- Volume 130, Issue 5, 1984

Novozym 234, a commercially available enzyme, has been used successfully to prepare protoplasts from yeast-like cells, mycelium and chlamydospores of the polymorphic fungus Aureobasidium pullulans. Use of the density gradient material Percoll after wall digestion resulted in preparations of high purity that were metabolically active. Size distributions of protoplasts were determined easily using Coulter analysis.

Extracellular membrane particles were isolated from the cell-free culture liquid of Acinetobacter calcoaceticus 69-V grown on n-alkanes. It was demonstrated by means of an antiserum raised against intact cells of A. calcoaceticus 69-V grown on hexadecane that the particles were probably derived from the surface membrane fraction of the bacteria. The accumulation by the bacteria of radioactivity originating from [14C]hexadecane solubilized in the particles was studied. The apparent K m value for the uptake of solubilized hexadecane was 33 µm and the V max value was 1·2 × 10−9 mol min−1 (mg bacterial protein)−1. The K m value for the uptake of free hexadecane was approximately 10 times higher, indicating the possible role of particles as transport vehicles for the insoluble substrate. Accumulation of radioactivity was inhibited by KCN and NaN3, suggesting an energy-dependent uptake process.

Inclusion of ammonia in germinant mixtures containing l-alanine and inosine stimulated germination of unactivated Bacillus cereus spores at rates equal to those obtained using heat-activated spores without ammonia. d-Alanine had little effect on germination of heat-activated spores, but severely inhibited germination of unactivated spores in the presence of ammonia. Ammonia did not replace the requirement for either l-alanine or inosine: all three compounds were required for rapid germination. Kinetic analysis suggested that the functions of ammonia and l-alanine were more closely related than the functions of ammonia and inosine. With rate-saturating concentrations of l-alanine and inosine, germination rates showed saturation kinetics for ammonia with a K m for NH4C1 of 5 mm. Comparisons of the effects of salts, amines and pH on germination rates suggested that NH4OH rather than NH4 + was the rate-limiting form of ammonia. In comparisons of various strains of B. cereus, stimulation of germination by ammonia occurred in all cases, although spores of most soil isolates germinated more rapidly than B. cereus T spores in the absence of ammonia.

Gas-liquid chromatography (GLC) and high-performance liquid chromatography (HPLC) were used to separate organic acids in spent culture media. Organic acid components were identified on the basis of their retention times and confirmed by gas chromatography/mass spectrometry (GLC/MS). The sensitivity of the analytical procedures varied considerably with each organic acid. GLC analysis of spent culture media esterified by the H2SO,4/methanol procedure failed to detect fumarate, malate and lactate. The BF3/methanol derivatization was particularly sensitive to pyruvate and lactate. GLC/MS, using the BF3/methanol technique, was insensitive to acetate. Cation-exchange HPLC, using a Bio-Rad acid analysis column, did not require prior derivatization of the sample, and proved to be a rapid and non-destructive assay technique for organic acids. This procedure was used to analyse the spent culture media of aerobically grown Azospirillum brasilense and those of Desulfovibrio desulfuricans grown on pyruvate fermentatively or with sulphate as the electron acceptor. Organic acid profiles of the spent culture media obtained by cation-exchange HPLC analysis were compared with those obtained by GLC analysis using both the H2SO4/methanol and the BF3/methanol esterification procedures.

Effects of very low concentrations of dissolved O2 on nitrogenase activity in Klebsiella pneumoniae were studied in a stirred chamber system which enabled simultaneous measurements of steady-state O2 concentrations, O2 consumption and C2H2 reduction. A strain carrying a chromosomal nifH:: lac fusion as well as the Nif+ pRDl, expressed nitrogenase activity with 80 nm-O2, a concentration known to inhibit nifH:lac expression by about 50%. Thus nitrogenase activity in vivo was no more sensitive to O2 than expression of nifH:: lac. When compared with anaerobic treatments, dissolved O2 near 30 nm apparently stimulated nitrogenase derepression and enhanced the activity of nitrogenase synthesized anaerobically. Thus, in this organism, N2 fixation occurs in microaerobic as well as anaerobic conditions.

Three strains of Gram-negative, obligately aerobic, moderately thermophilic, CO-utilizing bacteria have been isolated by enrichment at 45–55 °C in mineral medium under CO, H2 and air. The properties of the strains, designated C2, SI and FE, suggest that they should be included in a single species that we have named Pseudomonas thermocarboxydovorans. The isolates can grow on CO and a limited range of organic and amino acids; neither sugars nor H2/CO2 support growth and H2 is not oxidized. Strain C2 contains a soluble, inducible CO. phenazine etho-sulphate oxidoreductase, lacks hydrogenase and assimilates CO via ribulose bisphosphate carboxylase. The organism apparently contains a mechanism for assimilating phospho-glycollate.

