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The Isolation and Characterization of Mutant Strains of the Blue-green Alga Anacystis nidulans
Abstract
Procedures for mutagenesis and conditions for selection by penicillin enrichment in the blue-green alga Anacystis nidulans have been developed. The characterization of 19 mutant strains involving single or serially induced multiple markers was achieved. In addition to drug-resistant and morphological mutants auxotrophic strains requiring phenylalanine, methionine, biotin or acetate together with strains deficient in sulphate and nitrate reduction have been isolated.
... Although morphological and drug-resistant mutants of several species of blue-green algae have been isolated with relative ease, the isolation of auxotrophic mutants has proved more difficult (see review by Van Baalen, 1973) and several authors have commented on their failure to isolate mutants of this type (Asato & Folsome, 1969;Houghton, 1971 ;Kaney & Dolack, 1972). Mutants defective in the enzymes of nitrate assimilation have been recognized for some time in Anacystis nidulans (Van Baalen, 1965) and in Agmenellum quadruplicntum (Stevens & Van Baalen, 1970) and such mutants are the most common auxotrophic type produced by treatment with N-methyl-N'-nitro-N-nitrosoguanidine (Herdman & Carr, 1972). They are easily isolated, even on non-supplemented medium on which they grow as yellow colonies presumably because they lack phycocyanin, known to be a protein storage material (Allen & Smith, 1969). ...
... Auxotrophs of other phenotypes have been found only at very low frequency. Strains of Anacystis nidulans requiring phenylalanine, methionine, biotin, acetate or a reduced sulphur source, together with the more frequent mutants blocked in nitrate reduction, were described by Herdman & Carr (1972). Ingram, Pierson, Kane, Van Baalen & Sensen (I 972) described a tryptophan-requiring mutant of Agmenellum quadruplicatum which has a defective tryptophan synthetase A protein. ...
... The physical conditions for the mutagenesis and selection of a restricted range of mutant strains have been described (Herdman & Carr, 1972). This communication describes the growth of Anacystis nidulans on complex media, the development of a chemically defined complete medium which supports the growth of all auxotrophs tested, and the use of this medium in the isolation and rapid identification of mutant strains. ...
A complete medium of defined composition has been developed for quantitative growth of wild-type and auxotrophic mutant strains of Anacystis nidulans. This medium has proved to be more satisfactory than other complex media (for example casein hydrolysate, yeast extract) for both the isolation and the growth of auxo-trophs. Rigorous control of the pH of complete and other supplemented media is essential for quantitative growth on agar. Four diagnostic media are described which each contain a different combination of the supplements used in the complete medium and facilitate the identification of the nutritional requirements of mutants. By using these media a number of auxotrophs have been isolated including five with novel phenotypes which require respectively (i) thiamine, (ii) p-aminobenzoic acid, (iii) a combination of pyruvate or acetate plus malate or succinate or fumarate, (iv) serine or glycine and (v) adenine.
... Mutations are central to genetics, both to identify unknown genes that are involved in a particular process, and to elucidate the function of known genes. Spontane-ous mutants can be isolated (Astier et al. 1984;Montesinos et al. 1997), or mutations can be generated physically [e.g., with UV light (Singh and Tiwari 1969;Herdman and Carr 1972;Wolk et al. 1988;Vega-Palas et al. 1990)], chemically [e.g., with methyl sulfonates or N-methyl-N′-nitro-N-nitrosoguanidine (Herdman and Carr 1972;Currier et al. 1977;Vega-Palas et al. 1990;Buikema and Haselkorn 1991)] or biologically (e.g., with transposons or insertional mutagenesis). Because cyanobacteria often have on the order of 10 copies of their chromosome per cell andfor filamentous strains -many linked cells, and transposon mutants can be selected, random mutagenesis with transposons has often facilitated the search for mutants (Borthakur and Haselkorn 1989;Ernst et al. 1992;Wolk et al. 1991). ...
... Mutations are central to genetics, both to identify unknown genes that are involved in a particular process, and to elucidate the function of known genes. Spontane-ous mutants can be isolated (Astier et al. 1984;Montesinos et al. 1997), or mutations can be generated physically [e.g., with UV light (Singh and Tiwari 1969;Herdman and Carr 1972;Wolk et al. 1988;Vega-Palas et al. 1990)], chemically [e.g., with methyl sulfonates or N-methyl-N′-nitro-N-nitrosoguanidine (Herdman and Carr 1972;Currier et al. 1977;Vega-Palas et al. 1990;Buikema and Haselkorn 1991)] or biologically (e.g., with transposons or insertional mutagenesis). Because cyanobacteria often have on the order of 10 copies of their chromosome per cell andfor filamentous strains -many linked cells, and transposon mutants can be selected, random mutagenesis with transposons has often facilitated the search for mutants (Borthakur and Haselkorn 1989;Ernst et al. 1992;Wolk et al. 1991). ...
Cyanobacteria are oxygenic photosynthetic bacteria that have been used increasingly to study diverse biological processes, including photosynthesis and its regulation; cell differentiation and N 2 fixation; metabolism of nitrogen, carbon, and hydrogen; resistance to environmental stresses; and molecular evolution. Many vectors and other genetic tools have been developed for unicellular and filamentous strains of cyanobacteria. Transformation, electroporation, and conjugation are used for gene transfer. Diverse methods of mutagenesis allow the isolation of many sought-for kinds of mutants, including site-directed mutants of specific genes. Reporter genes permit measurement of the level of transcription of particular genes, and assays of transcription within individual colonies or within individual cells in a filament. Complete genomic sequences have been obtained for the unicellular cyanobacterium, Synechocystis sp. strain PCC 6803 and the filamentous, heterocyst-forming cy-anobacterium, Anabaena sp. strain PCC 7120. Genomic sequence projects are under way for Nostoc punctiforme strain PCC 73102 (ATCC 29133) and strains of the uni-cellular genera, Synechococcus, Prochlorococcus, and Gloeobacter. Genomic sequence data provide the opportunity for global monitoring of changes in genetic expression at transcriptional and translational levels in response to variations in environmental conditions. The availability of genomic sequences accelerates the identification , study, modification and comparison of cyano-bacterial genes, and facilitates analysis of evolutionary relationships, including the relationship of chloroplasts to ancient cyanobacteria. The many available genetic tools enhance the opportunities for possible biotechnological applications of cyanobacteria.
