Deamplification and deletion of amplified DNA in Streptomyces lividans and S. fradiae

Summary Streptomyces lividans arginine auxotrophs which show amplification of a 5.7-kb DNA sequence, arose at a very high frequency, varying from 10% to 25% of Cml^s spores. The amplifiable DNA sequence was shown to be stable over many generations. However, treatment of Cml^s arg mutants with subinhibitory concentrations of antibiotics such as spectinomycin, streptomycin, chloramphenicol, thiostrepton and kanamycin, either during sporulation or during vegetative growth of mycelia, led to the deletion of the entire amplified DNA sequence, including the left and right junction sequences. Depending upon the method of antibiotic treatment a reduction in the copy number of the amplified DNA was also observed. This reduction in copy number apparently occurred without drastically affecting the basic structure of the amplifiable unit of DNA. This phenomenon appears to be universal since deamplification and deletion were observed also in S. fradiae. Further, spontaneous arg mutants arose at much lower frequency from spectinomycin-pretreated Cml^s cells compared to untreated cells. These arg mutants isolated in the presence of spectinomycin did not show amplification of the 5.7-kb sequence. Southern blot analysis using the 5.7-kb probe showed that the entire DNA sequence homologous to the amplifiable DNA sequence had been deleted. Offprint requests to: K. Dharmalingam     

Analysis of a genetically unstable region inStreptomyces lividans 66-TK64

Genetic and molecular analyses of an unstable region encompassing the gene loci cml arg and a 5.7 kb amplifiable unit of DNA were done. Spontaneous mutants from Cm1^R →Cml^S and the revertants from Cml^S →Cml^R were analysed for mutations at arg locus and amplification of amplifiable unit of DNA. Twenty-one revertants were analysed. Two of these had large-scale amplification and one of these was also Arg^-. Nine of the revertants which were Arg⁺ had low-level or intermediate-level amplification of the 5.7 kb DNA sequence but no deletions of the flanking sequences were detected. Five of the CmI^R’ revertants, which were also Arg⁺, had lost one of the two copies from the doublet of amplifiable unit of DNA. The remaining five revertants did not show any other change. The amplifiable unit of DNA, therefore, not only undergoes amplification but can also suffer specific deletion of one copy. Thus, this region as a whole is characterized by instability and the events appear to take place at more than one locus concomitantly with a high frequency.     

Structure of an amplifiable DNA sequence in Streptomyces lividans 66

Summary Spontaneous chloramphenicol-sensitive mutants of Streptomyces lividans 66 had previously been shown to be very unstable and to yield arginine auxotrophic mutants at a frequency of 25% of spores; the Arg^- mutants had amplified a particular 5.7 kb DNA sequence to over one hundred tandem copies per genome. In this paper we report the cloning of the amplifiable region from amplified and wild-type strains. This showed that the amplifiable fragment is already present as a duplication in wild type cells. Hybridisation experiments also demonstrated that in the amplified strains there was a deletion of neighbouring DNA sequences to one side of the amplifiable element; sequences to the other side remain intact.     

DNA amplification affects protease production and sporulation inStreptomyces fradiae

Chloramphenicol resistance is an unstable character inStreptomyces fradiae, since spontaneous chloramphenicol-sensitive (Cml^s) mutants arose at very high frequencies. One such Cml^s mutant, DM14, showed DNA amplification as well. Extracellular protease activity was tenfold higher in DM14 when compared with its wild-type parent. Protease activity decreased considerably in DM14 when treated with spectinomycin, a treatment that reduces the copy number of amplified units of DNA. Sporulation in DM14 was delayed in the presence of spectinomycin at a concentration of 5 g/ml, whereas the wild type was unaffected at that concentration. The results strongly indicated that the amplified DNA affected the two secondary metabolic functions, viz., protease production and the onset of sporulation in the mutant.     

Properties of transposon SCTn1 of Streptomyces coelicolor A3(2)

Summary Chloramphenicol resistance (Cml^r) of Streptomyces coelicolor A3(2) behaves like a transposon locus, not being localisable in any region of the map and yet being transferable in crosses at a rate comparable to that of chromosomal markers. It can, also be transposed onto a plasmid (SCP1) and back to the chromosome. Some traits, such as arginino-succinate synthase production (ArgG), aerial mycelium formation (AmyA), resistance to tetracycline and to rifamycin C appear to be joined to Cml in three processes: co-mutation, i.e. simultaneous loss, post-mutation, i.e. spontaneous loss at high, frequency in subclones from Cml^s strains, co-transfer, i.e. joint transfer with the cml locus in crosses or during infection by the aggregate SCP1::SCTn1 plasmid. All these processes have been consistently observed with special attention to the argG locus.     

