- Volume 30, Issue 3, 1963

Galactokinaseless mutants of Escherichia coli are, in general, phenotypically constitutive for the remaining enzymes of the galactose pathway, a phenomenon which has been attributed to internal induction (Jordan, Yarmolinsky & Kalckar, 1962). One exceptional, kinaseless, mutant, though constitutive during stationary phase, is inducible during logarithmic growth. This mutant, W3092A, also produces a constitutive concentration of enzyme after confluent growth on solid medium and continues to do so even after 3--5 generations of subsequent logarithmic growth in liquid medium. This effect of growth on solid medium is peculiar to W3092A. It does not occur in other inducible strains, including galactose-fermenting revertants of W3092A, nor in W3092A itself grown as isolated colonies. Even in confluently grown organisms it is lost after storage at 4° for several days. In each case high degrees of enzyme synthesis can be prevented by an inhibitor of induction of the galactose enzymes, methyl-β-d-thiogalactoside, and are therefore attributed to internal induction. Though the enzyme concentrations decrease rapidly when the cultures resume logarithmic growth after stationary phase, high concentrations may persist up to the fifth generation after confluent growth on solid medium and then decline to reach the basal level only after the 12th generation. The inducibility of W3092A (A character) was shown to be independent of the mutation to kinaseless, since other kinaseless mutants derived from a galactose-fermenting revertant of W3092A still carried the A character.

A mutant strain, 72--44, of Aspergillus niger was selected after ultraviolet irradiation of the spores of A. niger strain 72--4. This mutant was capable of giving high yields of citric acid in shaken flask fermentations. A study was made of the effect of various lipids on the yield of citric acid produced by A. niger strain 72--44. Fatty acids with less than 15 carbon atoms inhibited growth and no citric acid was produced. Natural oils with a high content of unsaturated fatty acids and oleic acid itself, when added at 2% (v/v) to suitable fermentation media, increased the yield of citric acid by about 20%. Lipids which improved the yield of citric acid had no effect on the dry weight of mycelium. The possible mode of action of effective lipids is discussed. It is suggested that unsaturated lipids act as alternative hydrogen acceptors to oxygen during the fermentation and thus improve the yield of citric acid.

The nutritional requirements for high conversion of sugar to citric acid in shaken flask fermentations were investigated for the mutant strain 72--44 of Aspergillus niger. Single variable and factorial experiments were used to determine the effects of nitrogen, phosphorus, magnesium, manganese, copper, zinc and iron on the yield of citric acid. When 2% (v/v) peanut oil was added to the fermentation media it markedly improved the yield of citric acid. Two media were devised which yielded on average 10--10°5 g. anhydrous citric acid from 14 g. sugar, with occasional yields as high as 13 g. Both media contained 2% (v/v) peanut oil and the following trace metals (mg./l.): Fe2+ 2°0; Cu2+ 0°3; Zn2+ 0°1. In medium A the major nutrients were (g./l.): ion-exchange purified cane sugar, 140; KH2PO4, 1°0; MgSO4.7H2O, 0°25; NH4NO3, 1°87; and in medium B (g./l.): ion-exchange purified cane sugar, 140; KH2PO4, 0°3; MgSO4.7H2O, 0°15; NH4NO3, 1°4. Interactions were found between: zinc and iron, nitrogen and iron, nitrogen and phosphorus. The implications of these findings for the fermentation of crude sugar substrates in citric acid production are discussed.

Extrapolation from 10-year survival curves indicates that many lyophilized bacterial suspensions may be expected to yield viable organisms after centuries of storage, but that suspensions of Pseudomonadaceae may become sterile within one to several decades. The characters of Lactobacteriaceae and other bacteria appear unaltered as a result of lyophilization and storage for 5 years, but some Bacteroides isolates manifested apparently altered fermentation reactions under the same circumstances. Freezing bacteria in pellets is suggested as an alternative method of storage for those species which are particularly sensitive to lyophilization.