... Mutations are central to genetics, both to identify unknown genes that are involved in a particular process, and to elucidate the function of known genes. Spontane-ous mutants can be isolated (Astier et al. 1984;Montesinos et al. 1997), or mutations can be generated physically [e.g., with UV light (Singh and Tiwari 1969;Herdman and Carr 1972;Wolk et al. 1988;Vega-Palas et al. 1990)], chemically [e.g., with methyl sulfonates or N-methyl-N′-nitro-N-nitrosoguanidine (Herdman and Carr 1972;Currier et al. 1977;Vega-Palas et al. 1990;Buikema and Haselkorn 1991)] or biologically (e.g., with transposons or insertional mutagenesis). Because cyanobacteria often have on the order of 10 copies of their chromosome per cell andfor filamentous strains -many linked cells, and transposon mutants can be selected, random mutagenesis with transposons has often facilitated the search for mutants (Borthakur and Haselkorn 1989;Ernst et al. 1992;Wolk et al. 1991). ...
... Mutations are central to genetics, both to identify unknown genes that are involved in a particular process, and to elucidate the function of known genes. Spontane-ous mutants can be isolated (Astier et al. 1984;Montesinos et al. 1997), or mutations can be generated physically [e.g., with UV light (Singh and Tiwari 1969;Herdman and Carr 1972;Wolk et al. 1988;Vega-Palas et al. 1990)], chemically [e.g., with methyl sulfonates or N-methyl-N′-nitro-N-nitrosoguanidine (Herdman and Carr 1972;Currier et al. 1977;Vega-Palas et al. 1990;Buikema and Haselkorn 1991)] or biologically (e.g., with transposons or insertional mutagenesis). Because cyanobacteria often have on the order of 10 copies of their chromosome per cell andfor filamentous strains -many linked cells, and transposon mutants can be selected, random mutagenesis with transposons has often facilitated the search for mutants (Borthakur and Haselkorn 1989;Ernst et al. 1992;Wolk et al. 1991). ...
Cyanobacteria are oxygenic photosynthetic bacteria that have been used increasingly to study diverse biological processes, including photosynthesis and its regulation; cell differentiation and N2 fixation; metabolism of nitrogen, carbon, and hydrogen; resistance to environmental stresses; and molecular evolution. Many vectors and other genetic tools have been developed for unicellular and filamentous strains of cyanobacteria. Transformation, electroporation, and conjugation are used for gene transfer. Diverse methods of mutagenesis allow the isolation of many sought-for kinds of mutants, including site-directed mutants of specific genes. Reporter genes permit measurement of the level of transcription of particular genes, and assays of transcription within individual colonies or within individual cells in a filament. Complete genomic sequences have been obtained for the unicellular cyanobacterium, Synechocystis sp. strain PCC 6803 and the filamentous, heterocyst-forming cyanobacterium, Anabaena sp. strain PCC 7120. Genomic sequence projects are under way for Nostoc punctiforme strain PCC 73102 (ATCC 29133) and strains of the unicellular genera, Synechococcus, Prochlorococcus, and Gloeobacter. Genomic sequence data provide the opportunity for global monitoring of changes in genetic expression at transcriptional and translational levels in response to variations in environmental conditions. The availability of genomic sequences accelerates the identification, study, modification and comparison of cyanobacterial genes, and facilitates analysis of evolutionary relationships, including the relationship of chloroplasts to ancient cyanobacteria. The many available genetic tools enhance the opportunities for possible biotechnological applications of cyanobacteria.
... Recombination has been claimed by some authors (13,65,133), while these results have been questioned by others (102). Recently, transformation was also reported (45,46,121). The temperate cyanophages may allow for transduction studies, which can lead to extensive genetic investigations of the blue-green algae and cyanophages. ...
... Genetic studies of factors controlling bluegreen algal nitrate reduction have not been numerous but seem promising for the future. Mutants of A. nidulans (128) and Agmenellum quadruplicatum (136,327) lacking nitrate and nitrite reductases have been isolated from populations treated with nitrosoguanidine or ultraviolet light. ...
... strain PCC 7120 and Mutagenesis approaches developed for cyanobacteria have played fundamental roles in identifying unknown genes involved in a particular process and in elucidating the function of genes (Koksharova and Wolk 2002a). In particular, chemical or ultraviolet (UV) light-induced mutagenesis has been used to produce mutants of cyanobacteria (Buikema and Haselkorn 1991;Chapman and Meeks 1987;Currier et al. 1977;Herdman and Carr 1972;Singh and Tiwari 1969;Vega-Palas et al. 1990;). Low mutagenesis efficiency and the time-consuming nature for mapping the mutated genes have limited its application. ...
... strain PCC 7120 and Mutagenesis approaches developed for cyanobacteria have played fundamental roles in identifying unknown genes involved in a particular process and in elucidating the function of genes (Koksharova and Wolk 2002a). In particular, chemical or ultraviolet (UV) light-induced mutagenesis has been used to produce mutants of cyanobacteria (Buikema and Haselkorn 1991;Chapman and Meeks 1987;Currier et al. 1977;Herdman and Carr 1972;Singh and Tiwari 1969;Vega-Palas et al. 1990;). Low mutagenesis efficiency and the time-consuming nature for mapping the mutated genes have limited its application. ...
