- Volume 125, Issue 1, 1981

Proteolysis of leaf Fraction 1 protein, casein, azocasein and bovine serum albumin by the cell-associated proteinases of the rumen bacterium Bacteroides ruminicola R8/4 was investigated and the kinetic parameters V max and K m were evaluated for each substrate. A variety of proteinase inhibitors was used to show that the proteolytic activity comprises a mixture of at least three different classes of proteinase. With respect to substrate specificity and some inhibition characteristics, the proteolytic activity of B. ruminicola R8/4 was similar to that of rumen contents.

The cyanophycin granule polypeptide (CGP) is known to serve as a nitrogen reserve protein in cyanobacteria and is mobilized during nitrogen starvation. An exopeptidase, provisionally called cyanophycinase, which degraded CGP in vitro has been studied, and its product characterized, in two N2-fixing Anabaena species. The enzyme had a pH optimum of 8·5, had no requirement for monovalent or divalent cations and was inhibited by l-arginine and l-aspartic acid. The product of this enzyme was an aspartic acid-arginine dipeptide. A higher activity of both arg-poly(asp) synthetase and cyanophycinase was observed in extracts of heterocysts of both Anabaena species than in vegetative cells. This supports the view that CGP has a dynamic role in nitrogen metabolism of cyanobacteria as well as a storage function.

The phenol oxidases from mature fruiting bodies of Lentinus edodes (Berk.) Sing., a commercially cultivated mushroom, were studied. The major phenol oxidase was a laccase with a pH optimum near 4.0 and an apparent molecular weight of 100000. Catechol oxidase and tyrosinase were also present. The laccase investigated was primarily extracellular; the highest activity was in the pigmented rind of the pileus and in the stipe. Increased laccase activity was associated with rapid growth of non-pigmented aerial mycelium and formation of pigmented primordia and fruiting bodies. Possible functions of the laccase and its regulation during the development of fruiting bodies are discussed.

The growth and development of a Curtobacterium sp., a Mycoplana sp. and a Pseudomonas sp. in the rhizosphere of gnotobiotic wheat, barley and maize plants were studied. When inoculated singly in the wheat or barley rhizosphere, each of the three species grew exponentially during the first 48–72 h and slowly thereafter. However, in the maize rhizosphere the Curtobacterium sp. showed only a small increase in numbers and the Mycoplana sp. had a long lag phase, a slower growth rate and a smaller final population than when it was inoculated into the wheat or barley rhizosphere. Interactions between these three species in various combinations and in different rhizospheres are described and some of the possible mechanisms involved are discussed.

pLE2451, a 24·5 megadalton conjugative plasmid from Neisseria gonorrhoeae, was capable of efficiently mobilizing gonococcal β-lactamase plasmids between gonococci and from gonococci to Haemophilus influenzae and restriction-deficient Escherichia coli. Donor strains of N. gonorrhoeae carrying pLE2451 were also found to be capable of mobilizing a variety of non-conjugative plasmids originally derived from enteric bacteria or Haemophilus species when such plasmids were resident in E. coli. Nevertheless, pLE2451 was not detected physically in E. coli or H. influenzae transconjugants. This suggests that the plasmid is unstable in these hosts but survives transiently to provide transfer functions for mobilization. The proficiency of pLE2451 in promoting intraspecific and intergeneric mobilization was not paralleled by pUB701, pR1234 and pFR16017, a series of conjugative plasmids derived originally from Haemophilus species. These plasmids were incapable of mobilizing even Haemophilus -lactamase plasmids, such as RSF0885, between Haemophilus species.

No DNA polymerase I-like activity was found associated with the ultraviolet (u.v.)-protecting plasmids R205. R46 or pKM101 in either uninduced or u.v.-induced wild-type or DNA polymerase I-deficient strains of Escherichia coli. Nor was any plasmid-associated polymerase activity detectable in similar systems containing u.v.-irradiated DNA as template. However, plasmids R205, R46 and pKM101 still increased survival and mutagenesis of the polymerase I-deficient E. coli strain after u.v. irradiation.

