- Volume 18, Issue 3, 1958

Thirty-five substituted benzimidazoles, benzotriazoles and quinoxalines were tested as growth inhibitors on three mutants of Escherichia coli, strain W, having different purine requirements and on the ciliate Tetrahymena geleii. A number of the compounds, especially those with a nitro group in the benzene ring, were inhibitory. Both organisms were affected in a similar way by the compounds. Several of these analogues were tested for mutagenic activity at the purine and streptomycin loci in a purine-requiring mutant of E. coli. Only inhibitory compounds were found to be mutagenic; non-inhibitory concentrations of one analogue, 4-nitro-6-hydroxybenzimidazole (NHB), were, however, also mutagenic. No specific mutagenesis was demonstrated. NHB was found to increase the rate of mutation as determined by the papilla method. Evidence is presented which eliminates the hypothesis that selection rather than mutagenesis was involved. This analogue did not increase the frequency of mutants in non-dividing cells, and was not incorporated into bacterial DNA. The mechanism of this mutagenesis is thought to include the inhibition of enzymes concerned with DNA synthesis. DNA and RNA and their constituents, purine and pyrimidine precursors and analogues, vitamin B12 and other vitamins, were found to have no effect on the inhibition or mutagenesis produced by NHB. Several amino acid combinations and one non-mutagenic analogue, 4-hydroxy-6-nitrobenzotriazole, were, however, found to interfere with mutagenesis by NHB. These compounds did not affect the frequency of spontaneous mutants. The amino acid combination was effective only when present simultaneously with the mutagen and did not act by a selection mechanism. The mechanism of antimutagenesis by amino acids may involve their ability to increase the growth rate and also to modify the inhibited nucleic acid metabolism.

The mutagenesis by 5-nitroquinoxaline, a pterin analogue, was also found to be depressed by amino acids in the same purine-requiring mutant. The annulment of mutagenic activity by amino acids was strain and mutagen specific.

Twenty-five known inhibitors of nucleic acid synthesis and antagonists to inhibition were tested for mutagenic activity in five strains of Escherichia coli. Several, including 6-mercaptopurine, were found to be mutagenic; in addition, NHB, 5-nitroquinoxaline and 5-aminouracil were mutagenic in most of the strains tested. The mutagenic activity of 5-aminouracil was not annulled by thymine, thymidine or other nucleic acid components, but the mutagenesis of 6-mercaptopurine was annulled by purine bases and by ribosides.

SUMMARY: A flagellate amoeba, isolated from Ohio River water and identified as Naegleria gruberi (Schardinger), has been maintained in permanent cultures on a buffered sucrose nitrate agar with its original bacterial associate, identified as Proteus mirabilis. On Czapek agar plates, the growth curve of the amoeba at 25–27° was found to consist of a lag phase of about 4 hr., a log growth phase of about 60 hr., and a growth-encystment phase of 2–3 weeks. At 37° the lag phase was shortened by half and the log growth phase by about a third. The generation time was 4½ hr. at 25–27° and 2½ hr. at 37°. Continuous cultivation at 37°, however, resulted in occurrence of abnormal forms. Very poor growth of amoeba on nutrient and tryptose agars was attributed to their high organic nitrogen content, poor growth in liquid media to the difficulty of amoebae to engulf bacteria. Attempts to grow the amoeba on heat-killed bacteria were failures. Redox potential and pH determinations revealed that the mixed fluid cultures were aerobic and the amoebae perished at pH values more acid than 5·6. Replacement of Proteus mirabilis by other bacteria by Oehler’s technique was unsuccessful because of swarming but was successful with a Flavobacterium sp., which produced a yellow pigment whose antibiotic effect prevented swarming. Permanent cultures of the amoeba with the new bacterial associate were maintained after the amoeba overcame the antibiotic effect of the pigment. Replacement of the Flavobacterium sp. in the mixed culture by new species of bacteria was easily accomplished, and permanent cultures were maintained with Aerobacter aerogenes, Escherichia coli , 8 species of Salmonella and 2 of Shigella. The amoebae encysted earlier on all these species than on Proteus mirabilis.