... Recombination has been claimed by some authors (13,65,133), while these results have been questioned by others (102). Recently, transformation was also reported (45,46,121). The temperate cyanophages may allow for transduction studies, which can lead to extensive genetic investigations of the blue-green algae and cyanophages. ...
After an introduction the cyanophages are discussed in the following chapters. Characteristics of cyanophages; growth cycle; interaction between cyanophage development and the photoautotrophic metabolism of the blue green algal host; ecological considerations and genetics. The article is concluded by a literature list of 164 items.
... strain PCC 7120 and Mutagenesis approaches developed for cyanobacteria have played fundamental roles in identifying unknown genes involved in a particular process and in elucidating the function of genes (Koksharova and Wolk 2002a). In particular, chemical or ultraviolet (UV) light-induced mutagenesis has been used to produce mutants of cyanobacteria (Buikema and Haselkorn 1991;Chapman and Meeks 1987;Currier et al. 1977;Herdman and Carr 1972;Singh and Tiwari 1969;Vega-Palas et al. 1990;). Low mutagenesis efficiency and the time-consuming nature for mapping the mutated genes have limited its application. ...
Determining spatiotemporal gene expression and analyzing knockout mutant phenotypes have become powerful tools in elucidating the function of genes; however, genetic approaches for simultaneously inactivating a gene and monitoring its expression have not been reported in the literature. In this study, we designed a dual-functional gene knockout vector pZR606 that contains a multiple cloning site (MCS) for inserting the internal fragment of a target gene, with a gfp gene as its transcriptional marker located immediately downstream of the MCS. By using this gene knockout system, we inactivated ava_2679 from Anabaena variabilis ATCC 29413, as well as all2508, alr2887, alr3608, and all4388 from Anabaena sp. strain PCC 7120. The ava_2679 knockout mutant fails to grow diazotrophically. Morphological analysis of ava_2679 knockout mutant after nitrogen step-down revealed defective junctions between heterocysts and adjacent vegetative cells, and the heterocyst was 1.53-fold longer compared to wild-type heterocysts. The alr2887, all4388, and alr3608 mutant colonies turned yellow and showed lack of protracted growth when deprived of fixed nitrogen, consistent with the previous reports that alr2887, all4388, and alr3608 are Fox genes. The all2508 encodes a GTP-binding elongation factor (EF4/LepA), and its knockout mutant exhibited reduced diazotrophic growth. The heterocyst development of all2508 knockout was significantly delayed, and only about 4.0 % of vegetative cells differentiated to heterocysts after nitrogen deprivation for 72 h, decreased 49.6 % compared to wild-type. Thus, we discovered that All2508 may regulate heterocyst development spatiotemporally. Concurrently, the GFP reporter revealed that all five target gene expressions were up-regulated in response to nitrogen deprivation. We demonstrated that the pZR606-based specific gene knockout approach worked effectively for the five selected genes, including four previously identified Fox genes or Fox gene homolog, and a previously unknown function of gene all2508. Thus, gene expression and phenotypic analysis of mutants can be achieved simultaneously by targeted gene inactivation using the pZR606-based system. This combined approach for targeted gene inactivation and its promoter reporting with GFP may be broadly applicable to the study of gene function in other prokaryotic organisms.
... UV irradiation (Singh and Tiwari 1969)], chemical [e.g. exposure to N-methyl-N 0 -nitro-N-nitrosoguanidine (Herdman and Carr 1972)] or biological [e.g. transposon mutagenesis (Wolk et al. 1991)] treatments of the cells. ...
Synechocystis sp. PCC 6803 (Synechocystis) that is the first sequenced photosynthetic organism has two advantages, natural transformation and light-activated heterotrophic growth, and such characteristics have mainly promoted reverse genetic analysis. However, approximately 50% of genes are still annotated as "unknown protein" or "hypothetical protein" to date. Therefore, forward genetic analysis is required for identifying significant genes responsible for photosynthesis and other physiological phenomena from genes of unknown function. The in vivo transposon mutagenesis system is one of the major methods for random mutagenesis. However, present in vivo transposon mutagenesis systems for cyanobacteria face problems such as relatively low frequency of transposition and repeated transposition in the host cells. In this study, we constructed vectors based on a mini-Tn5-derived vector that was designed to prevent repeated transposition. Our vectors carry a hyperactive transposase and optimized recognition sequence of transposase, which were reported to enhance frequency of transposition. Using the vector, we succeeded in highly-frequent transposition (9 × 10(-3) per recipient cell) in Synechocystis. Transposon insertion sites of randomly selected ten mutants indicated that the insertion sites spread throughout the genome with low sequence dependency. Furthermore, one of the ten mutants exhibited slow-growing phenotype, and the mutant was functionally complemented by using our expression vector. Our system also worked with another model cyanobacterium, Synechococcus elongatus PCC 7942 with high frequency. These results indicate that the developed system can be applied to the forward genetic analysis for a broad range of cyanobacteria.
... A valine auxotroph (Val-) was derived from this EthR Het-mutant by further mutagenesis with NTG, and was isolated by the penicillin enrichment technique developed by Davis (1948) and adapted by Ingram et al. (1972) and Herdman & Carr (1972) for use with cyanobacteria. Following mutagenesis, cells were inoculated into a flask containing MM plus penicillin (150 pg ml-I). ...
... The original ampicillin resistant transformant colony gave on subculture a high frequency of ampicillin sensitive colonies with a slow growing, yellow chlorotic phenotype and aberrant, elongated colony morphology. A similar yellow phenotype has been observed previously in Nar mutants of Synechococcus strains PCC6301 (Herdman & Carr, 1972) and PCC7942 (Kuhlemeier et al., 1984a;Madueno et al., 1988). The Nar phenotype of Tf6 was confirmed by assays of nitrate reductase activity (Table 2). ...
