- Volume 20, Issue 3, 1959

SUMMARY: Parallel physiological and cytological investigations of a chlortetra-cycline-producing culture—Streptomyces aureofaciens, strain 536—indicate some intracellular causes of the dependence of chlortetracycline biosynthesis upon the inorganic phosphorus content of the nutrient medium.

Increase of inorganic phosphorus in the fermentation medium led to a formation of large nuclear elements containing much DNA (in corn steep medium) or ribonucleo-proteid complexes in the shape of large volutin granules (corn steep liquor ash medium). As compared with the control, the nuclear substance/cytoplasm ratio in the hyphae is increased. The ability of mycelium overloaded with nuclear elements or volutin granules for chlortetracycline biosynthesis is markedly decreased. The iufluence of phosphorus on the microscopical structure of mycelium and biosynthesis of antibiotic depends on the composition of medium. The poor quality of certain batches of corn steep liquor used for the production of chlortetracycline is largely due to their high content of inorganic phosphorus.

SUMMARY: A total of 978 cultures of heterotrophic organisms were isolated from twelve actively nitrifying soils; each isolate was tested for ability to form nitrite or nitrate in glucose peptone broth. None of the isolates yielded substantial amounts of nitrite; concentrations found did not exceed 2 μg. nitrite-N/ml. Almost 7 % of the isolates formed nitrite-N in excess of 0·2 μg./ml., while slightly over 2 % yielded more than 0·5 μg./ml. Fungus isolates were the most numerous and most active nitrite producers; fifteen of the fungi formed nitrate in addition to nitrite. Concentrations of 5–45 μg. nitrate-N/ml. were recorded for the active fungi. Further isolations of fungi from ten soils of diverse properties resulted in 353 cultures but only three of these formed nitrate. One of these cultures yielded 90 μg. nitrate-N/ml. after 14 days growth in the test medium.

Most of the fungi which produced nitrate (16 of the 18 active cultures) were identified as Aspergillus flavus. A Penicillium sp., and a Cephalosporium sp. also formed low concentrations of nitrate. Each isolate of A. flavus obtained from soil proved capable of nitrate formation. Strains of A. flavus were encountered which did not yield nitrate, but these had been obtained from culture collections, and had been carried on artificial media for many years. Some stock cultures of A. flavus, several cultures of A. flavus and one of A. glaucus, freshly isolated from wheat seed, also were active nitrate producers. Ammonium sulphate and urea media supported growth of soil fungus isolates but not nitrate formation. Other organic nitrogen substrates in order of increasing effectiveness in nitrate production were: yeast extract, peptone, Protone and casein.

SUMMARY: An enzyme which splits high molecular weight polymetaphosphate (PMP) to orthophosphate has been extracted from Corynebacterium xerosis, partially purified and some of its properties studied. A method for the preparation of bacterial PMP labelled with 32P is described. The enzyme hydrolyses bacterial or synthetic PMP at the same rate. No formation of short-chain phosphate polymers was detected during the reaction and the enzyme did not attack metaphosphates smaller than hexametaphosphate. It does not transfer phosphate from PMP to ADP to form ATP, nor does it synthesize PMP from orthophosphate (Pi) in the presence of ATP or other nucleotides. All the metal ions tested, including Mg++, inhibited the enzyme. Sodium ethylenediaminetetraacetate (EDTA; 0·2 mm) stimulated the rate of reaction; higher concentrations inhibited. The significance of polymetaphosphate and enzymes which split it in micro-organisms is briefly discussed.

SUMMARY: The fine structure of the yeast-like cells of Histoplasma capsulatum Darling and H. duboisii Vanbreuseghem has been studied and compared by electron microscopy. The micro-morphology of single and budding cells, as well as some details of the budding process, are described. No evidence of a capsule or slime layer on the outside of the cell wall of either species was demonstrated. In both species the cell walls were found to be laminated and in general slightly thicker in H. duboisii than in H. capsulatum. The possibility of the laminations being associated with the presence of chitin in the framework of the cell walls is discussed. The cells of the two species, strikingly similar in structure, were found to be highly differentiated, resembling animal cells in some of their micromorphological features, namely in the presence of endomembrane systems, in mitochondrial numbers and forms, and in nuclear fine structure.