Cytological observations include: (a) appearance of the chromatin material as short rods; (b) no evidence of thread-like structures radiating from the karyosome or chromatin patches in resting nucleus, nor the centrioles in mitosis; (c) karyosome origin of both the ‘interzonal body’ and the polar caps.

SUMMARY: Phase-contrast microscopic examination of Naegleria gruberi (Schar- dinger) during flagellate transformation and reversion revealed the following cytological features. Flagella were formed by filamentous extension from endoplasmic protrusion. One, two or three pairs of flagella were formed from a single protrusion. Occasionally a second protrusion gave rise to the 3rd pair of flagella. A chromatin body of cytoplasmic origin was always present at the base of the protrusion or of the flagella, and is believed to be the parabasal body. Occasionally one chromatin body was seen at the base of each pair of flagella. Alternating bands of light and dark areas were seen in the flagella soon after they were formed and persisted throughout the flagellate stage. Reversion from flagellate to amoeba stage was accomplished by absorption of the flagella, the shedding of one or more flagella and the absorption of the rest, or by casting-off a small part of the body to which the flagella were attached. The following effects of environmental factors on flagellate transformation were observed. As many as 35 % of the trophozoites underwent flagellate transformation in water when they were in metacystic stage or were harvested from plate cultures up to 12 hr. of incubation at 25–27°; as the cultural age advanced, the % amoebae undergoing transformation became progressively lower. Prolonged cultivation in a liquid medium at 25–27°, or on an agar medium at 37°, resulted in temporary loss of ability to form flagellates. The freezing of trophozoites up to 40 min. and of cysts up to 1 week at − 25° to − 30° stimulated flagellate transformation. An increase in water temperature from 25–27° to 37° or decrease to 8–10° suppressed the transformation, but after the flagellates were formed, the former temperature change shortened while the latter lengthened duration of the flagellate stage. Freezing of the amoebae in the flagellate stage at − 25° to − 30° exerted no adverse effect on their return to amoeboid form when the freezing time did not exceed 30 min. Cultures developed from single organisms isolated in the flagellate stage yielded amoebae showing flagellate transformation characteristics similar to those yielded by ordinary cultures.

Lactose-fermenting (lac+) mutants were isolated from a non-lactose-fermenting (lac-) strain of Escherichia coli, strain O 110, by the replica plating technique. When high dilutions of a 24 hr. broth culture of the strain were plated on Endo agar, to give discrete colonies, secondary lac+ colonies began to appear in the initially lac- colonies after 3 days' incubation; after 10 days, over 50% of colonies contained secondary lac+ colonies. Twenty serial subcultures were made in broth containing lactose, each subculture being incubated for only 6 hr. to prevent selection of lac+ mutants from exhaustion of other nutrients while exposing the bacteria to lactose during many generations. When heavy inocula from the final subcultures were plated on Endo agar, no more lac+ colonies appeared than from control platings of broth cultures which had not been exposed to lactose. An R variant of the lac- strain of E. coli, strain O 110, was found to assimilate lactose to a considerable extent without fermentation; the original S form possessed this capacity only to an insignificant extent.

SUMMARY: A population of Streptomyces griseus, strain W107, exposed for several growth cycles to a sterile culture filtrate of S. griseus, strain M142, acquired several genetic characteristics similar to those of strain M142. The changes observed were: (1) streptomycin sensitivity to resistance; (2) bacteriophage V1 sensitivity to resistance ; (3) absence to presence of soluble pigment; (4) presence to absence of pigment in the vegetative mycelium. The filtrate contained streptomycin and a temperate bacteriophage. The low concentration of streptomycin did not grossly inhibit the growth of strain W107 but streptomycin-resistant mutants were selected. Resistance to the temperate phage frequently conferred resistance to bacteriophage V1. The observed morphological changes were coupled with bacteriophage- and streptomycin- susceptibility. Vegetative hybridization was the result of selection of mutants rather than gene transfer.

