- Volume 135, Issue 11, 1989

The ability of selected bacterial cultures to synthesize ethylene during growth in nutrient broth supplemented with methionine or 2-oxo-4-methylthiobutyric acid (KMBA) was examined. Although most cultures transformed KMBA into ethylene, only those of Escherichia coli SPAO and Chromobacterium violaceum were able to convert exogenously added methionine to ethylene. In chemically defined media, E. coli SPAO produced the highest amounts of ethylene from methionine and KMBA. This capability was affected by the nature of the carbon source and the type and amount of nitrogen source used for growth. When glutamate was used as sole source of carbon and nitrogen for growth, the activity of the ethylenogenic enzymes was reduced to 25% of that observed with cultures grown with glucose and NH4Cl. Neither methionine nor KMBA significantly affected the ethylenogenic capacity of E. coli SPAO. Menadione and paraquat, compounds that generate superoxide radicals, stimulated ethylene synthesis by harvested cells, but not by cell-free extracts of E. coli SPAO. In addition, cells of Pseudomonas aeruginosa, which produced no ethylene in culture in the presence of exogenously added KMBA, yet possessed the necessary enzymes in an active form, were able to synthesize ethylene from KMBA when incubated with menadione or paraquat.

Summary: The filamentous fungus Sclerotium glucanicum excreted significant amounts of 1,3-β-glucanase, 1,6-β-glucanase and --glucosidase activities when the culture medium was depleted of carbon sources. During starvation small amounts of intracellular 1,3-β- and 1,6-β-glucanase and β-glucosidase activities were also detected. Very low levels of -glucanase activity remained bound to mycelium and some activity was found loosely attached to the cells and/or to water-soluble 1,3-β-1,6-β-glucan adhering to the cell walls. During active growth intracellular 1,3-β-glucanase and mycelium-bound 1,3-β- and 1,6-β-glucanase activities were detected in small or trace amounts. During hyphal growth very low levels of 1,3-β- and 1,6-β-glucanase activities were also found to be weakly associated with the cells and/or with water-soluble -glucan covering the hyphae. Cycloheximide inhibited the increase in intra- and extracellular 1,3-β- and 1,6-β-glucanase and -glucosidase activities. This indicated that de novo protein synthesis was involved in the intra- and extracellular appearance of these three enzyme activities in derepressed cells. The formation of the extracellular 1,3-β-glucanase, 1,6-β-glucanase and -glucosidase activities was regulated by catabolite repression. 1,3-β- and 1,6-β-glucanase activities were uncompetitively inhibited and -glucosidase activity noncompetitively inhibited by glucose and glucono-δ-lactone. Optimum pH and temperature values as well as thermal stabilities of the three extracellular enzyme activities were determined. Almost all of the -glucosidase activity but only one-third of the extracellular 1,3-β- and 1,6-β-glucanase activities were found to bind to Con A-Sepharose. Under conditions of carbon limitation almost 90% of the extracellular 1,3-β-/1,6-β-glucan excreted during fungal growth was degraded by the extracellular -glucanases.

Summary: Methylophilus methylotrophus produced viscous and non-viscous exopolysaccharides (EPS) when grown in batch culture. Both types contained glucose, galactose, mannose and an unidentified 6-desoxyhexose, and were substituted with pyruvate and acetate residues. When the organism was grown in continuous culture only the non-viscous EPS was synthesized; the rate of production was 18·5 mg h−1 (g biomass)−1 in methanol-limited cultures and increased by approximately 3- and 4-fold when growth was limited by oxygen or nitrogen respectively. The specific activity of methanol dehydrogenase in cell extracts was relatively low when bacteria were grown under conditions of methanol excess and increased 2-fold in carbon-limited cells, reflecting the need to scavenge the small amounts of available methanol. In contrast, the specific activities of several key enzymes of the ribulose monophosphate (RuMP) pathway were greater in cells grown under conditions of nitrogen or oxygen limitation than when growth was Limited by the availability of carbon, indicating the potential for increased carbon flux round the cycle when excess methanol was present in the growth medium. When methylotrophs are grown under conditions of methanol excess it is important that there is a mechanism to prevent the overproduction of formaldehyde, and we suggest that these changes in EPS production and in the specific activities of the key enzymes of the RuMP cycle are necessary for the efficient removal of this toxic metabolite of methanol.

Summary: Corynebacterium sp. XG is a facultative methylotroph. Determination of the activities of several enzymes and use of mutants unable to grow on methanol indicated that in this Gram-positive strain methanol was metabolized through the serine pathway. Synthesis of the enzymes of this pathway was induced when cells were grown in the presence of methanol or its catabolic products.