SUMMARY: A significant extension in the variety of gene-markers available in Saccharomyces has been achieved by exploiting resistance and sensitivity to a variety of poisons. Although resistance to many poisons was obviously heritable, gene- control could not be demonstrated, presumably because other genes modified the phenotypes. Gene-control of resistance and sensitivity for both nicotine and caffeine was demonstrated by tetrad analysis and by the observation of regular Mendelian segregation of the characters among the progeny of closely related hybrids.

SUMMARY: The synthesis and breakdown of poly-β-hydroxybutyric acid in growing cultures of selected strains of Rhizobium, Spirillum and Pseudomonas are described. The occurrence of the polymer in root nodules of tropical and temperate leguminous plants is reported.

SUMMARY: Protoplasts were prepared from cells of Bacillus megaterium, strain M, by lysozyme treatment in the presence of sucrose. The protoplasts were shocked osmotically and the lysate thus obtained was fractionated into membranous ‘ghosts’ and soluble protoplasm by means of differential centrifugations. The distribution of various enzymes between these two fractions was studied. The bulk of the cytochromes, the succinic dehydrogenase and the diphosphopyridine nucleotide oxidase of the lysate was recovered in the ‘ghost’ fraction. On the other hand, the soluble protoplasm contained most of the isocitric dehydrogenase, the catalase, the hexo- kinase and the acid phophatase of the lysed bacteria. Considerable amounts of malic dehydrogenase, lactic dehydrogenase and fumarase were found in both the ‘ghosts’ and the soluble protoplasm. None of the enzymes studied was localized in the ribonucleic acid-containing particles of the bacterial protoplasm.

SUMMARY: The growth characteristics of eight strains of Bordetella pertussis were studied in defined liquid media. Two of these strains (1017, 1018), which are both very virulent on intracerebral injection into mice, were found to have growth idiosyncrasies. Both these strains differed from typical virulent pertussis strains and from a degraded strain in that they needed more magnesium and a different gas phase (carbon dioxide and oxygen) for optimal growth in defined liquid medium. Both strains were inhibited by crystalline lysozyme (100 mg./ml.) in defined medium, whereas typical virulent strains and a degraded strain were insensitive to it.

SUMMARY: This paper describes a replica plating technique for a filamentous fungus and shows its effectiveness in the isolation of nutritionally-exacting mutants, including mutants unable to grow with certain sugars as sole carbon source.

SUMMARY: An organism which ferments the dextran of Streptococcus boms was isolated from the rumen of cows on a diet of fresh clover. The bacterium is a strictly anaerobic Gram-positive, non-sporing, branching rod that closely resembles Lactobacillus bifidus. It is of interest from the point of view of the metabolism of dextran in the rumen.

SUMMARY: α--Oxoglutaric, pyruvic and amino acids accumulated extracellularly when Mycobacterium butyricum was grown in a chemically defined medium containing glycerol as carbon source and ammonium sulphate as nitrogen source. The excretion of keto acids was diminished and that of amino acids was increased by raising the concentration of ammonium sulphate relative to that of glycerol. This change is consistent with the operation of a citric acid cycle which is performing synthetic functions. The labelling of the somatic amino acids formed in the presence of glycerol and either (14C) bicarbonate or (2-14C) acetate is also consistent with the occurrence of a citric acid cycle.

SUMMARY: The strain of Saccharomyces carlsbergensis 4228 studied differed from those previously used for the assay of vitamin B6 in that it required this vitamin for rapid growth in the absence of added thiamine. The rate, rather than the ultimate extent, of growth of the organism is proportional to the concentration of vitamin B6, and the size of the inoculum and its physiological age, the duration and temperature of incubation and the degree of aeration, must be strictly controlled. Non-phos-phorylated forms of the vitamin only are utilized, and natural materials are acid-extracted before assay. The organism responds a little less well to pyridoxamine than it does to pyridoxal and pyridoxine. A linear response curve is obtained over a range of pyridoxine concentrations (0·2 to 2 × 10−8 m) when incubation is in static culture at 37° (24 hr.) in a basal medium slightly modified from those previously used.

