Growth of cyanophage N-1 under the influence of heavy metal ions.

The growth of cyanophage N-1 in the cyanobacterium Nostoc muscorum under the influence of heavy metal ions, namely Co2+, Cr6+, Cu2+, Mn2+ and Ni2+ has been studied. One-step growth experiments revealed that heavy metal ions extended the latent period by 1-2 hrs with a concomitant decrease in the phage burst size. The latter was reduced in the order Cu2/Mn2+, Ni2+, Co2+ and Cr6+. The treatment of the phage-infected bacteria with heavy metal ions did not induce mutations affecting either the phage plaque morphology or burst size. The final phage titer after such a treatments was lowest with Co2+, Cu2+ and Cr6+. The inhibition of the phage growth under the influence of heavy metal ions is discussed in context with the interaction of cyanophage N-1 with the photosynthetic reactions in the host bacteria.

Nutrient stress causes akinete differentiation in cyanobacterium Anabaena torulosa with concomitant increase in nitrogen reserve substances.

Addition of nitrogen source (nitrate), carbon sources (acetate, citrate and fructose), depletion of nutrients (phosphate-free nitrate medium), dilution of medium (2, 4 and 8 times diluted nitrate medium) under unaerated conditions induced akinete differentiation in Anabaena torulosa. Aerated cultures under the same conditions did not differentiate akinetes. The amounts of reserve metabolites--glycogen and cyanophycin (multi-L-arginyl-poly-L-aspartic acid) granule polypeptide (CGP)--were determined in unaerated and aerated cultures, and at different stages of growth and akinete differentiation. The addition of nitrate, acetate, citrate and fructose under unaerated conditions resulted in the accumulation of glycogen and CGP in higher amounts after 4 d (akinete initiation); the CGP content further changed at mature free akinetes phase. Higher accumulation of reserve products was also observed under nutrient deficiency (phosphate-depleted or diluted media) after 4 d of cultivation. Under aerated conditions reserve product accumulation was considerably lower. Thus a low accumulation of reserve products in aerated cultures showed that aeration probably somehow relieves the organism from a nutritional stress.

Effect of nutrients and aeration on O2 evolution and photosynthetic pigments of Anabaena torulosa during akinete differentiation.

The addition of a nitrogen (nitrate) and carbon sources (acetate, citrate and fructose) and phosphate deficiency (nitrate medium deficient in phosphate) under unaerated conditions induced akinete differentiation in Anabaena torulosa. Aerated cultures of this organism in these nutrients did not differentiate akinetes. Oxygen evolution by aerated cultures was higher when compared to unaerated cultures, which concurred with high chlorophyll content of aerated cultures. Nitrate nitrogen supported high phycocyanin content in unaerated cultures; phycocyanin and allophycocyanin contents were low under aerated conditions. The contents of phycocyanin, allophycocyanin, phycoerythrin and carotenoids gradually decreased at the mature akinete phase. Under aerated conditions, chlorophyll content rose and the content of all the pigments increased with the growth rate of the organism.

Characterization of host-range mutants of cyanophage N-1.

Fifteen host-range (h) mutants of cyanophage N-1 were characterized with reference to their efficiency of plating, time of appearance, morphology and size of plaques on Nostoc muscorum and its three phage-resistant (Nm 1/N-1, Nm 2/N-1 and Nm 8/N-1) mutants. While phage N-1 did not adsorb to the three phage-resistant mutants, the h mutants differed one from the other in having lower or higher adsorption rate constants on N. muscorum or the phage-resistant mutants. The inability of majority of h mutants isolated on Nm 1/N-1 to grow in Nm 8/N-1 was shown to be due to a failure of adsorption. The h mutants also differed one from the other in their reversion (back mutation) frequencies. The lethal doses (LD37) required to kill 37% of free phage particles after UV-irradiation, heating and ethylenediamine tetraacetate (EDTA) treatment greatly varied. Most of the h mutants were found to be considerably more sensitive to UV and thermic inactivation than N-1 while they were resistant to EDTA. The h mutants except five of them were unable to multiply at 40 degrees C. The significance of these features is discussed.

