Stimulation of in vivo nitrate reductase activity in the thermal cyanobacteriumOscillatoria princeps under microaerobic conditions

Nitrate reductase activity in the thermal cyanobacteriumOscillatoria princeps was studied in vivo. Nitrite production byO. princeps grown microaerobically was four- to seven-fold higher than that in aerobic controls. Whereas microaerobic conditions caused instantaneous stimulation of nitrate reductase activity, addition of 40% O₂ in air immediately inhibited it. Exposure of another cyanobacteriumAnabaena doliolum to microaerobic conditions had no effect on NO ₂ ^– production.     

Regulation of assimilatory nitrate reductase in the cyanobacteriumAnabaena doliolum

The assimilatory nitrate reductase (NR) from the cyanobacteriumAnabaena doliolum was membrane bound and solubilized by sonication. The Km value of the enzyme was 870 µM for nitrate with dithionite-reduced methyl viologen (MV) as electron donor. The pH optimum was 10.5 in the MV assay. Nitrate acted as an inducer and ammonium as repressor of the enzyme synthesis. In the presence ofl-methionine-d,l-sulfoximine (MSX) or azaserine, inhibitors of the glutamine synthetase-glutamate synthase (GS-GOGAT) pathway, ammonium did not exhibit any inhibitory effect on the enzyme. The photosynthetic nature of NR was shown with PS II inhibitor, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). This enzyme fromA. doliolum has been shown to be a light-driven process, requiring de novo protein synthesis. It was inhibited by chlorate, the structural analog of nitrate;p-chloromercuribenzoate, a thiol reagent; sodium tungstate; and certain cations.     

Induction of Oligosporogeneous and Asporogeneous Mutants of Blue-green Alga, Anabaena doliolum by Acriflavine and Acridine Orange

Acriflavine and acridine orange were highly effective in producingmutants defective in spore differentiation in the blue-greenalga, Anabaena doliolum. Acridine orange produced both oligosporogeneous(Osp) and asporogeneous (Sp^–) mutants in high yield withthe frequency ranging from 2.4 to 21 per 10⁴ survivors, whereaswith acriflavine no oligosporogeneous mutants were detected,although it produced non-sporulating (Sp^–) mutants withthe frequency of 1.4 per 10⁴ survivors. Mutants isolated throughthese dyes were stable and showed no tendency towards spontaneousor induced reversion. Behaviour of various mutants indicatedtheir being blocked at different stages of sporulation withdifferent mutagenic sites. Induction of high numbers of Ospand Sp^– mutants after acridine dye treatment indicatesthe involvement of an extrachromosomal determinant in sporulationin Anabaena doliolum. Ohgosporogeneous mutants, asporogeneous mutants, Anabaena doliolum, blue-green algae, mutagenesis, acriflavine, acridine orange     

A comparative study of antioxidative defense system in the copper and temperature acclimated strains of <Emphasis Type="Italic">Anabaena doliolum</Emphasis>

This study provides first hand comparative account of growth and antioxidative defense system of the wild type, Cu²⁺ and temperature treated wild type and acclimated strains of Anabaena doliolum Bharadwaja against Cu²⁺ and high temperature. The acclimated strains showed perceptible growth at 250 μM Cu²⁺ and 47°C temperatures, respectively. In contrast to this the wild type strain on exposure to 50 μM Cu²⁺ and 47°C temperature depicted almost complete inhibition of growth. However, the peroxide content was significantly higher in the acclimated strains than the wild type. Superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) showed maximum activity at high temperature followed by Cu²⁺ acclimated and minimum in the wild type strains. The ascorbate (ASC) and glutathione (GSH) contents were increased by 2.3 and 43.3, and 15.5 and 36.5-fold in Cu²⁺ and 47°C acclimated strains, respectively. However, when the wild type strain was subjected to Cu²⁺ and temperature all antioxidative enzymes except SOD showed inhibition of their activity. In case of wild type the GSH content was inhibited by 0.39-fold at 50 μM Cu²⁺ but the ASC content registered increase by 2 and 2.7-fold on subjecting to Cu²⁺ and temperature, respectively. Thus increased activity of enzymatic antioxidants as well as accumulation of ascorbate and glutathione in both the acclimated strains suggests that enzymatic and non-enzymatic antioxidants help in the acclimation of A. doliolum Bharadwaja against Cu²⁺ and high temperature. However, inhibition of antioxidative defense system of wild type under Cu²⁺ and heat stress appears to be the reason for its non survival. In view of the appreciable increase in the level of antioxidants as well as greater inhibition of specific growth rate in temperature than Cu²⁺ acclimated strains, temperature (47°C) is proposed to be is more deleterious to the organism than copper (250 μM).     