SUMMARY: Five mutants of Escherichia coli which grow to a greater or less extent when the minimal medium is supplemented with vitamin B6 have been studied. They fall into two groups according to the further effect of serine + glycine on growth. (1) Two strains showed full and rapid growth with pyridoxine alone and serine + glycine neither replaced this vitamin nor enhanced growth in its presence. (2) Three strains showed delayed and suboptimal growth with pyridoxine alone. With these serine + glycine had two effects: (a) it decreased the lag and enhanced the final extent of growth when added with pyridoxine; (b) it permitted suboptimal growth in the absence of pyridoxine. The second effect was dependent on (two strains), or greatly increased by (one strain), the concurrent addition of glyeolaldehyde.

SUMMARY: Escherichia coli B166 synthesizes as much vitamin B6 when growing on serine or glycine plus glycolaldehyde as does its parent (E. coli B) on unsupplemented medium. Suspensions of strain B166 harvested after growth on serine+ glycine plus glycolaldehyde, and incubated with glucose and NH4 +, only synthesize vitamin B6 when glycolaldehyde is added; serine + glycine increases the synthesis. Strain 22–99 produces more vitamin B6 when grown on glycolaldehyde plus serine + glycine than when it is grown on the amino acids alone, while, depending upon the conditions of growth, vitamin B6 synthesis by suspensions is absolutely or partially dependent on serine+ glycine plus glycolaldehyde. However, serine + glycine plus glycolaldehyde have little effect on the biosynthesis of vitamin B6 by wild-type E. coli during growth, or in washed suspensions.

SUMMARY: Two extract preparations from each of twelve species of blue-green algae were tested for possible anti-pigmentation activities when applied to unpigmented cultures of Serratia marcescens. Extracts from eight of these species inhibited pigment formation, two of them completely. The two extraction methods gave similar results and there appears to be no difference in the effects produced by extracts from pure and impure algal cultures. Living or preserved samples of the algae containing the anti-pigmentation factor (APF) were also toxic to mice when whole or homogenized organisms were injected intraperitoneally. Numerous tests showed that the bacterial associates of the algae did not enter into the production of the APF or the toxic substance. The most effective extract concentration was 0·2 ml. extract/ml. broth, and within the range of concentrations employed, the degree of pigment inhibition was inversely proportional to the extract concentration. The possibility of using the anti-pigmentation test to determine the presence of toxic substances in blue-green algae is suggested.

SUMMARY: Twenty-three preparations of vaccinia virus and 5 of cowpox virus were examined by electron microscopy of spray droplets and by infectivity titrations at various stages of purification. Extraction of virus-containing tissues with a fluorocarbon compound was a useful early step in purification, but preparations of the highest purity were obtained by flocculation of the virus with m-NaCl. The average ratio of total particle count to infectivity for the vaccinia virus preparations was 12. The lowest observed ratio based on reliable particle counts was 3·4.

SUMMARY: Staphylococcal strains which showed a range of reactions with the phages 47 C, 52, 52 A, 80 and 81 were examined for changes in phage sensitivity following artificial lysogenization. The lysogenic phages used were isolated from strains of three different phage-typing patterns and differed in serology. The changes in phage reactions thus produced included: (1) immunity to the B phages, 52, 52 A and 80 produced by a B phage; (2) immunity to the A phage 81 produced by a serologically unrelated F phage; (3) immunity to phage 47 C and sensitivity to phages 52 and 52 A produced by a number of A phages. The mechanisms whereby a variety of typing patterns may be obtained within a related group of staphylococci are discussed.