Characterization of TS-mutants of cyanophage N-1 by their inactivation by physical and chemical agents.

The effect of temperature, ultraviolet (UV) light and ethylenediaminetetraacetic acid (EDTA) on the stability of cyanophage N-1, infecting the cyanobacterium Nostoc muscorum was studied. Complete inactivation of the phage occurred at 60 degrees C in 6 mins. All the temperature-sensitive (ts) mutants exhibited faster inactivation at 50 degrees C than the wild type. UV light readily inactivated the particles of the wild giving a survival of 3.44% at a dose of 60 secs. All the ts-mutants were found to be more sensitive to UV light than the wild type. 10(-4) mol/l EDTA inactivated 40% of the wild type in 60 mins. 5 x 10(-4) mol/l EDTA inactivated the wild type nearly completely within 2 mins, while a similar inactivation of ts-mutants required only 90 secs.

Isolation and characterization of temperature-sensitive mutants of cyanophage N-1.

Optimal conditions for the induction of temperature-sensitive (ts) mutants of cyanophage N-1 were established after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). A treatment with MNNG (400 micrograms/ml) for 2 hrs at pH 8.0 induced ts-mutants at a maximum frequency of 1.46 x 10(-3). A characterization of 10 such ts-mutants with regard to adsorption, one-step growth and temperature-shift experiments with Nostoc muscorum as host bacterium led to the identification of temperature-sensitive steps in the phage multiplication at the restrictive temperature (37 degrees C). All the mutants were found to be conditionally lethal at 37 degrees C since they resumed growth upon shifting to 28 degrees C.

Spontaneous and induced host-range mutants of cyanophage N-1.

Optimal conditions for the induction of host-range mutants of cyanophage N-1 by acridine orange were established. Induced host-range mutants were isolated with a frequency of 0.1 to 4.0 x 10(-5) over a spontaneous mutation frequency of 0.2-3.6 x 10(-11).

Biochemical studies on sporulation in blue-green algae. III. Effect of amino acids on glycogen accumulation.

The effect of 21 amino acids was studied on glycogen accumulation during sporulation in the blue-green alga Anabaena sp. All the amino acids enhanced the initial level of glycogen on the 4th day. The maximum amount of glycogen, on the 20th day, was noticed from L-methionine, L-tyrosine, glycine, and L-histidine supplemented cultures. Others like L-serine, L-valine, L-asparagine, DL-alanine, L-glutamic acid, L-phenylalanine, L-aspartic acid, L-arginine, and DL-lysine come next in the order. On the other hand, L-cysteine and L-cystine, although upto the the 16th day they exhibited higher values of glycogen, did not show much variation in glycogen content over nitrogen-free medium. Except in these two amino acids, in all others initiation of sporulation occurred on the 4th day, resulting in free spores on the 8th day. But in case of L-serine, L-asparagine, L-isoleucine, and L-phenylalanine free spores were noticed only on the 12th day while in DL-lysine they were seen on the 16th day. L-cysteine and L-cystine supplemented media showed free spores on the 20th day as in nitrogen-free control.

Biochemical studies on sporulation in blue-green algae. II. Factors affecting glycogen accumulation.

Inorganic nitrogens sources like nitrate, nitrite enhanced sporulation and glycogen accumulation in Anabaena sp. but ammonium chloride neither influenced sporulation nor glycogen accumulation. Acetate and citrate also stimulated early sporulation and glycogen level was higher over nitrogen free control. Nitrogen and carbon sources in combination proved to be useful in inducing early sporulation and increased content of glycogen. Phosphate and calcium also affected glycogen accumulation significantly, although, the sporulation was found to be of the same order as in nitrogen free medium. Sulphate initiated early sporulation, the mechanism of which is not known.

Biochemical studies on sporulation in blue-green algae. I. Glycogen accumulation.

Glycogen accumulation in vegetative cells of Anabaena sp. is demonstrated to be a light-dependent process. No glycogen accumulation is found in dark or in cultures supplemented with 10(-5) M DCMU in light. Large quantities of glycogen accumulate in cells undergoing sporulation and the amount increased with the onset of maturation of spores.