A nitrate reductase gene of the cyanobacterium Synechococcus PCC6301 inferred by heterologous hybridization cloning and targeted mutagenesis.
N-methyl-N′-nitro-N-nitrosoguanidine (NTG)-induced mutants of filamentous, heterocystous and nitrogen fixing blue-green alga Anabaena variabilis were classified in five groups: (i) Non-nitrogen fixing with normal trichome, (ii) non-nitrogen fixing with short trichome, (iii) non-sporulating and colony forming, (iv) non-sporulating and non-colony forming and (v) non-sporulating with gas vacuoles. All the mutants were stable and did not revert during successive subcultures. Induction of nif⁻ mutants exhibited maximum mutation frequency among all the mutants under reference. The salient features of the mutants have been described.
This chapter contains the description of ultrastructural plasticity manifestations in laboratory maintained strains of cyanobacteria with different metabolic traits during formation and functioning of model associations with cells, tissues, regenerated plantlets, and whole plants non-symbiotrophic species. A wide
array of examples supporting the tight coupling of the diversity of ultrastructural plasticity manifestations of sub-cellular, single-cell, and cell population levels with metabolic potential of the cyanobacteria studied and their capability of forming association with plants. Special attention is paid to the description of the structure
of stable model association of plant partners with free living Nostoc muscorum strains. These systems, as well as natural symbioses of Nostoc sp. with higher plants, are characterized by the maximum manifestation of ultrastructural plasticity of cyanobacteria. The extent of the manifestations correlates with the capability of a given species to form symbiotic associations with plants.
A preliminary characterization of 8 temperature-sensitive, high-fluorescent mutants of the blue-green alga S. cedrorum is presented. The mutants can be placed into 3 broad classes based primarily on fluorescence induction kinetics. Class 1 mutants have an increased variable fluorescence, grow less than 50% as fast as wild-type, but show few other photosynthetically-related changes. Mutants in class 2 are morphological growth mutants. They divide very poorly at 40.degree. C, but are similar to the wild-type in pigment composition and fluorescence kinetics. Class 3 is composed of mutants that are characterized by very high fluorescence and a smaller relative proportion of variable fluorescence. Mutants in this class have perturbed photosynthetic rates and variations in the low temperature fluorescence spectra. One of these mutants is also a distinct temperature-sensitive pigment mutation and has a high phycocyanin to chlorophyll ratio when grown at 40.degree. C. All of the mutants display specific alterations in their membrane protein composition when grown at the high temperature.
The wild type Nostoc muscorum (UW strain) has yielded various physiological mutants altered in utilization of sulphate, following mutagenic treatments with N‐methyl, N′‐nitro N‐nitrosoguanidine (NTG). One of the mutant strains designated as Sat‐20 failed to grow in a medium containing sulphate (MgSO4 · 7 H2O). However, the mutant strain could grow when supplemented with thiosulphate (Na2S2O3 · 5 H2O), while methionine could fulfil the sulphur requirement only partially. On comparative reasons, the wild type as well as the mutant showed preference for thiosulphate over other sulphur sources employed.
Under conditions in which two known mutagenic agents, ultraviolet irradiation and nitrosoguanidine (MNG), induced high proportions of mutations (chiefly affecting growth rate and colony form), PbCl2 produced no appreciable increase in the incidence of such mutant types above the spontaneous frequency in Platymonas subcordiformis.
A uracil-requiring auxotroph ofAnacystis nidulans was isolated after treat- mentwithN-methyl-N'-nitr o-N-nitrosoguanidine. Neither precursorsinthede novo pyrimidine pathway norcompounds of"salvage" ordegradative pathways could replace theuracil requirement. Thereversion frequency formutation toa nonuracil requirement forgrowth was 2.0x 10-8. Thecalculated averagerateof uracil utilization was 1.1x 10-17 molofuracil perunitcell mass/h. Theamount ofuracil required forthesynthesis ofa unitcell mass was 3.8x 10-'7 molof uracil.
The selection and analysis of mutants of the CO2-concentrating mechanism in cyanobacteria have greatly advanced our understanding of the physiological and genetic components of this mechanism. This paper reviews the processes for the creation of mutants and the properties of mutants that have been produced so far. In addition to this, consideration is given to developing new mutant selection protocols, based on our current knowledge of the operation of the CO2-concentrating mechanism. As a result, new screens are suggested for the isolation of transport mutants that are defective in either HCO3− or CO2 transport activity, and putative carboxysomal mutants that have altered carbonic anhydrase activities. The procedures for physiological analysis of mutants are also reviewed, with a conclusion that there is a great need for the further development of in vitro assays, particularly for the inorganic carbon transport process and carboxysome function. Particular consideration is given to the in vitro carboxysome assay, and it is concluded that many of the properties of a carboxysomal Rubisco may be difficult to resolve owing to increases in carboxysomal leakiness during isolation and the higher ratio of [CO2] to [HCO3−] experienced during assays compared with the in vitro situation. Finally, consideration is given to the genetic analysis of cyanobacterial mutants and the progress that will need to be made in the future, discovering what are the functions of the proteins coded for by the genes that are isolated. Key words: cyanobacteria, mutants, photosynthesis, carboxysome, CO2-concentrating mechanism.
The blue-green algae have been used in comparative studies of photosynthesis. Their prokaryotic nature, phycobiliprotein accessory pigments, and oxygen yielding photosynthesis set them apart from the diverse eukaryotic algae and photosynthetic bacteria. From an experimental standpoint, blue-green algae can be most recalcitrant, and selection and characterization of mutant forms has proceeded slowly. In the past few years, satisfactory growth conditions have been developed for plating coccoid and filamentous forms. This has allowed some standard chemical and physical agents to be tested for their effectiveness as mutagens. The classic penicillin technique for the enrichment of mutants has been applied to the blue-green algae, but there is little uniformity in the methodology. In this chapter, an overview is presented of the existing methodology for the induction of mutants, with comments on extant types of blue-green alga mutants. General growth conditions are described and mutagenic agents are discussed in the chapter. N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment conditions are also elaborated.