SUMMARY: Three temperate phages isolated from lysogenic staphylococci of phage type 52/52 A/80 were used to lysogenize strains of phage-type 80. Two of these phages belonged to serological group A and one to serological group F. Lysogeniza- tion of a strain of phage-type 80 with one of the phages (287′) resulted in a change in the typing pattern from 80/81 to 52/52 A/80. There was, therefore, a gain in sensitivity to phage 52 and 52 A and a loss in sensitivity to phage 81. Lysogenization of type 80 strains with the second phage (581′) caused a loss in sensitivity to phage 81, while lysogenization with phage 7287′ caused a gain in sensitivity to phage 52 and 52 A.

Loss in sensitivity to phage 81 after lysogenization with phage 287′ seems most probably due to specific prophage immunity since phages 287′ and 81 belong to the same serological group (A) and may be closely related. Loss in sensitivity to phage 81 after lysogenization with phage 581′ does not appear to be a case of specific prophage immunity since the two phages are serologically distinct and the phenomenon resembles examples of prophage interference observed in other organisms.

SUMMARY: The mutation rate from histidine auxotrophy to prototrophy in Escherichia coli was measured during growth and in the stationary phase at different temperatures. Dining growth the rate of mutation/mutable unit/hr. had the same temperature coefficient as the rate of growth. Therefore the rate of mutation/ mutable unit/generation was the same at all temperatures. The temperature coefficient of the rate of mutation/unit/hr. during the stationary phase may be the same as that during growth, indicating that although mutation is slower in this phase it may involve the same process as during growth.

SUMMARY: Disruption of cells in a Mickle disintegrator in the presence of n-octanol is described as a method for obtaining extracts from the non-capsulated strain R19 of Streptococcus pneumoniae, derived from a strain of capsular type II. Such extracts were fractionated with ammonium sulphate in the presence of Celite and the distribution in the various fractions so obtained of a number of enzymes concerned with uridine pyrophosphoglycosyl metabolism was studied. Celite in the presence of ammonium sulphate was effective in removing organic pyrophosphatases, phosphatases acting upon uridine pyrophosphoglycosyl compounds and a reduced tri-phosphopyridine nucleotide (TPNH) oxidase.

A reduced diphosphopyridine nucleotide (DPNH) oxidase was concentrated by fractionation with ammonium sulphate and the uridyltransferases acting upon uridine pyrophosphoglucose (UPPG) and uridine pyrophosphoacetylglucosamine (UPPAG) were obtained as partially separated entities. Uridine pyrophospho- galactose (UPPgalactose) and guanosine pyrophosphomannose (GPPM) were not pyrophosphorylysed by the unfractionated material or by any of the fractions obtained.

UPPgalactose-4-epimerase and phosphogalactosyl uridyl transferase were present in the extracts under study, these enzymes being involved in the conversion of galactose-l-phosphate to glucose-1-phosphate. Such extracts also converted mannose to glucose phosphate without the participation of UPPG.

Evidence is provided for the presence of a diphosphopyridine (DPN) linked UPPG dehydrogenase which is capable of forming uridine pyrophosphoglucuronic acid (UPPGA) from UPPG. The UPPGA so formed was separated by ion-exchange chromatography. The extracts used did not catalyse the epimerization of UPPGA to uridine pyrophosphogalacturonic acid (UPPgalacturonic acid) as do extracts of a strain of capsulated type I pneumococcus. An examination of the cellular content of uridine nucleotides in relation to the bacterial growth curve showed that UPPGA did not accumulate in significant amounts until the organisms of this strain had reached the stationary phase.

