Expression of c-myc and c-Ha-ras oncogenes in human lymphoblastoid cells (Namalva).

We have studied the expression of c-myc and c-Ha-ras oncogenes in this cell line. Daudi lymphoblastoid cells and normal human leukocytes served as a positive and a negative control, respectively. The Northern blot analysis using a c-myc probe revealed a 2.7 kb transcript and two larger transcripts greater than 23 kb. In the Northern blot hybridization using a c-Ha-ras probe, two transcripts with sizes of 1.8 and 6.1 kb were observed. The affinity of the Namalva RNA hybridization to c-myc probe was about 10-fold lower than that in Daudi RNA, whereas no difference in the c-Ha-ras hybridization was observed between the two cell lines. These data indicate that c-myc and c-Ha-ras are expressed in Namalva cells. It is noteworthy to consider that the difference in oncogene expression between Namalva and Daudi cells might be due to the difference in interferon properties between the two cell lines.

Plasmids of Azotobacter vinelandii.

Four laboratory strains and two isolates of Azotobacter vinelandii were found to contain plasmids. Twenty-five laboratory strains which could fix nitrogen did not have free, covalently closed circular plasmid DNA. The plasmids varied in size from 9 to 52 megadaltons, and each strain yielded only one plasmid. No discernible differences in ability to fix nitrogen were found between plasmid-bearing and cured cultures.

Growth of Azotobacter vinelandii on Soil Nutrients.

Azotobacter vinelandii cells grew well in a medium made from soil and distilled water which contained little or no carbohydrate. They utilized p-hydroxybenzoic acid and other phenolic acids, soil nitrogen, and water-soluble mineral substances. Seventeen soils which supported excellent growth of A. vinelandii contained 11 to 18 different phenolic acids each, including p-hydroxybenzoic, m-hydroxybenzoic, vanillic, p-coumeric, syringic, cis- and trans-ferrulic, and other unidentified aromatic acids. Three white, chalky "caliche" soils which were taken from areas where no plants grew failed to support the growth of A. vinelandii, and these contained no, two, and three phenolic acids, respectively. A. vinelandii did not fix nitrogen when growing in dialysates of soils which contained numerous phenolic acids. Growth was ample and rapid in most of the soils tested, but cell morphology was different from that usually seen in chemically defined, nitrogen-free media which contain glucose.

Survival of Azotobacter spp. in Dry Soils.

Dry soils stored in glass containers in the laboratory and protected from contamination for periods of 22 to 24 years yielded numerous colonies of Azotobacter chroococcum and other members of the family Azotobacteraceae. These results were compared with those reported in 1974, and the findings are uniformly consistent in terms of surviving populations. The data prove that these bacteria remain viable after prolonged periods of dormancy in much the same way as do the endospores of gram-positive bacteria.

True morphology of the Azotobacteraceae-filterable bacteria.

The history of bacteriology up to the 1950s contains many references to filterable forms of bacteria but misconceptions regarding the nature of filterability eventually made the entire phenomenon an unresolvable paradox. Although the existence of filterable azotobacter has often been reported, so much doubt in these results has been expressed that now Bergey does not even mention filterability. In 1965 we reported the resistance of soil azotobacter to gamma rays and concluded that the possibilty of a very small Azotobacter form could not be dismissed and as a result of studies on survival in soil, we suggested the possible existence in soil of a still undiscovered phase of the Azotobacter cyst. We report here the discovery of these small, soil azotobacter and provide evidence to support this claim.

Phosphate-limited culture of Azotobacter vinelandii.

Batch cultures of Azotobacter vinelandii grown in phosphate-deficient media were compared with control cultures grown in phosphate-sufficient media. Phosphate limitation was assessed by total cell yield and by growth kinetics. Although cell protein, nucleic acids, and early growth rate were unaffected by phosphate deficiency, cell wall structure, oxygen uptake, and cell viability were significantly affected. Also, phosphate-limited cells contained much larger amounts of poly-beta-hydroxybutyric acid but lower adenylate nucleotide energy charge than did control cells. The ratio of adenosine 5'-triphosphate to adenosine 5'-diphosphate was much lower in phosphate-deficient cells. The data indicate a substrate saving choice of three metabolic pathways available to this organism under different growth conditions.

Mechanism of lethal action of 2,450-MHz radiation on microorganisms.

Various bacteria, actinomycetes, fungi, and bacteriophages were exposed to microwaves of 2,450 +/- 20 MHz in the presence and in the absence of water. It was found that microorganisms were inactivated only when in the presence of water and that dry or lyophilized organisms were not affected even by extended exposures. The data presented here prove that microorganisms are killed by "thermal effect" only and that, most likely, there is no "nonthermal effect"; cell constituents other than water do not absorb sufficient energy to kill microbial cells.

The effect of temperature on phenol degradation in wastewater.

