- Volume 61, Issue 2, 1970

SUMMARY

Mutants (rod) have been isolated from one strain of Bacillus subtilis and two of B. licheniformis after treatment with i-methyl-3-nitro-i-nitroso-guanidine, by replica plating from media containing 0·8 m-NaCl to media of low salt content. When grown on the latter media these mutants appear as groups or strings of coccal bodies which, when examined in section under the electron microscope, show gross distortions in their walls and membranes; septum formation is greatly disorganized. When grown in media containing 0·8 to 1·0m-NaCl, or KC1, or ample supplies of organic nitrogen, considerable correction of the morphology of one class of these mutants occurs. The other class of mutants is not changed to rods by growth in media of high salt content but is so changed by growth on rich media containing yeast extract. All the mutants revert to the parent type, but at very different rates. The physiological characteristics of the mutants and the parents are in most respects identical.

SUMMARY: The morphological mutant rod-4 derived from Bacillus subtilis 168 trp can be changed from a round form to a rod by the addition to the growth medium of sufficient acid-hydrolysate of casein. The hydrolysate can be replaced by a mixture of amino acids, and the only individual amino acids giving similar results are l-glutamic acid, l-proline, l-arginine and l-ornithine. Since the lag in the action of l-glutamate was less than for the other amino acids, this amino acid is likely to be responsible for the effect of the mixture. Experiments with the l-glutamine analogue, γ- l-glutamylhydrazide, strongly suggest that l-glutamine is the active metabolite rather than the amino acid itself. The correcting effect of high ionic strengths of the growth medium on the morphology of this mutant seems to be mostly due to the increased effectiveness of l-glutamate or l-glutamine in the presence of high concentrations of salts.

SUMMARY

A soluble glucan was isolated from the mycelium of Phytophthora cinnamomi and found to be a β-1 →3-linked glucan with branches arising from residues substituted at both C-3 and C-6. This glucan is an important reserve material of the fungus; it accumulates in mycelium grown in glucose-rich medium and disappears upon incubation in media devoid of glucose.

SUMMARY

Mutagenesis of a transformable strain of pneumococcus by N-methyl-N′-nitro-N-nitrosoguanidine produced several mutants resistant to ampicillin. One of these was resistant to 0·1 μg. ampicillin/ml., but a purified solution of its DNA transformed the sensitive parent to three different levels of resistance at frequencies compatible with single, double and triple transformants. The higher levels of resistance were dependent on the presence of the genes conferring the lower resistances. Furthermore, transformants to the higher levels of resistance can be obtained at single or double frequency by using as recipient the strain already possessing the genes conferring the lower resistances. The expression times of the ampicillin genes are short (approximately 25 min.), but the actual times were difficult to determine since there was a delay of some 15 to 20 min. before the ampicillin exerted an effect on the sensitive strain under these conditions.

SUMMARY: The susceptibility of five strains of bacteria, recovered after storage in the aerosol state, to certain hydrolytic enzymes has been examined. Aerosolized Escherichia coli organisms, strains b and jepp, rapidly became susceptible to the bactericidal effects of lysozyme, ribonuclease, deoxyribonuclease and trypsin. E. coli strain commune and Serratia marcescens strain 8 UK organisms remained insensitive to all four enzymes, Aerobacter aerogenes strain h organisms developed sensitivity to lysozyme only. Raffinose, dextran, glucose, glycerol or sodium glutamate added to the bacterial suspensions increased their survival time as aerosols and decreased the sensitivity of survivors to lysozyme. These results support the hypothesis that changes in the outer wall structure of bacteria precede, and possibly contribute to, the death of organisms in the aerosol state.

Summary: More than 100 Gram-negative, strictly aerobic, methane-utilizing bacteria were isolated. All used only methane and methanol of the substrates tested for growth. The organisms were classified into five groups on the basis of morphology, fine structure, and type of resting stage formed (exospores and different types of cysts) and into subgroups on other properties. Methods of enrichment, isolation and culture are described.

SUMMERY: Three varieties of resting stage, an exospore and two types of cyst, were formed by methane-utilizing bacteria. Exospores were budded off by two types of organism, both of which underwent a change in morphology prior to spore formation. Exospores possessed some properties in common with endospores: staining properties, some structural features, mode of germination, desiccation and heat resistance, but contained no detectable dipicolinic acid. Some organisms formed desiccation-resistant cysts similar in morphology, staining properties and fine structure to cysts formed by Azotobacter species. Other strains formed non-desiccation-resistant bodies considered to be similar to immature azotobacter-type cysts. A desiccation-resistant cyst formed by one strain differed in structure and appearance from Azotobacter-type cysts and was referred to as a Tipid’ cyst.

