation in the amiA locus (Table 1) and its affinity for the cofactor (NADPH) is probably unaffected by mutation in this locus (Table 4). Dihydrofolate reductase activity in amiA5 CFE is considerably more thermolabile than that in wild-type CFE (Table 2).The enzyme in CFE of the high resistance strain amiD1 has the same affinity for the substrate, cofactor and antimetabolite as FH₂ reductase in wild-type CFE (Figs. 1–4, 8 and 9; Table 4). However, specific activity of the enzyme in amiD1 CFE is 11-fold higher than that in wild-type CFE (Table 1) and it is much more heat stable (Table 2).Some properties of FH₂ reductase in CFE of the high resistance recombinant strain amiA5amiD1 are intermediate between those in CFE of wild-type and amiD1.Preliminary results suggest that CFE of wild-type and amiA5 contain a factor, which is neither dialyzable nor heat sensitive, that has an inhibitory effect upon activity and stability of FH₂ reductase in amiD1 CFE (Tables 2 and 3).     

The effect of tra mutations on the synthesis of the F-pilin membrane polypeptide

Summary We had previously demonstrated that several F specific polypeptide bands could be detected in the membranes of Flac, but not F^- strains of Escherichia coli K 12, (Moore et al. 1981). One of these polypeptides co-migrated with F-pilin protein on polyacrylamide gels. We have now analyzed ³⁵[S]methionine labelled membrane preparations from a series of strains containing Flac tra mutant plasmids. The F-pilin polypeptide was absent from preparations of strains containing all traA mutants tested, confirming the importance of the traA gene in F-pilin biosynthesis. A polypeptide which migrated in the F-pilin position was still present, however, in membranes prepared from Flac strains carrying mutations in traL, traE, traK, traB, traV, traW, traC, traU, traF, traH or traG despite the inability of these mutants to elaborate F-pili filaments. Thus, all of these gene products may be concerned with F-pilus assembly and outgrowth rather than biosynthesis of the F-pilin subunit. The polar mutation tra-4 did, however, prevent the appearance of pilin polypeptide, indicating that at least one unidentified gene in the region between traE and traG must also be required in F-pilin biosynthesis.Our analysis also permitted the identification of a 100,000 dalton membrane protein as the product of traG. The appearance of an F specific 12,000 dalton protein was prevented by traD amber mutants. As expected, traJ mutants prevented the expression of all the tra operon products detected except the product of traT. The traT product band was reduced only to 50–60% of its normal intensity.     

Cooperative control of translational fidelity by ribosomal proteins in Escherichia coli

Summary Two spontaneous mutants of Escherichia coli strain KMBL-146 selected for resistance to the aminoglycoside antibiotic neamine show severe restriction of amber suppressors in vivo. Purified ribosomes from the mutant strains exhibit low neamine-induced misreading in vitro and a decreased affinity for the related antibiotic streptomycin.Biochemical analysis shows that the mutants each have two modified 30S ribosomal proteins, S12 and S5. In agreement with these results, genetic analysis shows that two mutations are present, neither of which confers resistance to neamine by itself; the mutation located in gene rpxL (the structural gene for protein S12) confers streptomycin dependence but this dependence is suppressed in the presence of the second mutation, located in gene rpxE (the structural gene for protein S5).     

Synergistic effect ofrib80 andhit Mutations on riboflavin biosynthesis and iron transport in the yeastPichia guilliermondii

Mutant strains of the yeastPichia guilliermondii, carrying bothrib80 andhit mutations in a haploid genome, were derived from previously obtained strains with defectiverib80 orhit genes, exerting negative control of the riboflavin biosynthesis and iron transport inPichia guilliermondii. The double mutant rib80hit strains exhibited an increased level of riboflavin biosynthesis and higher activities of GTP cyclohydrolase and riboflavin synthetase. Iron deficiency caused an additional increase in riboflavin overproduction. These results suggest the synergistic interaction of therib80 andhit mutations. A combination of both mutations in a single genome did not affect iron assimilation by the cells: ferrireductase activity, the rate of⁵⁵Fe uptake, and the iron content in cells of the double mutants remained at the level characteristic of the parent strains.