SUMMARY: The phosphorus-containing compounds of certain Gram-positive and Gram-negative organisms were estimated by a modification of the Schmidt & Thannhauser (1945) procedure. Another method Mitchell & Moyle, 1950, 1951a, b, 1954 in which pentosenucleic acid (PNA) was estimated from the optical absorption of the ‘PNA fraction’ at 260 mμ. was found to be inaccurate, because of the presence of ultraviolet-absorbing impurities. A more accurate method for estimating PNA has now been developed, in which the nucleotides are separated from impurities by paper electrophoresis. The results thus obtained show that non-nucleotide phosphorus-containing compounds are present in the ‘PNA fraction’ of both Grampositive and Gram-negative organisms. There is, therefore, no evidence to connect these compounds with the Gram staining properties of Gram-positive microorganisms (cf. Mitchell & Moyle, 1950, 1951a, b, 1954).

SUMMARY: Normal bacilli of Bacillus megaterium have large redox-active granules at the poles of the cells, and smaller ones at the periphery, in the region of the crosswalls. In non-septate filaments produced by growth in the presence of urethane the peripheral granules were not present. This is in accordance with the belief that these are the growing-points associated with the development of the cross-walls.

Summary: Direct observations on the occurrence and spatial distribution of planktonic bacteria were made by using a membrane filter technique. The membrane filters were treated to allow direct microscopic examination of bacteria on their surfaces. Deviations between direct counts on the membranes and plate (colony) counts of bacteria depended upon availability of organic matter in the natural waters studied, and can be accounted for by clumping effects and the occurrence of very small forms not visible on the membrane filter surfaces. Indirect evidence for these ‘dwarf’ forms was provided by later development of colonies with cells of normal size, after the membrane filter was placed on a nutrient medium. This response suggests that the organisms are zymogenous forms. Pure cultures of Bacillus subtilis and Pseudomonas fluorescens were used in some experiments to demonstrate the relationship between the clumping effect and concentration of dissolved nutrients in water. A concentration of 0·5 mg. peptone/1. in a tap-water medium caused accumulations of bacteria around clean chitin particles; accumulations of bacteria did not develop at the higher concentrations tested. This behaviour is explained by the local differences in concentrations of nutrients in the medium. It correlates with the observations on natural populations.

The conditions for luxuriant axenic cultivation of the trypanosomid flagellate Crithidia fasciculata in chemically-defined media were determined. Improvements over the hitherto published media include higher concentrations of amino acids, folic acid (where this is the sole source of pteridine) and purines and the inclusion of threonine and Tween 80. Increased oxygenation was obtained by incubating liquid cultures in tubes in a sloped position. Under these conditions consistently excellent growth (up to 2 × 108 organisms/ml.) was obtained and the useful incubation period could be decreased to 4 days. Using the improved medium, qualitative and quantitative studies on the nutritional requirements of the organism were carried out. Growth failed when any one of the following amino acids was omitted from the basal medium: histidine, phenylalanine, isoleucine, leucine, valine, lysine, arginine, tyrosine, methionine, tryptophan. The omission of threonine caused drastically decreased growth rates and lower yields. It was found that phenylpyruvic acid could replace phenylalanine; cysteine, cystathionine or homocysteine could replace methionine; and citrulline, but not ornithine, could replace arginine for C. fasciculata. In addition to the amino acid requirements a nutritional need for the following was demonstrated: a carbohydrate, haemin, a purine, thiamine, riboflavin, pantothenic acid, nicotinic acid or its amide, pyridoxal or pyridoxamine, biotin, folic acid and an unconjugated pteridine (biopterin). The natural purines, their nucleosides and nucleotides are nearly equivalent, on a molar basis, in growth promotion. Numerous substituted purines were also tested for their ability to supply the purine requirement. No exogenous source of pyrimidine for nucleic acid synthesis is necessary provided folic acid is present. Folic acid can be omitted from the growth medium provided thymine and methionine are present. The absolute requirement for biotin could not be demonstrated in the present medium without the use of avidin.