Genetic control of sporulation in the blue-green alga Anabaena doliolum Bharadwaja

Summary Two genetically distinct, non-sporulating mutant strains of Anabaena doliolum were isolated following exposure of the alga to ultraviolet radiation. Among the progeny of a cross between the two non-sporulating strains, some filaments were able to sporulate. The spores of such filaments were of two types. Type 1 upon germination produced the parental phenotype, type 2 gave rise to wild phenotype. This suggests the involvement of heterozygosity in genetic recombination in A. doliolum. The results further indicate that the formation of spores in this alga is under the control of more than one genetic determinant, and that nuclear segregation occurs during sporulation.     

Response ofAnabaena doliolum to bimetallic combinations of Cu,Ni and Fe with special reference to sequential addition

This study reports physiological features of a N₂-fixing cyanobacteriumAnabaena doliolum in response to metal mixtures. Exposure of the cyanobacterium to Cu, Ni and Fe individually, as well as in combinations (Cu + Ni, Cu + Fe, Ni + Fe), showed marked differences in growth inhibition, nutrient uptake (NH₄ ⁺ and NO₃ ⁻), photosynthesis, ATP content, nitrate reductase, glutamine synthetase and urease activities. The response to metal combinations was also dependent upon the order in which the metals were added. The Cu-Ni combination resulted in synergistic interaction, in contrast to the antagonism of Cu-Fe and Ni-Fe. Pre-addition of Fe protected the cyanobacterium against Cu and Ni toxicity. Statistically significant (P < 0.005) inhibition of all the processes following metal supplementation was observed. This study suggests that carbon fixation is the most suitable variable for assessing heavy metal toxicity.     

Loss of hydrazine-induced mutability in wild-type and excision-repair-defective yeast during post-treatment inhibition ofcell division.

The fate of presumed premutational DNA lesions induced by hydrazine was studied under a variety of post-treatment conditions in wild-type and in excision repair-defective (rad2-1) strains of Saccharomyces cerevisiae. In all strains the full extent of hydrazine-induced, forward mutability from CAN1 to can1 (canavanine resistance) was dependent upon post-treatment cell division in mutagen-free synthetic or complex growth medium before plating on canavanine-containing selective agar and could be blocked by inhibitors of DNA synthesis (hydroxyurea) or protein synthesis (cycloheximide) contained in the growth medium. Following the growth-inhibitory period, cells were permitted to grow in fresh medium lacking inhibitors to determine the level of induced mutation remaining. Nearly all induced mutability was lost after a one-day growth inhibition, compared with mutagen-treated control samples subsequently grown twice in medium lacking inhibitor. In the wild type, half the induced mutability was lost after 3 h. The data suggest that premutational DNA lesions induced by hydrazine were removed, or possibly rendered non-mutagenic, by some error-free repair process that acted before mutation fixation by base mispairing during DNA replication. Since rad2-1 and RAD strains both exhibited loss of mutability, this process is not dependent upon the activity of an intact pyrimidine dimer excision-repair system.     

Variable expression of the ssb-1 allele in different strains of Escherichia coli K12 and B: Differential suppression of its effects on DNA replication, DNA repair and ultraviolet mutagenesis

Summary We have transduced the mutant allele ssb-1, which encodes a temperature-sensitive single-strand DNA binding protein (SSB), into several Escherichia coli strains, and have examined colony-forming ability, DNA replication, sensitivity to ultraviolet light (UV) and UV-induced mutability at the nonpermissive temperature. We have found: 1) that the degree of ssb-1-mediated temperature-sensitivity of colony-forming ability and of DNA replication is strain-dependent, resulting in plating efficiencies at 42° C (relative to 30° C) ranging from 100% to 0.002%; 2) that complete suppression of the temperature-sensitivity caused by ssb-1 occurs only on nutrient agar, and not in any other medium tested; 3) that strains in which ssb-1-mediated temperature-sensitivity is completely suppressed show moderate UV sensitivity and normal UV mutability at 30° C, but much more extreme UV sensitivity and drastically reduced UV mutability at 42° C; and 4) that defects in excision repair or in other Uvr⁺-dependent processes are not responsible for most of the UV sensitivity promoted by ssb-1. We discuss our results in relation to the known properties of SSB and its possible role in the induction of DNA damage-inducible (SOS) functions.     