SUMMARY: The flagellation and motility of Salmonella typhimurium were unaffected by proteolytic enzymes; but when washed stationary-phase bacteria were briefly exposed to acid (e.g. pH 2·6 for 30 sec. at 43°) the proportion of motile, and of flagellated, bacteria was decreased from more than 50 % to less than 1 %, with little or no decrease in viable count. When 5. typhimurium or Vibrio metchnikom grown on stiff agar were rubbed for several minutes on the agar, the proportion of motile and of flagellated bacteria was decreased from c. 90 % (found in control bacteria, soaked off without rubbing) to c. 1 %, without decrease in viable count. High-speed stirring in a blendor also deflagellated and destroyed the motility of 5. typhimurium in suspension, without killing the bacteria or retarding their growth. Blendor treatment sufficient to deflagellate 5. typhimurium had no effect on the movements of certain non-flagellated motile organisms (Leptospira canicola, Treponema pallidum and T. duttoni), except when it caused gross morphological changes, attributed to cell damage. When log-phase broth cultures of 5. typhimurium deflagellated in the blendor were incubated, bacteria with flagella (at first all short) and motile bacteria soon reappeared. At first many bacteria rotated; this suggests that flagella shorter than c. 0·3 μ cause rotational, but not translational, motility. The rate of increase of mean summed flagellar length/bacterium was compatible with the hypothesis that flagella are not (spontaneously) shed in broth cultures of 5. typhimurium and that the rate of growth of flagella is unaffected by deflagellation. Up to one generation time after deflagellation the mean number of flagella/bacterium was, however, less than before deflagellation. It is inferred that in a log-phase culture about half the flagella present at any one moment are ‘fully-grown’, incapable of being regenerated when broken off. All the results obtained are compatible with the hypothesis that the motility of flagellated bacteria is due to active movement of their flagella.

During work on the genetics of motility and flagellation in salmonella a nonlethal method of removing flagella from bacteria was needed. No satisfactory way of stripping selected individual bacteria of their flagella has been found, but it appeared that whole cultures might be deflagellated, without killing the bacteria, by several different methods. Such methods should provide a test of the hypothesis that the motility of flagellated bacteria results only from the active movement of their flagella, for total removal of the flagella would then necessarily result in loss of motility; whereas if normal motility in salmonella, etc., results from alterations of the shape of the bacterial body (Pijper, 1948, 1957), a treatment which removed all flagella might still leave motility unaffected. The effect of the application of similar treatment to motile bacteria which do not possess flagella (Leptospira, etc.) was also investigated, as a partial ‘control’ on the effects, other than deflagellation, of one of the treatments used. The manner and rate of the reappearance of flagellar

Summary: Solanine toxicity was assessed by its effect on the viability of spores of Fusarium caeruleum. Factors which were found to influence toxicity included solanine concentration, period of exposure, hydrogen-ion concentration, spore density, the Na ion/Ca ion ratio and the previous history of the spores. The toxicity of some related compounds was determined, and the toxicity of solanine to some other micro-organisms. The effect of solanine on the growth of F. caeruleum in culture was investigated. The nature of the toxic action of solanine on fungus spores and the similarity of spore toxicity to haemolytic activity are discussed. It is concluded that the presence of solanine is unlikely to be an important factor in preventing fungal infection of those tissues in which it is found.

Summary: With a strain of Fusiformis nigrescens as test organism, the potencies of compounds of the vitamin K group were estimated, and the results compared with those from animal assay techniques. The compounds showed in the main the same relative activity for the micro-organism as for higher animals, with two exceptions: 1 : 4-naphthoquinone was highly active for F. nigrescens but is inactive for animals; phthiocol, which is active for animals, inhibited the response of F. nigrescens to menaphthone, i.e. phthiocol acted as an anti-vitamin K for the micro-organism. Most of the compounds tested were toxic in the range 1–5 µg./ml.; vitamins K1, K2 and a water soluble form of menaphthone (menaphthone bisulphite) were relatively less toxic.

Summary: The early events in the infection of tobacco and Nicotiana glutinosa by tobacco mosaic virus occur sooner when the inoculum is the nucleic acid of the virus than the whole virus. In plants at 28° newly formed virus becomes detectable between 6 and 8 hr. after inoculation with the nucleic acid and after 8–10 hr. with whole virus. Although the latent period is lengthened by lowering temperature, the difference between the lengths of the latent periods given by the two inocula is little changed. Infective centres initiated by nucleic acid also become resistant to hot- water treatment (a 30 sec. dip in water at 50°) about 2–4 hr. sooner than do those initiated by whole virus.

Exposure of inoculated plants to 37° decreases the number of lesions produced by the nucleic acid much more than by whole virus; resistance to this treatment develops from 30 to 120 min. after inoculation with the nucleic acid, depending on the temperature at which the plants are kept.