Summary: A modification of Fontana’s silver-plating method was devised and used for the eytological study of 214 isolates of Pseudomonas spp. Many features, such as flagella, cross-walls or septa, capsules and slime were thereby clearly demonstrated simultaneously and easily. Comparisons were made with other published methods for staining flagella, capsules and slime, and good agreement was obtained. No totally non-flagellate isolate was found, nor were flagella lost during maintenance for 3–5 ½ years in the laboratory. The number of flagella/rod and also bipolar flagellation were found to be characters of little value for species differentiation. The flagellation of young cultures was not found to be significantly different from that observed in older cultures; the polar position of the flagella was confirmed. Flagella curvature (wavelength and amplitude) as seen in silver-plated organisms prepared under reasonably standardized conditions, was noted and considered to have no usefulness for subgeneric classification.

Summary: Of a total of 739 actinomycetes freshly isolated from soil samples from China, 515 (69·7 %) were antibiotically active. Their antibiotic spectrum was determined against 14 test micro-organisms, including bacteria, yeasts and fungi, and 386 of them were characterized by paper chromatography using agar blocks, by behaviour on agar plates incorporating ion-exchange resins and by u.v. absorption spectrum (to detect polyene substances). Of these strains, 195 (50·5 %) produced a mixture of antibiotics of both polyene and non-polyene type. On the basis of these results the active strains have been classified in various ways.

Summary: Mutant spores of Neurospora crassa incubated on minimal medium showed two main types of behaviour. One mutant type survived incubation for a week on minimal medium and then responded to the addition of growth substance. The second mutant type died after incubation for one or two days. Strains which died were characterized by an ability to germinate in minimal medium and to begin immediately synthetic processes such as the synthesis of protein and nucleic acid. Addition of ethionine to minimal medium prolonged the survival of strains which committed ‘suicide’. Non-germinated Neurospora conidia withstood freezing and storage at −10° for extended periods, whereas conidia which had been incubated in growth medium died, indicating a difference between dormant and synthetically active spores in their response to the environment. The suicide phenomenon is reminiscent of the cell death due to ‘unbalanced growth’ reported for the thymine- requiring mutant of Escherichia coli. In this ease, however, cells requiring substances other than nucleic acid constituents display the effect.

Summary: Investigations on the size of inhibition zones in agar tube cultures of Staphylococcus aureus (Mayo) due to diffusion of streptomycin have confirmed the formula previously suggested. No significant deviation from expectations has been found over a temperature range of 27–40°, nor with variations of inoculum size from 2 × 103 to 5 × 108 organisms/ml. The theoretical basis of the formula has been further elucidated and the meaning of critical population, critical concentration and critical time clarified. These constants are determined by the necessity of absorbing a critical amount of antibiotic on the organisms to inhibit growth within the zone. To maintain this absorption a minimum inhibitory concentration must remain in the surrounding medium.

Summary: By the use of a staining technique which enables the flagella and cell walls to be demonstrated simultaneously, the distribution of flagella on the component cells of motile Gram-positive cocci has been examined. By comparison of these preparations with others made by the technique of Pennington (1950), which indicates the physiological age of cells according to the resistance of their basophilic elements to the action of formaldehyde, it is apparent that such multicellular cocci divide in the manner already suggested for rod-shaped bacteria, not by simple fission but by a process analogous to budding, so that different cells in a group are at different physiological ages or stages of development.

Summary: Organisms of a strain of Flavobacterium peregrinum and an Achromobacter strain, capable of decomposing 2:4-dichlorophenoxyacetic acid (2:4-D) and 4-chloro-2-methylphenoxyacetic acid (MCPA) respectively, have been obtained by growing cultures on peptone agar plates containing either 2:4-D or MCPA. Glucose did not prevent adaptation. Organisms of the F. peregrinum strain were adapted to oxidize 2:4-D when grown on peptone agar containing either MCPA or 2-chloro-4-methylphenoxyacetic acid; they did not decompose MCPA. The effects of some related compounds on adaptation to 2:4-D by these bacteria were examined. Growth of the Achromobacter strain on peptone agar containing either 2:4-dichlorophenol or 5-chloro-2-cresol gave organisms which were adapted to oxidize both 2:4-D and MCPA as well as the inducing compound.