Mutagenic DNA repair in Escherichia coli

Summary Escherichia coli K12 Hfr H Tsx^s Str^s and F^- Pro^- Tsx^r His^- Arg^- Str^r bacteria were conjugated in the absence of arginine with or without glucose. The efficiency of conjugation, measured by the frequency of Pro⁺ and His⁺ recombinants was not affected. Arginine starvation alone did not affect the tsx ^s gene expression which occurred in all the zygotes which had received the gene. In contrast, argine and glucose starvation allows tsx ^s expression only in those zygotes in which the donor gene had been integrated in the genome. As the glucose starvation brings on a destabilization of the messenger RNA synthesized by the F^- cells in absence of arginine, the results can be interpreted as follows: the transferred tsx ^s genes are transitorily expressed in all the zygotes at the unintegrated state. After this transient period, only those genes integrated in the chromosomes of the zygotes continue to be expressed.     

Modulation of Serine/Threonine Protein Kinase Activity in Chloramphenicol-Resistant Mutants of Streptomyces avermitilis

A mutation to chloramphenicol resistance (Cml^r) stimulates production of macrolide avermectin in Streptomyces avermitilis; production starts in the early stationary phase. By labeling in vivo, the Cml^r mutation was shown to stimulate phosphorylation of Ser and Thr in several proteins in the same growth phase. Autophosphorylation of active protein kinases (PK) was analyzed in gel after one- or two-dimensional PAGE for the original S. avermitilis strain ATCC 31272, its Cml^r mutant, and a Cml^s revertant. An increase in in vivo phosphorylation was associated with an increase in autophosphorylation of Ser/Thr-PK 41K, 45K, 52K, 62K, and 85K and complete suppression of autophosphorylation of PK 66K. Comparison of the PK molecular weights and pI with the parameters deduced for putative PK encoded by S. avermitilis genes identified the 41K, 45K, 52K, 62K, and 85K proteins as pkn 24, pkn 32, pkn 13, pkn12, and pkn5, respectively. Prenylamine lactate, a modulator of calmodulin-dependent processes, substantially reduced the avermectin production, impaired the Cml resistance, and selectively inhibited Ca²⁺-dependent PK 85K in the Cml^r mutant. It was assumed that PK 85K is involved in regulating the avermectin production.     

Genes required for both gliding motility and development in Myxococcus xanthus

Myxococous xanthus cells can glide both as individual cells, dependent on A dventurous motility (A motility), and as groups of cells, dependent upon S ocial motility (S motility), Tn5-lac mutagenesis was used to generate 16 new A^- and nine new S^- mutations. In contrast with previous results, we find that subsets of A^- mutants are defective in fruiting body morphogenesis and/or myxospore differentiation. All S^- mutants are defective in fruiting body morphogenesis, consistent with previous results. Whereas some S^- mutants produce a wild-type complement of spores, others are defective in the differentiation of myxospores. Therefore, a subset of the A genes and all of the S genes are critical for fruiting body morphogenesis. Subsets of both A and S genes are essential for sporulation. Three S::Tn5–lac insertions result in surprising phenotypes. Colonies of two S^- mutants glide on ‘swim’ (0.35% agar) plates to form fractal patterns. These S^- mutants are the first examples of a bacterium in which mutations result in fractal patterns of colonial spreading. An otherwise wild-type strain with one S^- insertion resembles the frz^- sglA1^- mutants upon development, suggesting that this S^- gene defines a new chemotaxis component in M. xanthus.     

A jumping gene in Streptomyces coelicolor A3(2)

Summary The difficulty in mapping the gene for chloramphenicol resistance (cml ^R) in Streptomyces coelicolor A3(2) stock strains is possibly due to its location on different sites of the chromosome in various mixed subelones. Fresh isolates from Cml^R strains show single unequivocal locations of cml ^R. The same holds for Cml^R strains derived as revertants from Cml^S variants. The two best established sites for cml ^R are one between cys A and met A, the other at right of arg A, possibly in the right empty arc of the map (Fig. 2). The cml ^R gene was assumed to be on a transposon (SCTn1), together with a gene for arginine-succinate synthase (argG), a gene for chromosome transfer (tra) and a gene for aereal mycelium formation (amy). In a Cml^R revertant, the cml ^R gene appears disjoined from argG (Fig. 5), thus showing the ability of SCTnl to be split and partially transposed. The possible wide occurrence of transposons in the genus Streptomyces is discussed.     