SUMMARY

Methane-utilizing bacteria were examined by electron microscopy and found to possess complex membranous structures within the cytoplasm. Two types of membrane organization were recognized. One type consisted of pairs of membranes which either extended throughout the organism or were arranged at the periphery where they ran parallel to the cytoplasmic membrane. The other type consisted of vesicular discs of membrane organized into bundles which were distributed throughout the organism. Bacteria utilizing C2 to C4 gaseous n-alkanes and C11 to C18 liquid n-alkane mixture did not possess such extensive membranous structures. The former contained membranous bodies of the mesosome type, whilst the latter possessed only a cytoplasmic membrane. These structural differences add to the growing list of properties separating CH4-utilizing bacteria from those utilizing C2 and higher n-alkanes.

SUMMARY: Methods were developed for growing protoplasts or L bodies of Bacillus subtilis into L colonies on membrane filters. Only some types of filters permitted growth; for optimum growth the filters usually had to be extracted with 2 % (v/v) ethanol in water. Scoring was greatly improved by staining. Growth on the filters induced reversion of the protoplasts to the bacillary form. Reversion was greatly enhanced when B. subtilis wall was added to the filter. A lesser enhancement occurred when wall was added to protoplasts growing in agar. The stimulation of reversion by wall was non-specific since similar stimulation could be obtained by intact autoclaved B. subtilis, Escherichia coli, pseudomonads and yeast. Stimulation of reversion probably depended on physical contact between the naked protoplasts or L bodies and the surface provided by the filter, wall, or killed organism. The present status of induced reversion in B. subtilis is discussed.

SUMMARY

A mycobacterium, two pseudomonads and a torula were isolated from soil using selective enrichment techniques in mineral salts-kerosene media. The properties of the organisms and their possible identity are outlined. Hydrocarbons, fatty acids, n-alcohols, dicarboxylic acids and acyl amides able to support growth were determined. Growth of the torula on hydrocarbons increased the ability of the organisms to oxidize other alkanes and fatty acids; the organism was, however, able to oxidize these substrates, at a slow rate, irrespective of the substrate of growth. When the torula was grown on an alkane with an even number of carbon atoms, there was no evidence that the induced enzymes favoured the oxidation of fatty acids with even rather than odd numbers of carbon atoms.

Growth on hydrocarbons increased the amount of isocitric lyase present in all four micro-organisms, suggesting that β-oxidation of fatty acids derived from alkanes gives rise to acetyl-CoA and that this pathway and the glyoxylate bypass are important in the metabolism of alkanes.

d-Glutamic acid (d-glu) inhibited strongly the growth of two strains of Neurospora crassa in a minimal medium. The inhibition was completely annulled by equivalent concentrations of l-glutamic acid (l-glu) or l-gluta-mine. d-G1u also inhibited glutamate dehydrogenase (GDH) and was only antagonized completely by 10 equivalents of l-glu. d-G1u in media containing l-glu increased GDH-activity, presumably by de-repression. d-G1u showed no effect on glutamine synthetase and γ-glutamyl transferase activities. Inhibition of growth of N. crassa by d-glu thus seems due to the interference with glutamate synthesis by inhibition of GDH-activity.

Summary: We have isolated auxotrophic mutants of Streptomyces coelicolorwhich can grow on a minimal medium without growth factors when the gas phase is supplemented with CO2. Usually they have an alternative requirement for a specific growth factor such as arginine, purines or vitamins. Some of the CO2 mutants resemble those already known in Neurosporacrassa and Escherichia coli but others present novel phenotypes.

SUMMARY: An Escherichia coli k12 strain, j5-3 (R313), was presumed to carry an fi − R factor, R313, which determined resistance to the drugs tetracycline (Tc), streptomycin (Sm) and Sulphonamide (Su), and also determined a host specificity, hsll. When this strain was used as a donor of drug resistance, separation of R factor-carried determinants was observed in ex-conjugants. Resistance to Tc and hsll character were not separated, nor was resistance to Sm separated from resistance to Su, but separation of these two pairs of characters was observed. Tetracycline-sensitive segregants, obtained by penicillin selection, were resistant to Sm and Su, but had lost the hsll character. Similarly, segregants for selected sensitivity to Su were sensitive to Sm, but resistant to Tc and hsll+. The same pattern of separation of characters was also observed when drug resistance was transduced with phage P1, with the additional finding that the Sm and Su resistant trans-ductants lacked sex factor activity. The resistance to Sm carried by these transductants could be mobilized by an fi − R factor, R143. Explanations of this behaviour are considered, including the possibility that the strain J5-3 (R313) had carried two fi − R factors. This explanation would also require that the transduction of an R factor by phage P1 is not always complete.

SUMMARY

Two strains of Escherichia coli K12 carried R factors associated with the hsII host specificity determinants although they were apparently fi +, while all other R factors carrying this host specificity have been found to be fi − When these two strains were used as donors of drug resistance, separation of two R factors from each strain was observed. The separated R factors were tested for fi character, and it was found that in both cases the original strains carried an fi+ and an fi − R factor, and it was the fi − R factor which carried the hsII determinants.