Factors modulating differentiation of spores: Characterization of mutants defective in sporulation in the cyanobacterium Anabaena doliolum (AdS strain)

Summary Mutants of Anabaena doliolum (AdS strain) altered with respect to the time of initiation and degree of sporulation were isolated following mutagenesis with N-methyl-N-nitro-N-nitrosoguanidine and hydroxylamine. The non-sporulating mutant showed a high phycocyanin (Pc): chlorophyll a (chl a) ratio (ca. 7.2) as compared to sporulating strains (Pc:chl a, 4.7–5.3). Also this strain seemed to have higher RNA pools per unit of genomic material as reflected in a higher RNA:DNA ratio. The data suggest that degradaton of phycocyanin and controlled RNA synthesis are prerequisites for sporulation. Mutants exhibiting non-sporulation and delayed initiation of sporulation accumulated more nitrogen through nitrogen fixation, probably indicating nitrogenase function over an extended vegetative phase.     

Restoration of mutability in non-mutable Escherichia coli carrying different plasmids.

Summary N and I group plasmids, which increase methylmethane sulfonate (MMS) mutagenesis in lexA ⁺ strains of E. coli WP2 may be divided into two classes: those restoring part of the mutability of lexA ^- strains (class I) and those leaving lexA ^- strains non-mutable (class II). Almost complete restoration of MMS mutability is obtained by class I plasmids in a partially suppressed lexA rnm strain, while class II plasmids cause far fewer MMS revertants in this strain than in lexA ⁺. A pair of class I and II plasmids in lexA ^- shows a synergistic effect on mutability. These two classes do not coincide with plasmid division into incompatibility groups.     

Effects of physical culture parameters on bacteriocin production by Mexican strains of Bacillus thuringiensis after cellular induction

We have shown previously that in the presence of inducer Bacillus cereus 183, significant increases in bacteriocin production and bactericidal activity of B. thuringiensis occur when the latter is cultivated at pH 7.2, 28°C, and 180 rpm. Here we show that this activity can be further improved when B. thuringiensis is induced with B. cereus 183 and then cultivated with modification of pH, temperature, and agitation. Five native strains of B. thuringiensis, LBIT 269, LBIT 287, LBIT 404, LBIT 420, and LBIT 524 which synthesize, respectively, morricin 269, kurstacin 287, kenyacin 404, entomocin 420, and tolworthcin 524, were cultivated in four different fermentation media. Of these, fermentation in tryptic soy broth (TSB) yielded the highest level of bacteriocin activity (~100–133 FU). Bacteria grown in TSB were induced with B. cereus 183 and cultivated at different pH (6.0, 7.2, 8.0), temperature (26, 28, 30°C), and agitation (150, 180, 210 rpm). Full factorial design was performed and results were analyzed with analysis of variance (ANOVA) and Tukey multiple comparison tests at significant level of α ≤ 0.05 to study the influence of the three variables on bacterial growth and bacteriocin production. Our data show that the highest bacteriocin activity was found with LBIT 269 and LBIT 404 with an increase of ~95–100% compared with induced B. thuringiensis strains cultivated under fixed conditions (pH 7.2, 28°C, 180 rpm), for which the data were set at 0%. The optimal conditions for morricin 269 and kenyacin 404 production were, respectively, pH 8, 30°C, 210 rpm and pH 7.2, 26°C, 210 rpm.     