A phenylalanine-requiring strain of the unicellular blue-green bacterium Synechococcus cedrorum was isolated by means of penicillin enrichment following mutagenesis with nitrosoguanidine. Assays of pertinent enzymes indicated that prephenate dehydratase activity was absent in the mutant.
Previous attempts to isolate auxotrophic mutants of Anacystis nidulans produced only a limited range of phenotypes. The frequency of recovery of auxotrophic mutants has been quantified following different mutagenic and selective treatments, and their yield has been improved by using (1) a complete medium, (2) additional mutagens, (3) multiple cycles of penicillin enrichment and (4) altered pre-enrichment starvation conditions. These modified induction and selection conditions permitted the isolation of mutants defective in nitrate reductase, nitrite reductase or malate dehydrogenase, unable to reduce sulphate, or deficient in the synthesis of biotin, thiamine, paminobenzoate, serine, glutamate, adenine or uracil.
Evidence is presented which confirms the existence of genetic transformation in the blue-green bacterium, Anacystis nidulans. This process has been demonstrated for three mutations: streptomycin resistance, a phenylalanine requirement and an ornithine requirement. The optimal conditions under which transformation occurs are also investigated, and the potential of this system for genetic mapping is discussed.
The pre-emergence rice-field herbicide machete (2-chloro-2′, 6′-diethyl-N (butoxymethyl) acetanilide), is apparently toxic and lytic to parental, het+ nif11−, and Em-R het−nif2− strains of Nostoc muscorum. The intensity of the biological effects induced by the herbicide increases with the growth-promoting efficiency of various inorganic nitrogen sources. The herbicide has no effect on heterocyst differentiation, and induced biological perturbations are not reversed by glucose. Machete appears to be strong mutagen as 5–6% of the het+nif11− and Em-R het− are prototrophic revertants.
The survival curve after ultraviolet (u.v.) irradiation of Anabaena L-31 showed a pronounced initial shoulder, a narrow region of exponential lethality and a long tail, indicating a multiple hit two component pattern. A major portion of the u.v.-induced lethality was prevented by photoreactivation in white light. Survival was qualitatively and quantitatively similar for growth in nitrogen-fixing and nitrate-supported conditions.Prolonged u.v. irradiation caused the induction of giant cells, misalignment of cells in the filament and true branching. These u.v.-induced morphological abnormalities were not stable and disappeared in most cases after the fifth subculture in aerated liquid media. The induction of true branching appears to be of significance in the evolution of algae belonging to the Stigonematales.Low u.v. fluences did not affect growth or induce morphological abnormalities, but stimulated heterocyst differentiation.
The wild type Nostoc muscorum (UW strain) has yielded various physiological mutants altered in utilization of sulphate, following mutagenic treatments with N-methyl, N'-nitro N-nitrosoguanidine (NTG). One of the mutant strains designated as Sat-20 failed to grow in a medium containing sulphate (MgSO4.7 H2O). However, the mutant strain could grow when supplemented with thiosulphate (Na2S2O3.5 H2O), while methionine could fulfil the sulphur requirement only partially. On comparative reasons, the wild type as well as the mutant showed preference for thiosulphate over other sulphur sources employed.
This chapter reviews the cyanobacterial genome, the machineries of its expression, and the ways in which that expression appears to be controlled. It explains that cyanobacteria are of molecular biological interest for a number of independent reasons, each sufficient to justify their more intensive investigation: evolutionary position, autotrophic physiology, differentiation, ecology, and photosynthesis and nitrogen fixation. Cytochemical and ultrastructural studies showed that the frequently centrally located cyanobacterial “nucleoplasm” is not membrane bounded and contains loosely organized DNA fibrils. Attempts to identify structural analogs of eukaryotic chromosomes within it became less frequent with the growing recognition that cyanobacteria are not, after all, merely simple plants. A “histone-like” DNA binding protein is present in cyanobacteria although cytochemically identifiable histones may be absent. This protein is immunologically indistinguishable from the Escherichia coli (E. coli) histone-like protein HUE and very similar to it and a thermoplasrna “histone” in amino acid composition. There is preliminary evidence that the DNA of can be isolated as an RNA- and protein-containing “nucleoid” analogous to that of E. coli.
Auxotrophic mutants of the filamentous cyanobacterium Anabaena variabilis were isolated by a method in which, after mutagenesis and before penicllin enrichment, mutant and wild-type cells were separated by cavitation. Auxotrophs were identified by their inability to grow on minimal medium, and they were partially characterized by replica plating to media supplemented with single nutrients or specific groups of nutrients. Of the 83 auxotrophs isolated, 65 required an inorganic source of nitrogen for growth. In addition, auxotrophs were isolated that required methionine (six), uracil (two), adenine (one), biotin (two), and nicotinic acid (two). (The number of isolates of each type is indicated in parentheses.) The nutrient requirements of five auxotrophs appeared complex and were not determined. A large proportion of the mutants requiring inorgainic fixed nitrogen was altered in the differentiation of heterocysts. The following morphological aberrancies were observed: abnormally high and abnormally low frequencies of heterocysts; thick, uneven heterocyst envelopes; incompletely developed pore regions; very distinct pore regions; and protoplasts separated from the envelope of the heterocyst. Spontaneously occurring, N2-fixing, prototrophic revertants of mutants with aberrant heterocysts have been isolated at a frequency of 2 X 10(-8) to 4 X 10(-8) of the cells plated. That most such revertants produced morphologically normal heterocysts is consisten with the idea that heterocysts play an essential role in aerobic N2 fixation.