During growth of Escherichia coli ML308 on pyruvate in a continuous culture (turbidostat) or batch culture, flux of carbon into the cells exceeds the amphibolic capacity of the central pathways. This is balanced by diversion of carbon flux to acetate excretion which in turn diminishes the efficiency of carbon conversion to biomass [g] dry wt (mol substrate)−1]. However, restriction of carbon supply in a chemostat diminishes flux to acetate excretion and at a dilution rate (D = µ) of 0·35 h−1 or less, no flux to acetate excretion was sustained thus permitting perfect balance between carbon input on the one hand, and the output to biosynthesis and energy generation on the other. This, in turn, improves the efficiency of carbon conversion to biomass. Inclusion of 3-bromopyruvate (an inhibitor of pyruvate dehydrogenase) at a concentration which diminishes growth rate (µ) to 0·35 h−1 or less also prevented flux to acetate excretion. Furthermore, in a family of fluoroacetate-resistant strains, excessive flux of pyruvate was balanced by diversion of carbon flux to lactate excretion rather than acetate and a higher growth rate (µ = 0·63 h−1) was sustained.

Binding-protein-dependent sugar transport has been investigated in Agrobacterium radiobacter and A. tumefaciens. A. radiobacter contained two high-affinity glucose-binding proteins (GBP1 and GBP2) that additionally bound d-galactose (K D 0·26 μm) and d-xylose (K D 0·04 μm) respectively and were involved in the transport of these sugars. Partial sequencing of GBP1 and GBP2 showed that GBP2 exhibited significant homology with both the arabinose-binding protein (ABP) and the galactose-binding protein (GalBP) from Escherichia coli, whereas GBP1 exhibited significant homology only with ABP. Antiserum raised against GBP1 cross-reacted with GBP1 but not with GBP2, and vice versa. Anti-GBP1 and anti-GBP2 also cross-reacted with proteins corresponding to GBP1 and GBP2 respectively in A. tumefaciens, but little or no cross-reaction was observed with selected members of the Enterobacteriaceae, Rhizobiaceae and Pseudomonadaceae families grown under glucose limitation. GBP1 was less strongly repressed than GBP2 following batch growth of A. radiobacter on various carbon sources. The growth of A. radiobacter for more than approximately 10 generations in continuous culture under galactose or xylose limitation (D 0·045 h−1) led to the emergence of new strains which exhibited increased rates of glucose/galactose or glucose/xylose uptake, and which respectively hyper-produced GBP1 (strain AR18a) or GBP2 (strain AR9a). Similarly, growth of A. tumefaciens for more than approximately 15 generations under glucose or galactose limitation produced new strains which exhibited increased rates of glucose/xylose or glucose/galactose uptake and which respectively hyperproduced proteins analogous to GBP2 (strain AT9) or GBP1 (strain AT18a). It is concluded that growth of Agrobacterium species under carbon-limited conditions leads to the predictable emergence of new strains which specifically hyperproduce the transport system for the limiting nutrient. The GBP1-dependent system of A. radiobacter is unique amongst these transport systems in that the mutations that lead to its hyperproduction under carbon limitation render it least susceptible to repression by excess glucose during ammonia limitation, with the result that succinoglucan exopolysaccharide is produced from glucose at an enhanced rate.

Staphylococcus aureus H growing exponentially was labelled with N-acetyl[14C]glucosamine, which became incorporated into the peptidoglycan. The portion of peptidoglycan not linked to teichoic acid (60–75% of the whole) was degraded with Chalaropsis muramidase to yield disaccharide-peptide monomers and dimers, trimers and oligomers formed by biosynthetic cross-linking of the monomers. The degree of O-acetylation of these fragments was also examined. Pulse-chase experiments showed that the proportion of label initially in the monomer fraction immediately after the 1 min pulse declined rapidly during a 3 min chase, while the oligomer fraction (fragments greater than trimer) gained the radioactivity proportionately. The radioactivity of the dimer and trimer fractions remained virtually unchanged. At 4 min after the commencement of labelling (i.e. approx. one-tenth of a generation time) final values had been reached. The O-acetylation of all fragments had achieved final values even at 1 min, except for the monomer fraction, which showed an increase from 40% to 60% during the first 3 min of chase. Although O-acetylation was clearly a very rapid process, no O-acetylated peptidoglycan lipid-intermediates could be detected.