Ultrathin sections of vegetative hyphae of Rhizopus sexualis and R. homothallicus examined by electron microscopy revealed structure essentially similar to that reported for some other filamentous fungi and yeasts. The cell wall in section consists of elongated elements tangentially orientated. The nucleus is surrounded by a double membrane interrupted by pores and contains a denser body identified as the nucleolus. The cytoplasm contains well-defined mitochondria, vesicles (cisternae), unidentified spherical bodies, oil drops and vacuoles. There is a complex system of cytoplasmic membranes or membrane-like layers including the outer layer or plasmalemma, membranes (tonoplasts) surrounding the vacuoles and, in the zone of extension growth behind the hyphal tip, a more or less continuous, convoluted ‘cortical membrane’ separating a central core of cytoplasm from a peripheral zone. The possible significance of such a membrane in relation to cytoplasmic streaming is discussed.

1250 Gram-positive and catalase-positive cocci were isolated from bacon, pig and human skin and dust, and their morphology, physiology and biochemical characters examined. The genera Staphylococcus, Micrococcus and Sarcina were recognized. Staphylococci were distinguished by their ability to form acid from glucose anaerobically and sarcinas by the formation of cubical packets. 570 isolates could be placed in the genus Staphylococcus, 677 in the genus Micrococcus and 3 in the genus Sarcina. Six subgroups were recognized within the genus Staphylococcus and seven within the genus Micrococcus. The relationship of these subgroups to previously defined genera and species is discussed.

A regular pattern of morphogenetic responses to factorially arranged variations in concentrations of NH4Cl, K2HPO4 and glucose was found common to ten strains of Memnionella and Stachybotrys. Three phases of morphogenesis were observed: sterile swollen mycelium, sterile filamentous hyphae, and hyphae with conidiophores. The morphological status of the mature colony was found to be related both to the absolute concentrations of NH4Cl and K2HPO4, and to the ratio of these concentrations. At high values of the ratio the mycelium remained in the sterile swollen phase; at intermediate values the mycelium was mainly filamentous and sporing occurred; at low values the hyphae were filamentous but less fertile. Increase in concentration of K2HPO4 favoured filamentation whereas increase in the concentration of NH4Cl favoured swelling of the cells and suppressed sporulation. Glucose concentration determined mainly the extent of growth and intensity of sporulation, although some interaction between the effects of glucose and NH4Cl on sporulation were observed. A general set of conditions is proposed for the propagation of Memnionella and Stachybotrys in the sporing phase. It is suggested that the factorial design of nutritional experiment is appropriate for cultural studies fundamental to the taxonomy of fungi.

A total of 3919 brucella cultures was examined for lysis by five brucella phages, using a routine test dilution (r.t.d.) and 10,000 x r.t.d. All cultures of Brucella abortus examined were lysed by all five phages at both dilutions. In addition, all cultures with the oxidative metabolic pattern characteristic of B. abortus, irrespective of their properties as determined by conventional typing methods, were lysed by both phage dilutions. Cultures of B. suis were not lysed by phages at r.t.d. but all showed lysis by phages at 10,000 x r.t.d. Cultures of B. melitensis and those with the oxidative metabolic pattern characteristic of B. melitensis, irrespective of their properties as determined by conventional typing methods, were not lysed by phages at either dilution. All five phages used (Tb, 10/I, 24/II, 212/XV and 371/XXIX) displayed an identical host range; this was confirmed by neutralization tests with antiphage sera.

The swelling of spores of Bacillus cereus and B. subtilis during germination and outgrowth was followed by measuring changes in packed cell volume (pcv) and by photomicrographic measurements of single organisms. Three stages of swelling were recognized: (1) germination swelling involving a rapid increase of about 20% in pcv as the spore germinated; (2) pre-emergence swelling of up to 100% increase in pcv before emergence from the spore coat; (3) elongation. Germination swelling was due mainly to an increase in the breadth of the spore. It occurred in the absence of oxygen and was not inhibited by nisin whereas pre-emergence swelling was oxygen-dependent and was inhibited by nisin. Post-emergence swelling occurred during elongation of the new vegetative form. The significance of these findings is discussed.