A uracil-requiring auxotroph of Anacystis nidulans was isolated after treatment with N-methyl-N'-nitrosoguanidine. Neither precursors in the de novo pyrimidine pathway nor compounds of "salvage" or degradative pathways could replace the uracil requirement. The reversion frequency for mutation to a nonuracil requirement for growth was 2.0 times 10(-8). The calculated average rate of uracil utilization was 1.1 times 10(-17) mol of uracil per unit cell mass/h. The amount of uracil required for the synthesis of a unit cell mass was 3.8 times 10(-17) mol of uracil.
Analysis of cell-free extracts of Anacystis nidulans disclosed the absence of both thymidine phosphorylase (EC 2.4.2.4) and thymidine kinase (EC 2.7.1.21) activities. Thymine and thymidine were incorporated inefficiently by intact cells of A. nidulans either in the presence or absence of deoxyguanosine (250 mug/ml). Deoxythymidine monophosphate incorporation was also inefficient. Radioactive deoxyadenosine, at a minimally toxic level (3 mug/ml), was incorporated effectively into the deoxyribonucleic acid (DNA). A cesium chloride-ethidium bromide gradient analysis of the DNA revealed that both the plasmid DNA and the principal DNA of the A. nidulans genome were labeled effectively in cells exposed to [8-14C]deoxyadenosine.
DNA probes from the narG gene of Escherichia coli, which encodes the large polypeptide of respiratory nitrate reductase, show cross-hybridization at low stringency to a single region of the genome of the cyanobacterium Synechococcus PCC6301. This segment of cyanobacterial DNA was cloned as the insert of plasmid pDN1 and characterized. RNA complementary to pDN1 was shown to be substantially more abundant in nitrate grown cells of Synechococcus PCC6301 than in ammonium grown cells, thus parallelling the nitrate induction and ammonium repression of nitrate reductase activity in cultures of this cyanobacterium. A mutant of Synechococcus PCC6301 deficient in nitrate reductase activity was obtained after a potentially mutagenic transformation treatment using pDN1 as a donor. This mutant was restored to the wild type phenotype following stable integrative transformation with pDN1 DNA. Taken together these data suggest that pDN1 might encode a polypeptide of nitrate reductase. pDN1 is distinct from three clones of genes involved in nitrate assimilation that were isolated previously from the related cyanobacterium Synechococcus PCC7942 (Kuhlemeier et al., 1984a, J. Bact. 159, 36-41, and 1984b, Gene 31, 109-116).
Genetic transformation experiments have been performed in Anacystis nidulans using donor material from two sources, namely chemically extracted DNA and extracellular nucleic acids. A high proportion of the transformants became mutant at sites which were wild type in both parental strains. Linkage was less extensive in transformation mediated by chemically extracted DNA, and this increased frequency of recombination was associated with enhanced mutation frequencies. The frequencies of recombination and mutation were varied to the same extent by changing the DNA concentration, and both processes were prevented by pretreatment of donor DNA with DNase. Mutational events are, therefore, closely associated with recombination in A. nidulans.
Since the discovery of the mutagenic activity of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in 1960, this compound has become one of the most widely used chemical mutagens. The present paper gives a survey on the chemistry, metabolism, and mode of interaction of MNNG with DNA and proteins, and of the genotoxic effects of this agent on microorganisms, plants, and animals, including human cells cultured in vitro. Data on the carcinogenicity and teratogenicity of MNNG as well as on the genotoxic effects of homologs of MNNG are also presented.
Complementation of an E. coli mutant auxotrophic for the branched-chain amino acids (BCAA)--valine, leucine, and isoleucine--by the ilvG gene ( slr2088) of the cyanobacterium Synechocystis PCC6803 indicates that this gene encodes an active alpha-acetohydroxy acid synthase. Differences of response of the recombinants to the addition of the essential amino acids suggested a lower specificity for the initial reaction of the valine/leucine chain than for the isoleucine one. Inactivation of ilvG in Synechocystis led to a leaky phenotype, suggesting a capacity to compensate the auxotrophies by other processes. This observation is discussed in view of the general difficulty of obtaining auxotrophs in cyanobacteria.
The cell division cycle of Synechococcus sp. strain PCC 6301 in light is characterized by the sequential and orderly appearance of macromolecular synthesis periods. In the dark, macromolecular synthesis and cell division are severely curtailed. When dark-incubated cultures are reexposed to light, a new cell cycle is initiated. The pattern of the cell events displayed by Synechococcus in light and the absence of sustained growth in dark incubation conditions suggests that light-activated regulatory molecules control macromolecular synthesis and the cell division cycle. For example, ribosomal RNA synthesis is stimulated by a light-activated DNA binding factor in light but not in the dark. Light/dark conditions induce cell synchrony in Prochlorococcus. Distinct G1, S and G2 phases characterize cell cycles of marine Synechococcus and Prochlorococcus. Cell division in Synechococcus elongatus PCC 7942 and marine Synechococcus is controlled by circadian oscillators.
Some physiological characteristics of a mutant (E(1)) of Anacystis nidulans R(2), incapable of growing at air level of CO(2), are described. E(1) is capable of accumulating inorganic carbon (C(i)) internally as efficiently as the wild type (R(2)). The apparent photosynthetic affinity for C(i) in E(1), however, is some 1000 times lower than that of R(2). The kinetic parameters of ribulose 1,5-bisphosphate carboxylase/oxygenase from E(1) are similar to those observed in R(2). The mutant appears to be defective in its ability to utilize the intracellular C(i) pool for photosynthesis and depends on extracellular supply of Ci in the form of CO(2). The very high apparent photosynthetic K(m) (CO(2)) of the mutant indicate a large diffusion resistance for CO(2). Data obtained here are used to calculate the permeability coefficient for CO(2) between the bulk medium and the carboxylation site of cyanobacteria.