The immediate activities of the aminoglycoside antibiotic tobramycin were investigated in Pseudomonas aeruginosa PAO1. The lethal action of a low concentration of tobramycin (8 μgml−1) occurred rapidly (1–3 min) and was associated with leakage of certain cellular components into the supernatant. The presence of magnesium at the time of initial exposure protected cells by preventing uptake of tobramycin; however, magnesium addition following a brief exposure did not restore viability. Analyses of supernatant material revealed a rapid 2-fold increase in protein released following tobramycin treatment. A prominent 29 kDa protein, observed by SDS-PAGE in the released material was identified as the periplasmic β-lactamase. Brief exposure to tobramycin did not result in major morphological damage or cell lysis as observed by transmission electron microscopy, and release of LPS was not a primary event. Although activity at the ribosomal level was observed by 2–3 min, leakage was detected after only 1 min. These data indicate that leakage of cellular components, particularly β-lactamase, occurs simultaneously, if not prior to inhibition of protein synthesis by tobramycin.

The morphology of Candida albicans cells was determined from their maximum length, maximum diameter and septal diameter in a mathematical ratio, the morphology index (Mi), which usually ranged from approximately 1 for spherical yeast cells to approximately 4 for true hyphae, with elongated yeast cells and pseudohyphae giving intermediate values. Mi could be determined with high reproducibility for C. albicans grown in a variety of environments. The highest mean Mi was seen with cells grown in serum and Eagle's medium at 37°C, the lowest with cells grown in Sabouraud glucose broth at 26°C. Variant strains of C. albicans gave Mi values that remained constant in a variety of growth environments. The Mi facilitated detection of two variants that grew exclusively in the yeast form, one that grew as elongated yeasts but could be induced to form pseudohyphae in serum, and one consistently pseudohyphal variant. Cells with a mean Mi up to 2·5 could be easily separated at septal junctions by mild ultrasonication, whereas cells with a mean Mi greater than 3·5 tended not to separate under these conditions. The chitin content of C. albicans cells was almost twice as great in cells with a Mi approaching 4 as in cells with a Mi close to 1. The wide range of Mi distributions for a single C. albicans isolate in different environments demonstrates that the fungus does not undergo abrupt changes of morphological phase: rather there are continual changes in morphology between spherical yeasts and true hyphae at the extremes. The study shows that Mi can be used reliably in place of subjective descriptions of morphology to indicate the shape of a C. albicans cell. It should facilitate the detection of molecular and cellular markers specific for morphogenesis in the fungus.

The obligately methylotrophic bacterium Methylophilus methylotrophus hydrolyses aliphatic amides to ammonia and aliphatic acid using a cytoplasmic amidase. Physiological regulation of amidase activity was investigated by growing the organism under various conditions in batch, fed-batch and continuous culture. The results showed that synthesis of the enzyme was induced by various amides (acrylamide > acetamide) and repressed by ammonia. Growth of the wild-type organism in acetamide-limited continuous culture at very low dilution rate (D 0·025 h−1) led to the selection of a hyperactive strain (MM6), the subsequent growth of which under acrylamide limitation led to the selection of another strain (MM8) which showed even higher activity. The amidase activities of strains MM6 and MM8 were respectively approximately four and twelve times higher than that of the wild-type organism following growth under similar conditions, whereas the concentrations of the enzyme as determined by SDS-PAGE and scanning densitometry were approximately four times higher than the wild-type organism in both strains. The amidase in strain MM8 exhibited a K m for acrylamide that was approximately one-third lower than that of the wild-type organism or of strain MM6. It is concluded that the hyperactivity of strain MM6 was due predominantly to the production of more wild-type enzyme, whilst the hyperactivity of strain MM8 was due to the production of approximately the same amount of enzyme as strain MM6 (up to 25% of the total cell protein depending on the nature of the limiting amide) but with a substantially enhanced catalytic activity (K cat). These changes were apparently the result of spontaneous mutations that occurred in response to growth at extremely low amide concentrations, giving the novel strains a strong selective advantage under these conditions (possibly by enhancing the rate of diffusion of amide into the cell).