Interaction of super-repressible and dominant constitutive mutations for the synthesis of galactose pathway enzymes in Saccharomyces cerevisiae

Summary Two dominant uninducible mutant alleles in the gal80 locus were identified. The GAL80 ^s-1 and GAL80 ^s-2 mutants showed novel phenotypes in response to the newly isolated GAL81-1 mutant allele, a dominant constitutive mutation linked to the gal4 locus; the GAL80 ^s-1 GAL81-1 strain was inducible and the GAL80 ^s-2 GAL81-1 strain was uninducible. Many galactose positive revertants from the GAL80 ^s-2 GAL81-1 strain were isolated. It was proved that each revertant was due to a secondary mutation either in the gal80 or GAL81 locus, whereas revertants due to mutation at the supposed controlling site for the structural gene cluster of the galactose-pathway enzymes have not been isolated.This study was supported in part by grant no. 048164 to Y. Oshima from the Scientific Research Fund of the Ministry of Eduction, Japan     

Mutant regulatory subunit of 3'',5''-cAMP-dependent protein kinase of yeast Saccharomyces cerevisiae

Summary Four mutants with amino acid substitution(s) at or near the putative phosphorylation site (Arg¹⁴² Arg¹⁴³ Thr¹⁴⁴ Ser¹⁴⁵) of the regulatory subunit of cAMP-dependent protein kinase were obtained by site-directed mutagenesis. Three mutants, BCY1 ^{Ala 145} (Ser¹⁴⁵ to Ala), BCY1 ^{His 143} (Arg¹⁴³ to His) and BCY1 ^{Asn 144, Ala 145} (Thr¹⁴⁴ to Asn and Ser¹⁴⁵ to Ala) complemented a bcy1 mutant, whereas BCY1 ^{Gly 143} (Arg¹⁴³ to Gly) did not. In addition, mutant, BCY1 ^{Asn 144, Ala 145} exhibited a dominant coldsensitive phenotype, which can be most easily explained by the functional alteration of the regulatory subunit of cAMP-dependent protein kinase by the mutations. Analyses of these mutant genes revealed that phosphorylation of the regulatory subunit is not a prerequisite for the regulation of the cAMP-dependent protein kinase activity in responding to the cAMP level.     

Sporulation of mitochondrial respiratory deficient mit- mutants of Saccharomyces cerevisiae.

Summary The role of mitochondrial protein synthesis, electron transport, and four specific mitochondrial gene products on sporulation were studied in respiratory deficient mit ^- mutants. These mutants were isolated in an op1 strain and localized on the mitochondrial genome by petite deletion mapping. All 153 mutations studied could be assigned to the four mitochondrial regions OXI1, OXI2, OXI3 and COB, known to affect cytochrome c oxidase and cytochrome b. The specific loss of one mitochondrially translated polypeptide was found in some mutants of each locus: OXI1—cytochrome c oxidase subunit 2, OXI2 — subunit 3, OXI3 — subunit 1, and COB — cytochrome b.The ability of diploid mit ^- mutants to sporulate was systematically investigated. About one third of the mutants, representing three loci, were incapable of forming spores. All other cultures produced either respiratory competent mit ⁺ tetrads, both mit ⁺ and mit ^- tetrads, or only mit ^- tetrads. Mutants forming mit ^- tetrads mapped in all four loci. These results demonstrate that in contrast to petite mutants some mit ^- mutants have retained the ability to perform meiosis and sporulation.     