The cyanobacterium Anabaena torulosa required sodium and molybdenum for nitrogenase activity and diazotrophic growth. Addition of sodium or molybdenum to cultures deficient in either element restored nitrogenase activity. Heterocyst differentiation was unaffected by sodium but molybdenum deficiency enhanced differentiation. The non-heterocystous cyanobacterium Plectonema boryanum 594 also required sodium for nitrogenase activity but synthesized presumptive nitrogenase component I and II proteins during sodium deficiency. The results show that in cyanobacteria nitrogenase is synthesized even in the absence of sodium but functions only in its presence.

When a histochemical stain for β-galactosidase activity is applied to growth of Gram-negative bacteria on agar medium, the pigmentation is non-uniform and capable of revealing internal colony organization into different cell types. Use of an Escherichia coli strain with a thermo-sensitive lac repressor indicates that colonies expand by addition of new cells at the periphery and that older cells which have synthesized-galactosidase early in development remain in the centre. Mixed inocula of different strains show clonal exclusiveness as they proliferate outwards. Mudlac transposons can create genetic fusions that place β-galactosidase expression under a variety of regulatory systems. Stained surface cultures of E. coli and Pseudomonas putida strains carrying Mudlac insertions in plasmids reveal a variety of flower-like staining patterns. These patterns display both clonal (i.e. sectorial) and non-clonal (circular and radial) features which are heritable within a given strain. The non-clonal aspects of the patterns reflect phenotypic differentiation without genetic change. These observations indicate that bacterial growth on agar surfaces is a highly regulated process similar, in many respects, to the development of specific multicellular tissues and organisms.

Changes in the lipids of Sclerotium rolfsii were followed during its growth in agar and liquid synthetic media. The fungus was grown either aerobically on a cellophane membrane placed on a synthetic agar medium, or under submerged conditions in the same liquid medium, using a rotary shaker, with sclerotial formation induced by pouring the culture into Petri dishes. In the aerobically-grown fungus, lipid content decreased from 119·7 µg (mg dry wt)−1 in the mycelium to a low content of 7·4 µg (mg dry wt)−1 in mature sclerotia. The non-polar lipids were mainly utilized at the sclerotial initiation stage, while the polar lipids were utilized during sclerotial maturation. Total lipid content in submerged mycelium was 10-fold less than in aerial mycelium; however initial and mature sclerotia produced from either the submerged or aerial mycelium were similar in their lipid content and in the composition and distribution of fatty acids. Increasing glucose concentrations in the growth medium increased the fungal biomass but did not change lipid content or composition in the two growth systems. A supplement of 10−2 m-l-threonine to the growth media significantly increased the number of sclerotia produced in submerged culture. l-Threonine also prolonged the lag phase of a submerged culture and caused a delay in lipid accumulation. Moreover, l-threonine increased the lipid level but did not change the lipid or fatty acid distribution. This may help in elucidating the role of lipids in the mycelium during sclerotial formation.

The localization of chitin synthase in the cells of Mucor rouxii was studied by a method which combined permeabilization of the cells with toluene/ethanol and incubation with the radioactive substrate UDP-[3H]GlcNAc followed by high resolution autoradiography. By this technique it was demonstrated that most of the chitin synthesized by these cells was located within the cytoplasm, and only a small amount of the enzyme product appeared at the cell surface. It was concluded that most of the chitin synthase of M. rouxii is located in the cytoplasm of the cells.

A laboratory-grown strain of Neisseria gonorrhoeae, selected in vivo, BS4 (agar), is susceptible to complement-mediated killing by fresh human serum but is relatively resistant to killing by human phagocytes. It can be induced to serum resistance by incubation with a small molecular weight fraction of guinea pig serum. The serum-susceptible and induced-resistant forms show differences in pyocin sensitivity tests. This indicates either differences in the structure of their lipopolysaccharides or masking of some determinant(s). The pyocin sensitivity pattern of BS4 (agar) is only slightly different from that of a closely related strain, BSSH, which is more susceptible to killing by human phagocytes.

A previously unrecognized bacterial flagellar structure present in Campylobacter jejuni and Campylobacter fetus is described. The structure is located just below the insertion of the flagellar apparatus through the bacterial wall. It is disc shaped, approximately 100 nm in diameter and has eleven spokes radiating from a central hole. The function of this new structure and its possible importance in the taxonomy of campylobacters and other spiral micro-organisms are discussed.