A method is described for the preparation, on a laboratory scale, of staphylococcal alpha toxin having a purity of about 70% and in a yield of 40%. The method entails ammonium sulphate fractionation followed by curtain electrophoresis. The toxin so obtained contains two impurities, one of which can be removed electrophoretically, and the other ultracentrifugally. The toxin itself is a protein of molecular weight 44,000. It contains most of the usual amino acids which vary in amount from 4 residues for histidine to 44 for aspartic acid. Although electrophoretically heterogeneous its electrophoretic components produce the biological effects heterogeneous associated with alpha toxin. As by-products of purification, two other proteins were isolated in crystalline form. Their significance for pathogenicity, if any, remains to be determined.

The taxonomic position of organisms belonging to the rickettsiae and psittacosis-lymphogranuloma groups is controversial. Although, like viruses, all pathogenic forms of these organisms are obligate intracellular parasites, in other respects they resemble bacteria. Since a cell wall, deriving its rigidity from mucopeptide, which contains the amino sugar muramic acid as a key constituent, has so far only been found in bacteria and the closely related blue-green algae, the presence of muramic acid in an organism may be used as a taxonomic criterion. The mucopeptides of bacterial cell walls are also often sensitive to lysozyme, so that dissolution by this enzyme serves as an indication of the presence of mucopeptide. Organisms of the rickettsiae and psittacosis-lymphogranuloma groups have been examined for the presence of muramic acid. Critical chemical tests have shown that this substance is present in organisms of both groups. Cell walls of Rickettsia burnetii were dissolved by lysozyme. In the light of these and other results the taxonomic position of these groups of organisms is discussed.

The chemical instability and reactivity of certain biologically important sulphur derivatives are recalled and their relevance to the study of sulphur auxotrophs is pointed out.

Glucose and certain other sugars accelerated hydrogen sulphide production from l-cysteine by washed cells of Escherichia coli (strain Crookes) which had been grown in the presence of l-cysteine. On the other hand, glucose or some other sugars in protein hydrolysate media containing l-cysteine suppressed the synthesis of an enzyme(s) which mediates the formation of hydrogen sulphide from l-cysteine. Glucose accelerated sulphide formation from l-cysteine by sonicated preinduced cells, although activity was unstable to such treatment. Both effects of glucose were influenced by the amino acid content of the medium. Sulphide production probably resulted through the action of cysteine desulphydrase; certain evidence suggested that a transaminase linked to β-mercaptopyruvate desulphurase also may have functioned. Apparently glucose repressed the induction of one or more enzymes concerned with cysteine degradation.

When a column of air-dried soil was perfused with a mineral salt solution containing ‘Dalapon’ (Dow Chemical Company, Michigan), the concentration of dichloropropionate (Dalapon's herbicidal component) became minimal in 15--17 days. The soil was reperfused and the dichloropropionate became minimal in 4--5 days.

Three strains of the genus Pseudomonas, exhibiting a requirement for dichloropropionate, were isolated from the perfusate. Two strains attacked dichloropropionate when allowed to perfuse through sterilized soil.

A further seven micro-organisms, with a similar requirement, were isolated from pasture soils treated with Dalapon.

Autoradiographs were prepared from organisms of Paramecium aurelia which were grown in an axenic medium to which one of the following compounds was added: [carboxy-14C]glycine, [8-14C]adenosine, [8-14C]-guanosine, [2-14C]cytidine, [3H]thymidine, and [3H]uridine. Trichloro-acetic acid extracts of organisms grown with the isotopically labelled glycine, adenosine and cytidine were prepared and the radioactivity found either in the protein or in the acid-soluble fractions was determined in a scintillation counter. P. aurelia was unable to incorporate glycine into the nucleic acids of the macronucleus; however, this compound was utilized in the formation of the cytoplasmic proteins. Thymidine was preferentially taken up by the macronucleus; uridine was found mainly in the cytoplasm. The other purine and pyrimidine ribosides were found both in the macronucleus and cytoplasm.