Two mutations in the modC locus of Podospora anserina, which abolish the process of cell lysis resulting from the combination of the R and V non-allelic incompatibility genes, have previously been shown to produce complete suppression of a laccase exoenzyme. In the present study it was found that, in addition, the modC mutants show a marked increase in the activity of an amino-acid oxidase (an exo- and endoenzyme), most of the increase (some 9- or 15-fold) being in the extracellular fraction. The modC mutant strains are hypersensitive to cycloheximide in vivo, but biochemical and genetical investigations suggested that the hypersensitivity is not due to effects on the ribosome. The modC(l) strain also shows increased resistance to chlorate, a specific sensitivity to acid (6.5) and neutral (7.5) pH, and significant hypersensitivity to methylammonium and thiourea. ModC cells accumulate a higher amount of the urea analogue [14C]thiourea. The pH sensitivity associated with the modC(l) mutation is specifically suppressed by the combination of the R and V incompatibility genes, and the RVmodC(l) strain is resistant to sorbose. The specific consequences of the modC(l) mutation and the suppression of some of its effects by the combination of the R and V incompatibility genes lead to the suggestion that protoplasmic incompatibility might be induced by action of the R/V initiating complex in association with the plasma membrane.

The phagocytic capacity of peritoneal macrophages from resistant C3Hf mice and sensitive C57Bl/6 mice was studied in vitro using a virulent and an avirulent strain of Salmonella typhimurium. Virulent and avirulent 3H-labelled bacteria opsonized with normal mouse serum were killed to an equal extent (about 40 %) by macrophages from C3Hf mice and C57Bl/6 mice within 5 min after contact. Killing of both bacterial strains by macrophages from C3Hf mice continued at a lower rate for the next 30 min until about 40% of the remaining bacteria were killed. In this later phase macrophages from C57Bl/6 mice killed avirulent S. typhimurium to an extent comparable with the killing by macrophages from C3Hf mice, whereas macrophages from C57Bl/6 mice were unable to kill virulent S. typhimurium. Cytochalasin B did not inhibit the rapid initial killing of bacteria opsonized with normal mouse serum, but completely inhibited the slower phase of killing. From these results it is concluded that the resistance of the mice to infection with S. typhimurium correlates with the bactericidal activity of their peritoneal macrophages, and that killing of the bacteria occurs in an early extracellular phase followed by an intracellular phase. It is only the latter phase which reflects the animal’s resistance to infection.

The chemiluminescence response of macrophages to opsonized live S. typhimurium was independent of the susceptibility of the mice from which the macrophages were taken. Cytochalasin B and 2-deoxy-D-glucose reduced the chemiluminescence generated by opsonized or non-opsonized S. typhimurium. Comparison of the kinetics as well as inhibition, by cytochalasin B and 2-deoxy-d-glucose, of chemiluminescence and killing of S. typhimurium showed that the killing reaction of the peritoneal macrophages was not related to their chemiluminescence response.

During growth in liquid culture containing a single cellulosic or non-cellulosic carbon source, a newly isolated strain of Aspergillus fumigatus released cellulases into the medium; the amounts produced depended on the nitrogen source, the type and concentration of the carbon source, pH and temperature. Extracellular cellulolytic activity was still increasing after incubation for 60 d with 1 % (w/v) CF11 cellulose, (NH4)2SO4 as nitrogen source and a starting pH of 7. The activities of the new isolate were compared with those of A. fumigatus IMI 143864 and Trichoderma reesei QM6a (ATCC 13631) and it was shown to be a good producer of β-glucosidase.

Growth, hydrogen production and cellulose digestion by Acetivibrio cellulolyticus strain CD2 were considerably greater when the culture pH was maintained at 7·0 than when the pH was not controlled. Furthermore, if the pH of the growth medium was controlled the number of viable organisms was 6-fold greater after 3 d incubation and 100-fold greater after 6 d incubation compared with equivalent cultures in which the pH was not controlled. The differences were due to the combined effect of low pH values and acetic acid accumulation. The number of viable organisms was 2- to 3-fold lower after 12 h incubation in substrate-free medium containing 40 mm-acetic acid at pH 5·5 than in the same medium at pH 7·0. Addition of 90 mm-acetic acid during growth in a cellobiose-containing medium lowered the growth rate by 30% and the rate of hydrogen production by 40%. Exposure of A. cellulolyticus to oxygen for up to 2 h did not affect viability measurements provided that the organisms were subsequently transferred to reduced media.