The effect of the iron concentration in the medium on the concentration of toxin and coproporphyrin in the culture filtrate of a strain of Corynebacterium diphtheriae has been found to differ quantitatively from that previously reported in the case of the P.W. 8 strain. Only 50-60% of the iron present in the medium, at all concentrations tested, was recoverable from the organisms. Of the total iron present in the medium, only 9% was found to be present in the organisms as a haem complex capable of measurement as its pyridine haemochromogen. On the basis of their iron contents protohaemin and iron coproporphyrin III are less active than ferrous sulphate in their effect on toxin yield. These findings do not support the hypothesis that diphtheria toxin is the protein moiety of the cytochrome b of the cell.

Several cations have been shown to affect protein and porphyrin formation by the G 12/6 strain of Corynebacterium diphtheriae. Cobaltous ion (Co++), at concentrations only slightly inhibiting growth, caused a marked inhibition of toxin formation. This effect was not produced by vitamin B12 or annulled by increases of cysteine or histidine concentration. Only a small proportion of added Co++, at concentrations above 0·2 μg./ml., was found in washed organisms, and the effect of Co++ was more marked than that of Fe++ when based on the weight of metal in the organisms. No appreciable proportion of the Co++ existed as a porphyrin complex. The growth inhibition caused by 5 μg. Co++/ml. was annulled by 0·6 μg. Fe++/ml. Co++ caused no increase in the cytochrome b content of the organism, nor did it affect the iron concentration required for maximum toxin formation. The combined toxin-inhibitory effect of Fe++ Co++ was considerably less than the sum of their independent effects. The bearing of these findings on the thesis that diphtheria toxin is the apoenzyme of cytochrome b is discussed.

Primary infections were obtained by growing cabbage seedlings in a modified Hoagland's solution in which resting spores of Plasmodiophora brassicae Woron. were suspended. Seeds were germinated on filter paper wet with tap water, and after 2 days the plants were transferred to small glass tubes bent in the form of a shallow U or to small vials containing solution and spores. Zoosporangia were formed after several days growth at 25° in the dark. They were stained in aceto-carmine. A roughly linear relationship was found between the logarithm of number of infections/root and the logarithm of spore concentration in the medium. Numbers of infections were usually greater at 1/5 or 1/25 dilution of the culture solution than at the standard concentration, but were very much fewer or none in more dilute 1/125 or more concentrated (x 5) solutions. The concentration which permitted maximal infection tended to vary from one experiment to another. Infection was not affected by changing from pH 5 to 6 but was greatly decreased at pH 8.

Summary: Studies of the β-oxidation of the fatty acid side-chain of certain ω-aryl- and ω -aryloxy-n-alkylcarboxylic acids by Nocardia opaca (strain T16) and Nocardia sp. (strain P2) show that y-phenylbutyric is more rapidly oxidized than y-phenoxy- butyric acid. The effect of the oxygen bridge is even more striking when γ-(1-naphthyl)- and γ -(1-naphthyloxy)-butyric acids are compared. The rate of β -oxidation (by strain T18) of 3-, 4-, and 2-isomers decreases in that order. This applies to mono-chloro- and monomethylphenoxybutyric acids and to monochlorophenoxypropionic acids. Substitution in position 2 has by far the greatest effect and chlorine exerts a bigger influence than a methyl group in all positions. The conversion of γ-(2-methyl- 4-chlorophenoxy)-butyric acid (MCPB) and γ -(2:4-dichlorophenoxy)-butyric acid (2:4-DB) is very slow and proceeds only to β-hydroxy acids. On the other hand, ϵ-(2-methyl-4-chlorophenoxy)-, ϵ -(2:4-dichlorophenoxy)- and ϵ -(2-chlorophenoxy)-caproic acids are relatively rapidly converted to their corresponding butyric acid derivatives. With strain T16 ω-(2-naphthyloxy)-butyric and propionic acids are more rapidly converted to their corresponding acetic acids and phenols respectively than the ω-(1-naphthyloxy) compounds. The rate of β-oxidation of γ-phenyl-, γ-(3-indolyl)- and γ -(1-naphthyl)- butyric acids (by strain P2) decreases in that order. It has been shown that β-hydroxy acid intermediates are formed from ω-aryloxybutyric acids and those from MCPB, γ-(2-methyl-4-chlorophenoxy)-crotonic and γ-(2-naphthyl-oxy)-butyric acids have been isolated and identified.