β-nitropropionic acid and nitrite in relation to nitrate formation by Aspergillus flavus

Summary -nitropropionic acid (BNP) was converted to nitrate in media inoculated with A. flavus spores or with replacement cultures of mycelium pregrown in glucose-peptone medium. Conversion by replacement cultures was rapid: 8–30% in 2 days; influenced by pH: most rapid at pH 3.5; and extensive: as much as 80% BNP nitrogen appeared as nitrate after 14 days. Nitrite was detectable in BNP replacement cultures at low levels or not at all, and nitrate was formed in BNP replacement media with or without glucose. Nitrite was not oxidized in growing cultures inoculated with spores, but replacement cultures oxidized over 50% of added nitrite to nitrate in 8 days. No nitrite or nitrate appeared in replacement systems with pyruvic oxime, oxalacetic acid oxime, acetoxime, ketoglutaric acid oxime, or hydroxylamine.Of the three non-nitrifying mutants of A. flavus obtained, all formed nitrate from BNP in replacement but only one oxidized nitrite to nitrate. No accumulation of free or bound hydroxylamine or of nitrite could be detected in the mutants. BNP was detected by qualitative test in cultures of the wild type but not the mutants. Evidence indicates that the pathway in A. flavus is BNPNO₃ ^- rather than BNPNO₂ ^-NO₃ ^-.     

Cloning and DNA sequence analysis of the mercury resistance genes of Streptomyces lividans

Summary A broad-spectrum mercury resistance locus (mer) from a spontaneous chloramphenicol-sensitive (Cm^s), arginine auxotrophic (Arg^–) mutant of Streptomyces lividan 1326 was isolated on a 6 kb DNA fragment by shotgun cloning into the mercury-sensitive derivative S. lividans TK64 using the vector pIJ702. The mer genes form part of a very large amplifiable DNA sequence present in S. lividans 1326. This element was amplified to about 20 copies per chromosome in the Cm^s Arg^– mutant and was missing from strains like S. lividans TK64, cured for the plasmid SLP3. DNA sequence analysis of a 5 kb region encompassing the whole region required for broad-spectrum mercury resistance revealed six open reading frames (ORFs) transcribed in opposite directions from a common intercistronic region. The protein sequences predicted from the two ORFs transcribed in one direction showed a high degree of similarity to mercuric reductase and organomercurial lyase from other gram-negative and gram-positive sources. Few, if any, similarities were found between the predicted polypeptide sequences of the other four ORFs and other known proteins.     

A method for measuring the settling velocity distribution of large biotic particles

A simple method for measuring the settling velocity (V _s) distribution of pollen and spores 30–100 μm in diameter is detailed and evaluated. The method is called the ‘settling tower' and consists in taking sequential pictures of particles falling under gravity in calm air. The scene is illuminated by a cold light source, while a camera takes 15 pictures per second. Between 20,000 and 100,000 images are analysed to obtain the distribution of V _s for a given set of particles. The method was validated using two standard particles with mean diameters of 68 and 108 μm, respectively, as well as Lycopodium spores, with a mean diameter of 35 μm. For each set of particles, the theoretical V _s distribution was estimated from the particle diameter distribution and the volumetric mass using a non-Stokian law, as the Reynolds numbers of the particles were large. The mean V _s was measured with the ‘settling tower' with less than 12% error, while the standard deviation of the V _s distribution was estimated with less than 51% error. The maximum error on the mean V _s was 12% for the Lycopodium spores and less than 2% for the two larger particles. The mean V _s of Lycopodium spores was 4.2 cm s⁻¹, and its standard deviation was 0.7 cm s⁻¹. The reason for the small overestimation of V _s for Lycopodium spores by the ‘settling tower' method is discussed. Preliminary measurements shows that, the ‘settling tower' could be of great practical interest for measuring the distribution of V _s of maize pollen as well as other types of pollen or spores.     

Identification of the primary lesion in a protoporphyrin accumulating mutant of Chlamydomonas reinhardtii

Summary A group of chlorophyll deficient mutants (br _s mutants) of Chlamydomonas accumulates protoporphyrin and has poorly developed chloroplast membrane systems (Wang et al. 1974). In order to determine whether a poorly developed chloroplast membrane system is the reason for, or the result of, the inability of the br _s mutants to metabolize protoporphyrin to chlorophyll, a second mutation was selected which restored chlorophyll synthesis in br _s mutants. One such double mutant (br _s-2 g-4) was analyzed. The double mutant br _s-2 g-4 has partially restored chlorophyll synthesis, but has defective photosystem II and photosystem I electron transport as well as abnormal chloroplast ultrastructure. Since these defects are not present in cells carrying only the g-4 mutation, they are presumed to be caused by the br _s-2 mutation. It is concluded that a defect in chloroplast membrane development resulting from the br _s-2 mutation causes an apparent defect in magnesium chelation by protoprophyrin. This is consistant with evidence that chlorophyll biosynthesis from magnesium protoporphyrin to chlorophyll takes place on the chloroplast membranes.     