The use of a continuous Percoll gradient in a procedure for the rapid isolation of bacteroid- containing vesicles from French bean nodules is described. The purified vesicles appeared to be free from contamination by naked bacteroids and plant mitochondria and suitable for physiological studies. The metabolite uptake activities of vesicles isolated by this method were compared with those of free bacteroids. Succinate was transported through both peribacteroid and bacteroid membranes: the K m, values were 320μm. and 57μm, respectively. For glucose K m, values were 142μm. for the peribacteroid membrane and 102μm. for the bacteroid membrane, indicating that glucose could act as an energy-yielding substrate in functioning nodules. Experiments with inhibitors showed the involvement of ATPases in these transport processes and suggested that a proton-motive force was probably associated with them. The regulatory role of the peribacteroid membrane in the movement of the metabolites from the plant cytosol to bacteroids was demonstrated by its impermeability to glutamate, aspartate, citrate and benzoate.

A lysogenic strain, Rhizobium leguminosarum biovar trifolii UK-1: φU, was isolated from a wild white clover nodule. It was symbiotically effective on white clover. A lysogenic phage (U-mole) was induced from this strain by treatment with either UV irradiation or mitomycin C. Phage U-mole had an icosahedral head (40 nm wide), a short tail (9 nm long) and tail fibres (15 nm long). Phage U-mole was induced with mitomycin C both from bacterial cells in infection threads and from bacteroids in nodules. Southern hybridization using EcoRI fragments of phage U-mole as probe indicated that phage U-mole DNA was integrated into the chromosome of strain UK-1: øU at a site involving the 60 kb EcoRI fragment of phage U-mole. Phage U-mole also lysogenized the wild-type strain R. leguminosarum biovar trifolii 4S. The resulting lysogenized strain, 4S:øU, had lost its 315 kb Sym plasmid and its nodulation ability.

The data from an integrated numerical classification was used to construct identification schemes for some fasciculate penicillia. The identification schemes were presented as a synoptic key and a frequency matrix for computer-assisted identification. Statistical testing of the frequency matrix showed that although character separation values were generally low, only four pairs of taxa showed overlap greater than that expected for a rectangular distribution. The identification schemes were tested practically with 52 previously studied strains and 51 further cultures. A synoptic key based on 10 and 90% cutoff limits was used to correctly identify 44 of the 51 additional strains, although this proved very sensitive to single test discrepancies. The frequency matrix was used to correctly identify 45 of the additional strains with a Willcox probability score and this was compared to identifications based on the modal likelihood fraction.

Polyacrylamide gel electrophoresis of extracellular pectinase and amylase isozymes of 170 mainly terverticillate Penicillium strains was undertaken. The data were coded and subjected to numerical analysis. Variation in intensity of isozymes was observed in repeat analyses of some strains, although most were consistent. Variation was also observed between some representative strains of species. P. viridicatum was more variable than P. brevicompactum and P. hordei for intensity of pectinase activity. There was a correlation between the grouping of the strains on the basis of the isozymes and the species concepts only in some cases. The method proved useful for the identification of strains producing intense activity which provided clear patterns, for example, P. brevicompactum and P. chrysogenum and to a lesser extent P. solitum var. crustosum and P. hordei. The method was also exclusionary in that some species were restricted to a particular cluster or subcluster. Amylase patterns confirmed that strains referred to as single species are not all homogeneous genetically, and that some strains are not simply haploid homokaryons. The genetic heterogeneity of the strains explains some of the problems in the systematics of the terverticillate penicillia.

Twenty-five strains classified as Mycoplasma mycoides subsp. mycoides LC or subsp. capri have been compared by one-dimensional SDS-PAGE of their cellular proteins. A computerized numerical analysis revealed that the protein patterns of all but two aberrant strains formed one large phenon that separated clearly from representatives of the four other members of the ‘M. mycoides cluster’ at a similarity level (S) of 66% and which remained undivided at up to 78% S. At higher similarity levels, these strains fell heterogeneously into mixed sub-phenons containing strains of both subspecies. Serological comparisons by immunofluorescence largely confirmed the subspecies designations of the test strains, but also showed that some were serologically intermediate between subsp. mycoides and subsp. capri, being cross-reactive with both. These results confirm and enlarge upon those of our earlier studies indicating the protein-pattern inseparability of subsp. capri and subsp. mycoides LC strains and their distinctiveness from the classical M. mycoides subsp. mycoides SC strains and other members of the ‘M. mycoides cluster’. As also recognized by other workers, subsp. mycoides LC and subsp. capri strains appear to comprise one large group, wherein those most readily identifiable as either type lie at either end of a serological spectrum that also contains serologically cross-reactive strains. Our observations therefore suggest the lines along which the three groups classified at present within the species M. mycoides (SC and LC strains of subsp. mycoides; subsp. capri) might eventually be reclassified, subject to direct genomic comparisons.