Potential of ochratoxin A production by Aspergillus carbonarius strains isolated from grapes at different ecological factors

Aspergillus carbonarius is known to colonize and produce ochratoxin A (OTA) on grapes and its derived products which is harmful to humans. We screened and tested A. carbonarius strains which isolated from grapes for production of OTA and selected three high OTA producing strains (ACSP1, ACSP2, ACSP3) for this study. These strains were further tested for their ability to produce OTA at different ecological factors [temperature 15, 25, 30, 35°C; water activity (a_w) 0.98, 0.95, 0.90, 0.88; and pH 4.0, 7.0, 9.0, 10.0]. Out of the three strains tested, A. carbonarius ACSP3 produced high levels of OTA than ACSP2 and ACSP1 in all the ecological factors. At 30°C A. carbonarius strains produced the highest OTA compared with other temperature regimes. With reference to water activity, a_w 0.98 favoured mycelial growth and accumulation of more OTA with all the three A. carbonarius strains. Further, pH 4.0 was encouraged the greatest production of OTA in all the strains. No growth was observed at a_w 0.88 and pH 10.0 in all the three strains except the strain ACSP3 at high pH. Our work demonstrated that temperature 30°C, a_w 0.98 and pH 4.0 is optimum for growth and production of OTA by A. carbonarius strains. Maximum amounts of OTA were found at earlier growth stages (7–9 days of incubation) in all the strains of A. carbonarius. The present study revealed that different ecological factors had great impact on OTA production by A. carbonarius which is useful for understanding OTA contamination and to develop proper management practices in future research programmes.     

Pigment variations in ultraviolet-treated strains of the blue-green alga Anabaena doliolum

Summary Two kinds of cultures were raised from clones of Anabaena doliolum surviving on selective medium following exposure of spores to ultraviolet radiation. The pigments of these cultures have been characterized with respect to those of controls.     

Nitrogenase activity of the antarctic cyanobacteriumNostoc commune: Influence of temperature

Nitrogenase activity (C₂H₂ reduction) ofNostoc commune isolated from Schirmacher Oasis (Antarctica) was compared withNostoc muscorum, N. calcicola, Anabaena doliolum andGloeocapsa sp. The temperature profile of acetylene reduction (5–30 °C) forN. commune revealed (a) that the highest rate of nitrogenase activity was at 25±1 °C, (b) that it was low (69 %) in comparison withN. muscorum, and (c) that nitrogenase activity continued at lower temperatures, which was not evident for other cyanobacteria. The results suggest thatN. commune is adapted to lower temperatures in terms of nitrogen fixation.     

Characterization of salinity-tolerant mutant of Anabaena doliolum exhibiting multiple stress tolerance

Results show that an isolated mutant of the cyanobacterium Anabaena doliolum is a fast-growing strain. It exhibits approximately twofold higher NaCl tolerance than the wild type. It also reveals cross-resistance against the herbicide 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), drug bacitracin, and LiCl. Further, an improved LiCl tolerance property of both the mutant and wild-type strains at high concentration of NaCl (40 mM) may be interpreted in terms of competitive inhibition of the Li⁺ uptake by Na⁺ ions, whereas bacitracin resistance in these organisms is described to be the result of an alteration in the drug transporting channels of membrane. The multiple stress tolerance property of the A. doliolum may be attributed to altered membrane characteristics in the mutant strain, leading to reduced intake of such toxicants. Received: 6 November 2001 / Accepted: 14 December 2001     

A comparative study of the wild and mutated heavy metal resistant Klebsiella variicola generated for cadmium bioremediation

Ten indigenous heavy metals resistant bacteria were isolated from the discharged effluent of Biological Sciences building at Obafemi Awolowo University, Ile-Ife, Osun State, Nigeria. The bacterial strains were isolated using enrichment method in tryptone soya agar (TSA) supplemented with 100 mg/L of Pb, Cd, As, and Ni. All the isolated bacteria showed multiple tolerances to the heavy metals; however, strain GBB 220, which showed a maximum tolerable concentrations (MTCs) of 2000, 1200, 4700, and 1000 mg/L for Pb, Cd, As, and Ni, respectively, was selected for further studies. The bacterium was identified by 16S rRNA sequences as Klebsiella variicola and was subjected to mutational enhancement by acridine orange and ethidium bromide. Eight mutants were recovered, strains K. variicola MutAa–Ad showed improvement in their MTCs of 2500, 2200, and 5000 mg/L for Pb, Cd, and As whereas K. variicola MutEa–Ed had same MTCs as the wild type except for cadmium which increased from 1200 to 1500 mg/L. The antibiotic susceptibility patterns showed that all the strains of K. variicola had multiple resistances to some of the antibiotics. The K. variicola mutants had their optimum pH at 5 and 8 while the wild type had optimum pH at 7 and 8. The cadmium removal efficiency of 97.9% at pH 7, 97.7% at pH 8, and 99.4% at pH 7 was observed for K. variicola Wt, K. variicola Mut Eb, and K. variicola Mut Ac, respectively. These findings suggested that the artificially mutated strains of K. variicola may be applied to remove cadmium from polluted environment.     