Transduction in Streptomyces hygroscopicus mediated by the temperate bacteriophage SH10

Summary The temperate actinophage SH10 mediates generalized transduction in Streptomyces hygroscopicus at low frequency. The efficiency of transduction depends on the average phage input, age of outgrowing spores of the recipient and on the selective marker. The highest EOT was found for the auxotrophic mutants 21 (phe^-) and 5(try^-)(4.1x10^-6 and 2.7x10^-6, respectively). Transduction of the thermosensitive mutant NG14-216 ts 35 was two orders of magnitude lower (2.5x10^-8). The transductant colonies segregated into stable and unstable clones. Stable transductants were never found to be lysogenic for phage SH10.     

Genetic suppression and phenotypic masking of a Myxococcus xanthus frzF- defect

An insertion of transposon Tn5-lac, ω4519, generates a lacZ fusion with a Myxococcus xanthus promoter expressed during both vegetative growth and development. Sequence analysis of the junction of ω4519 with M. xanthus DNA shows that the insertion is in frzF, a homologue of cheR from Salmonella typhimurium. When frzF^- (or frzCD^-) cells are starved for nutrients at modest densities, they aggregate to form a radial pattern and produce fewer than 1% of the wild-type complement of spores. At higher densities, frzF::ω519 cells form‘frizzy’aggregates and produce 80–90% of the wild-type complement of spores. In contrast, when cells with both a frzF^- (or frzCD^-) and an sgtA1 mutation are allowed to develop at either low or high cell densities, they produce frizzy aggregates containing a near wild-type complement of heat-resistant spores. In addition to suppressing the density dependence of fruiting-body morphogenesis, the sglA1 mutation also suppresses the sporulation defect caused by two different frzF^- mutations and a frzCD^- mutation. In contrast, a mutation in a different S motility gene, sglG1, does not suppress the frz^- mutations. Thus, the suppression of frz^- mutations by sgl^- mutations is allele-specific, and depends on the sgl allele, but not the frz allele. Because the phenotypes of frz^- mutations have been determined in a (suppressing) sglA1 genetic background, the frz genes may play more central roles in development than initially recognized.     

Reducing blood glucose level in TIDM mice by orally administering the silk glands of transgenic hIGF-I silkworms

Protein O-mannosyltransferase 1 (POMT1) and its homolog, POMT2, are responsible for the catalysis of the first step in O-mannosyl glycan synthesis. Mutations in their genes are associated with a type of congenital muscular dystrophy called Walker-Warburg syndrome. Arg⁶⁴, Glu⁷⁸ and Arg¹³⁸ in the N-terminus region of ScPmt1p, a POMT homolog in Saccharomyces cerevisiae, are important for transferase activity. Arg¹³⁸ is also essential for complex formation with ScPmt2p. Here we examined the effects of replacing the corresponding residues in human POMT1 and POMT2 with Ala on complex formation and enzymatic activity. The human POMT1 mutants lost almost all transferase activity while the POMT2 mutants retained enzymatic activity. Neither mutant lost its ability to form complexes with the native counter component. These results indicate that ScPmtps and human POMTs have different mechanisms of complex formation. They also suggest that human POMT1 and POMT2 have discrete functions since the effect of amino acid substitutions on enzymatic activity are different.     

Selection and characterization of nuclear mutations affecting mitochondria in Paramecium.

Summary Escherichia coli pel ^- mutants inhibit the penetration of bacteriophage lambda DNA into the cell. Using P1 mediated cotransduction, we mapped pel ^- mutations between markers fadD and eda in the interval of minute 40 of the revised E. coli K-12 map. This places pel in the same region as genes kdgR and ptsM. Mutations in kdgR usually do not alter the Pel phenotype, and vice versa. In contrast, about 30% of ptsM ^- mutants are also pel ^-, and all pel ^- mutants isolated are ptsM ^-. These results suggest that pel and ptsM are one and the same gene. This interpretation would identify the bacterial product required for injection of phage DNA as a component of the phosphoenolpyruvate-dependent phosphotransferase system specific for mannose, glucosamine, glucose and fructose. However, the experimental results do not exclude an alternative explanation: that pel and ptsM identify two closely linked genes which would be simulataneously affected at high frequency by a particular mutational event.