Some properties of a mutant (RK1) of Synechococcus PCC7942, which requires high CO(2) for growth, are described. The photosynthetic affinity for inorganic carbon (C(i)) in RK1 was about 40 times lower than that in the wild type (WT) when grown at 3% CO(2) (H-cells) and did not change during 10 hours of exposure to low CO(2) (air containing 0.04% CO(2)). The gas exchange of WT and RK1 cells was measured using an open gas-analysis system. All the measurements were performed at a CO(2) concentration of 400 microliters per liter under the conditions where photosynthetic CO(2) fixation is inhibited. When the suspension of H-cells of WT or RK1 was illuminated, the rate of CO(2) influx from the gas phase into the suspension was low and addition of carbonic anhydrase during illumination released only a small amount of CO(2) from the medium into the gas phase. The rate of CO(2) influx and the amount of CO(2) released by carbonic anhydrase were increased in WT during low CO(2) adaptation. These changes did not occur in RK1 during exposure to low CO(2). Cytoplasmic membrane from H-cells of WT or RK1 contained small amount of 42-kilodalton polypeptide. Exposure of RK1 to low CO(2) did not have significant effect on the amount of 42-kilodalton polypeptide, while the same treatment on WT resulted in a large increase of this polypeptide. The RK1 mutant appears to be defective in its ability to utilize the intracellular C(i) pool for photosynthesis and also to transmit a low CO(2) signal for inducing the functional and compositional changes observed in WT during low CO(2) adaptation.
Filamentous mutants were induced in a coccoid blue-green alga, Agmenellum quadruplicatum strain BG1, after treatment with N-methyl-N'-nitro-N-nitrosoguanidine (NTG). The mutants fall into two general classes: filaments with cross walls and filaments without cross walls. All mutants of these general types derived from BG1 are stable and have growth rates the same as or very similar to the wild type under a variety of conditions. Detailed examination of one mutant, 53SB2, revealed no difference in deoxyribonucleic acid content nor in base ratios. Mutant 53SB2 did not revert to the normal cell size and shape when grown under different physical conditions nor upon the addition of potential reversing agents to the basal medium. It is our general experience that filamentous mutants such as those described here in BG1 are commonly induced in other coccoid blue-green algae after NTG treatment.
Inactivation of the blue-green alga, Anacystis nidulans, with nitrosoguanidine was concentration-dependent. Nitrosoguanidine-induced mutation frequencies were: 1.8 · 10−3, 2.98 · 10[su−3], 3.7 · 10−3, 1.0 · 10−3 and 4.8 · 10−4 for yellow and blue, minute-colony-forming, filamentous or snake, and polymixin-B-resistant mutants, respectively. UV-irradiated damage within the experimental conditions utilized was completely photoreactivity by visible light. When a UV-irradiated culture was incubated in the dark for 9 h, a survival curve with slope was observed. A small shoulder was evident, and the extrapolation of the asymptote of the curve to the ordinate showed an inactivation number of 2. Minute-colony-forming and snake mutants were also induced by UV irradiation if photoreactivation was prevented. The inability to isolate auxotrophic mutants of Anacystis nidulans was considered to be due to intrinsic properties and therefore presents a major problem in blue-green algal genetics.
N-methyl-N′-nitro-N-nitrosoguanidine (NTG) induces at least one mutation per treated cell under conditions permitting over 50 per cent survival. The procedure for obtaining these results is to take logarithmic phase cells from either nutrient broth or minimal medium, wash them, and treat them with 100 μg of NTG per ml of buffer at pH 6.0 for 15 to 30 minutes. Prior to plating, the cells are again washed, and - if auxotrophs are sought - are diluted into nutrient broth and permitted to undergo two division cycles.With this procedure the yield of induced auxotrophs is 11 per cent; at 1000 pg NTG per ml the yield is over 40 per cent, but 95 per cent of the cells are killed.
Two U.V. induced mutants of nitrogen-fixing Anabaena doliolum have been isolated: 1. 5M 16 - This strain characterised as glucose-requiring nitrogen-fixing mutant, does not grow photo-autotrophically in the basal medium but does so when supplemented with glucose. Its growth rate is rather slow on AA-1, AA-3 and AA-4 but it grows very fast on AA-2. 2. L Y-5M 8/5M 12 - This strain characterised on the basis of periodic occurrence of massive lytic property, characteristic of each supplemented medium, grows initially for some time and then the whole population undergoes lysis.
IN the course of measuring the resistance of blue-green algae to γ-radiation from a cobalt-60 source, initially contaminated blue-green algae were found to be free from bacteria after irradiation. Since difficulties are encountered in the purification of Cyanophyta1 (less than 30 per cent of the organisms in the Indiana University Collection are bacteria-free2), γ-radiation may afford an easy, reliable method for purifying algae from bacteria and be of advantage in physiological and biochemical investigations of these organisms. Practical conditions for the purification of a group of typical Cyanophyceae were, therefore, determined.
Following the induction of synchronous growth of Anacystis nidulans by light and CO2 deprivation, cell mass and RNA and DNA content during two cell cycles were measured. Both RNA and DNA synthesis were discontinuous and marked variation in survival to ultraviolet light was related to the state of replication. A model is presented which accounts for the proportion of cells (66%) induced into synchronous genome replication which is also related to the state of replication at the onset of pre-synchrony treatment.
After exposure of cells to N-methyl-N-nitro-N-nitrosoguanidine a series of stable mutants of the marine coccoid blue-green alga, Agmenellum quadruplicatum, were isolated.1.
Two of these mutants were blocked at nitrate reductase and required nitrite or ammonium ion for growth.
2.
Four of the mutants were blocked at nitrite reductase, required NH4
+ for growth but had a functional nitrate reductase. These mutants readily reduced NO3
- to NO2
-.