Data are presented which show that the microbiological degradation of phenol in industrial wastewater is affected by temperature in an unexpected manner. The rate of degradation is unaffected by temperature changes in the range from 24 to 10 degrees C but falls off rapidly at temperatures below 10 degrees C. In the interval from 10 to 2 degrees C the rate of degradation is a function of temperature and is proportional to the rate of growth of the bacteria studied. By contrast, the increase in rate of phenol degradation is much greater than the increase in growth rate as the temperature increases from 10 to 24 degrees C. Field and laboratory studies show that increased efficiency of phenol degradation in wastewater is due to increase in temperature rather than to shifts in microbial populations.

Physiology of L-asparaginase synthesis in recombinants of Escherichia coli A-1.

A mating between Escherichia coli 4318 (thi leu Las- Hfr) and E. coli A-1 (Met- Las+ F-) resulted in the formation of prototrophic recombinants having L-asparaginase activities at three distinct levels. The physiology of L-asparaginase synthesis in these recombinants is decribed. One class of recombinants produced significantly more L-asparaginase than E. coli A-1. L-Asparaginase synthesis in the recombinants was inhibited by the presence of dissolved oxygen in the medium and was transiently repressed by the presence of glucose in the same manner as that observed in the parental strains. L-Asparaginase activity was increased by the addition of oxalacetate as well as other members of the tricarboxylic acid cycle.

Effect of culture conditions on synthesis of L-asparaginase by Escherichia coli A-1.

The nutritional requirements and culture conditions affecting biosynthesis of L-asparaginase in a mutant of Escherichia coli HAP designated strain A-1 were studied. Asparaginase activity was increased by the addition of L-glutamic acid, L-glutamine, or commercial-grade monosodium glutamate. The rate of enzyme synthesis was dependent on the interaction between the pH of the culture and the amount of oxygen dissolved in the medium. A critical oxygen transfer rate essential for asparaginase formation was identified, and a fermentation procedure is described in which enzyme synthesis is controlled by aeration rate. Enhancement of L-asparaginase activity by monosodium glutamate was inhibited by the presence of glucose, culture pH, chloramphenicol, and oxygen dissolved in the fermentation medium.

Survival of Azotobacter in dry soil.

Detection of viable Azotobacter in soils stored in the laboratory for more than 10 years suggests that these bacteria can remain dormant in nature for prolonged periods of time. Studies of dried cultures show that cysts of A. vinelandii 12837 remain viable for at least 10 years, whereas vegetative cells do not survive for even 1 year under the same conditions.

Cysts of Azotobacter vinelandii with double coats.

Cyst germination was studied in cultures of Azotobacter vinelandii ATCC 12837. The existence of cysts enclosed by double coats is described.

Improved staining of extracellular polymer for electron microscopy: examination of Azotobacter, Zoogloea, Leuconostoc, and Bacillus.

Phase contrast, ultraviolet microscopy, and freeze-etching were used to determine the amount of exocellular polymer surrounding unfixed cells of four genera of bacteria: Azotobacter vinelandii, Zoogloea ramigera, Leuconostoc mesenteroides, and an acid-tolerant, floc-forming Bacillus species. Thin-sectional electron microscopy was employed to measure the effectiveness of a modified ruthenium red staining method. The results obtained with this modification of ruthenium red staining technique were compared to results obtained when previously proposed ruthenium red methods of fixation were employed. The results of these relations were then compared to the amounts of exocellular material as determined with phase-contrast microscopy, ultraviolet microscopy, and freeze-etching. The data obtained indicate that improved fixation of exocellular polymer is achieved when cells are pretreated with ruthenium red as described herein. In addition, the modified methods also reveal cytological detail not apparent when other methods of ruthenium fixation are employed.

Effect of peptone on Azotobacter morphology.

Pleomorphism in cultures of Azotobacter vinelandii was induced by addition of Difco peptone to the growth medium. Under the conditions of the study, 5% peptone gave rise to transient forms described as "fungoid cells" which were osmotically fragile. After some 48 hr of culture, they became osmotically stable and resumed a more typical morphology. It was shown that the pleomorphism-inducing principle in peptone was glycine.

Freeze-etching of Azotobacter vinelandii: examination of wall, exine, and vesicles.

The structure of the cell wall, the arrangement of the cyst exine, and the origin and distribution of intine vesicles in Azotobacter vinelandii ATCC 12837 were examined by freeze-etching and conventional electron microscopic techniques. In the vegetative organism the cell wall appears to have a woven texture which disappears during cyst formation. The exine is composed of two different types of material: the outer layer is a fibrous, amorphous layer, and the numerous inner layers form the basic hexagonal structures which unite to form the cyst coat. The presence of intine vesicles in the encysting organism was confirmed in frozen-etched cells. The appearance of frozen-etched cells and cysts and the distribution of capsular material indicate that extracellular polysaccharide is an important factor in cyst formation.