Size selection by continuous-flow centrifugation was used to prepare synchronous cultures of Bacillus subtilis. A selection of 5–10% was required to yield cultures which gave synchrony indices of about 0·45. Respiration rates [nmol O2 min−1 (ml culture)−1] of cells growing in the presence of glucose rose discontinuously as two steps in each cell cycle with the mid-points of the rises centred at 0·37 and 0·87 of a cycle. An asynchronous control culture exhibited no such periodicities. Two inhibitors of energy conservationN,N′-dicyclohexylcarbodi-imide and 4-chloro-7-nitrobenzofurazaninhibited respiration maximally at 0·0 and 0·6 of a cycle whilst an inhibitor of respiration2-heptyl-4-hydroxyquinoline-N-oxideexerted maximal effect only at 0·4 of a cycle. These results are compared with other temporal events which have been reported to occur during the cell cycle of B. subtilis.

In aerobic chemostat culture Thiobacillus A2-GFI grew autotrophically on formate and heterotrophically on glucose with maximum specific growth rates (μmax) of 0·21 and 0·33 h−1, respectively. At dilution rates of 0·1 and 0·18 h−1, it grew mixotrophically on formate + glucose mixtures, completely consuming both substrates. Ribulose-1,5-bisphosphate carboxylase and formate dehydrogenase were present at high specific activity in autotrophic and mixotrophic cultures, but were repressed in cultures on glucose alone. A greater proportion of added glucose was assimilated in mixotrophic culture than in heterotrophic culture. Raising the dilution rate of a mixotrophic culture from 0·1 or 0·18 to 0·3 h−1 resulted in washout (with an apparent μ max for mixotrophic growth of 0·25 h−1) and the establishment of a culture dependent on glucose for growth. Growth yields on formate and glucose were, respectively, 3·3 and 100 g dry wt (mol substrate consumed)−1. Steady state biomass production in mixotrophic culture indicated additive growth yields. The biomass produced in cultures on formate + glucose at a dilution rate of 0·3 h−1 suggested that growth only occurred on glucose, but organisms still contained high activities of ribulose-1,5-bisphosphate carboxylase and formate dehydrogenase. At a formate: glucose ratio (mm) of 100:1, some formate was oxidized and CO2 was fixed, but formate was not used when this ratio was 50:5. Formate-glucose mixotrophy benefits Thiobacillus A2-GFI when substrates are limited at low growth rates (<μ max for formate), but is characterized by a μ max below that possible on glucose. Physiological behaviour at high growth rates was influenced by the formate:glucose ratio, resulting under some conditions, at least, in loss of mixotrophy and the establishment of heterotrophic growth.

The distribution of methane mono-oxygenase (MMO) activities between particulate and soluble fractions of cell-free extracts of Methylosinus trichosporium OB3b was dependent upon growth conditions. Particulate activity was associated with the presence of intracytoplasmic membranes observed only under oxygen-limiting conditions in shake flask cultures. Particulate and soluble activities showed substantially different sensitivities to a range of potential inhibitors. The particulate enzyme was inhibited by metal-chelating agents, thiol reagents and amytal, whereas the soluble MMO was not inhibited by these compounds; both activities were sensitive to KCN, ethyne and 8-hydroxyquinoline. NAD(P)H was the only suitable electron donor. The activities were unstable at 0° C but the soluble enzyme could be partially stabilized by several compounds. The particulate and soluble MMO activities are compared with previously reported particulate and soluble MMO enzymes from this species and other methane oxidizers.

The obligate methylotroph Methylosinus trichosporium OB3b was capable of growth on methanol as sole carbon source at concentrations as high as 4% (v/v), and viability was maintained over many successive transfers. Methane mono-oxygenase, detected by epoxidizing and hydroxylating activity, was retained. The gross morphology of the organism on this substrate was dependent on culture conditions. It varied from organisms containing extensive peripheral membrane systems to those with extensive inclusions, the latter representing a poorly developed membrane system which predominated under most growth conditions.