The isolation and characterization of ngm2, a mutation that affects nitrosoguanidine mutagenesis in yeast

Summary We have isolated and characterized a new mutant of Saccharomyces cerevisiae, carrying a single mutant allele that we designate ngm2-1, which is defective with respect to induced mutagenesis. This mutant was isolated by screening mutagenized clones for reduced frequencies of reversion of the his1-7 allele, induced by N-methyl-N-nitro-N-nitrosoguanidine. As judged by the reversion of his1-7 and ilv1-92, ngm2-1 mutant strains are also deficient with respect to mutability induced by methyl methane sulfonate, ethyl methane sulfonate and, at least partially, by UV. UV-induced reversion of the ochre mutation arg4-17 and the frameshift mutation his4-38 was not much affected by ngm2-1, however. Like rev3 and rev7 mutations, ngm2-1 also has little influence on the reversion of the proline missense allele, cyc1-115. Ngm2-1 mutants are only at best very slightly more sensitive to the toxicity of the four mutagens used, and homozygous diploids sporulate normally.     

Temperature shift-induced changes in the antioxidant enzyme system of cyanobacteriumSynechocystis PCC 6803

The 24 h effect of low (20°C) and high (43°C) temperature on the antioxidant enzyme activities and lipid peroxidation was investigated in intact cells of the cyanobacteriumSynechocystis PCC 6803 grown at 36°C. At low temperature treated cells, the superoxide dismutase, catalase and glutathione peroxidase activities were significantly higher and the protein content lower than in high temperature treated cells. The increase of hydroxyl free radical level and malonyldialdehyde formation, when algal cells were exposed to low temperature, were due to the stimulated production of superoxide radicals O₂ ⁻ and hydrogen peroxide (H₂O₂).     

The uvsC and uvsE genes of Aspergillus nidulans are not required for the mutagenic repair of UV damage

The uvsC gene of Aspergillus nidulans is a homolog of the RAD51 gene of Saccharomyces cerevisiae. However, with respect to its effects on UV mutagenesis, it differs from the yeast gene, since it seems to be required for UV mutagenesis; however, this conclusion is based only on data from resting conidia. To further clarify the functional role of the uvsC gene, we tested the UV mutability of strains bearing a uvsC mutation in resting as well as in germinating conidia, by the p-fluoro-phenyl-alanine resistance test. We also evaluated the mutability of the uvsE mutant which belongs to the same epistatic group. Our results show that the uvsC and uvsE genes do not have a significant role in the mutagenic UV-repair pathway. Received: 20 January 1998 / Accepted: 22 April 1998     

Linoleate isomerase activity occurs in lactic acid bacteria strains and is affected by pH and temperature

Aims: To investigate the ability of lactic acid bacteria (LAB) to convert linoleic acid (LA) and α‐linolenic acid (α‐LNA) to conjugated linoleic acid (CLA) and conjugated linolenic acid (CLNA), respectively. To assess pH and temperature influences on CLA and CLNA production by Lactobacillus sakei LMG 13558. Methods and Results: A screening of 48 LAB yielded one Lactobacillus curvatus, five Lactobacillus plantarum and four Lact. sakei strains displaying linoleate isomerase (LAI) activity. CLNA conversion percentages varied largely (1–60%). CLA conversion, occurring in three strains, was lower (2–5%). The LAI gene sequences of the ten LAI‐positive strains shared 75–99% identity with the LAI gene sequence of a Lact. plantarum AS1.555. At pH 6·2, CLA and CLNA production by Lact. sakei LMG 13558 was higher at 30°C than at 20 and 25°C. At pH 5·5 (30°C) or 37°C (pH 6·2), LA was not converted and α‐LNA only slightly converted. Conclusions: LAB show strain‐dependent LAI activity. Production of CLA and CLNA is affected by pH and temperature, as shown for Lact. sakei LMG 13558. Significance and Impact of the Study: Several LAB produce CLA and/or CLNA, as shown for Lact. sakei and Lact. curvatus for the first time. These findings offer potential for the manufacturing of fermented functional foods.