3.
Two of the mutants had altered phycocyanin/chlorophyll a ratios, together with slightly impaired growth rates.
4.
Mutant AQ-19 responded to casamino acids and possibly represents an amino acid auxotroph.
Mutant strains of Anabaena cycadeae Reinke have been isolated after ultra-violet irradiation. All the four mutants described appear to be stable. They have been identified on the basis of their pigment composition, nutritional requirements, photoautotrophic growth and reaction to light. Strain 10 M
1L is a non-nitrogen-fixing mutant as indicated by its inability to grow on basal medium (AA) deficient in combined nitrogen. Strain 10 M
1L/10 M
1D is apochlorotic, and grows very slowly on medium AA-3 both in light and dark but comparatively better under the latter condition. Strain 10 M
1L/10 M
2D is deficient in β-carotenoid, photosensitive and able to grow in dark only on AA-3 medium while strain 10 M
1L/10 M
3D is a photoheterotrophic nitrogen-fixer.
The unicellular blue-green alga Anacystis nidulans was exposed to ultraviolet radiation during several successive subcultures. The ultraviolet-trained strain so obtained was then compared with the untrained control and found to be relatively more resistant to ultraviolet, penicillin, and streptomycin, and more sensitive to isoni-azid. It was further characterized by decreased carotenoid/chlorophyll ratio and by not having fully overcome the division-inhibiting effect of ultraviolet radiation. It was able to grow better than the untrained strain in a medium containing high concentrations of nitrite as the sole nitrogen source.
1.
Two mutant strains have been derived from Escherichia coli TAU-bar; one selected for resistance to nitrosoguanidine at pH 7.5 and the other for resistance to diazomethane. The comparative study of these strains shows that diazomethane, a decomposition product of nitrosoguanidine, is the principal agent involved in both lethal and mutagenic effects of nitrosoguanidine above pH 5. Nitrosoguanidine is the lethal agent at pH 5 and it is possibly a lesser mutagen itself.
2.
There are two types of chromosomal effects, lethal (susceptible to repair replication) and mutagenic (non-repairable), both due mostly to diazomethane.
3.
The expression of mutations was found to be independent of DNA replication or repair. The optimal conditions for nitrosoguanidine mutagenesis are discussed.
Evidence has been presented that blue-green alga Anacystic nidulans can undergo genetic transformation. DNA from erythromycin-, streptomycin-resistant of filamentous strains has been found to transform appropriate markers to a wild type or some other recipients. Favourable conditions for transformation have been described with respect to the revealing of transformants, the concentration of DNA and the competence of cells.
KUMAR1 reported the results of an investigation which seemed to demonstrate, for the first time, genetic recombination in the blue-green alga, Anacystis nidulans. This conclusion was based on an experiment in which a streptomycin-resistant and a penicillin-resistant strain were cultured together in the absence of antibiotics. Subculture of this mixture into liquid medium containing both antibiotics resulted in the growth of an apparently doubly resistant, recombinant strain. Because resistant clones had not been isolated, these results did not unequivocably demonstrate genetic recombination. Pikálek2 repeated Kumar's experiments and contested his conclusions by showing that the medium in which the apparently recombinant cells were growing lacked penicillin. He concluded that the observed growth was due to non-metabolizing streptomycin-resistant cells which were able to survive in penicillin for the short time before the decay of this antibiotic.
SEVERAL authors1-3 have induced mutations in blue-green algae by ultraviolet irradiation, but the mutation reported here seems particularly significant because it obscures the criteria for delimitation of taxa in the Cyanophyceae4. True branching is a distinctive feature of the algae comprising the order Stigonematales4 and is absent in Nostocales, although algae of the family Scytonemataceae of the latter order show false branching. This is the first report of a mutation induced by ultraviolet irradiation leading to true branching in the typically unbranched genus Nostoc of the order Nostocales.
Cells of Anacystis nidulans in the logarithmic phase of growth were briefly treated with the mutagen, N-methyl-N'-nitro-N-nitrosoguanidine, and plated under conditions suitable for single-cell growth. Selection of aberrant colonies and examination of the cultural characteristics of these clones suggest that mutants of at least several types are easily derivable in Anacystis.
-nitro-N-nitrosoguanidine in Escherichia coli
- N Adelberg
ADELBERG, N’-nitro-N-nitrosoguanidine in Escherichia coli. Biochemical and Biophysical Research Communications 1 8 7 788-795
clature in bacterial genetics
- M Demerec
- E A Adelberg
- A J Clark
DEMEREC, M., ADELBERG, E. A., CLARK, A. J. & HARTMAN, clature in bacterial genetics. Journal of General Microbiology 50, 1-14
Ultraviolet induced mutations to growth factor requirement and penicillin resistance in a blue-green alga
SRIVASTAVA, B. S. (1969). Ultraviolet induced mutations to growth factor requirement and penicillin
resistance in a blue-green alga. Archiv fur Mikrobiologie 66, 234-238.
Mutations of Anacystis nidulans that affect cell division A phylogenetic comparison of mutation spectra Short-trichome mutants of Plectonema boryanum Attempt to find genetic recombination in Anacystis nidulans
- R Cohen-Bazire
- G Archiv
- S I Redei
- G P Gowans
- E Shilo
KUNISAWA, R. & COHEN-BAZIRE, G. (1970). Mutations of Anacystis nidulans that affect cell division. Archiv
LI, S. I., REDEI, G. P. & GOWANS, C. S. (1967). A phylogenetic comparison of mutation spectra. Molecular
PADAN, E. & SHILO, M. (1969). Short-trichome mutants of Plectonema boryanum. Journal of Bacteriology 97,
PIKALEK, P. (1967). Attempt to find genetic recombination in Anacystis nidulans. Nature, London 215,
666-667.