- Volume 134, Issue 5, 1988

The rumen anaerobic fungi Neocallimastix frontalis, Sphaeromonas communis and Piromonas communis were grown in the presence of cellulosic substrate (sisal fibres) and the properties of the glycosidases and polysaccharide-degrading enzymes produced by the organisms were studied. β d-Glucosidase (EC 3.2.1.21), β d-fucosidase (EC 3.2.1.38), β d-galactosidase (EC 3.2.1.23), β1,3-glucanase (EC 3.2.1.6), β1,4-glucanase (EC 3.2.1.4) and βxylanase (EC 3.2.1.8) had pH optima of 6.0 and temperature optima under the conditions of assay of 50–55 °C, whereas for βxylosidase (EC 3.2.1.37) the optima were 6·5 and 39 °C respectively. The apparent K m values of individual enzymes secreted by the three fungi were similar. The results show that S. communis, P. communis and N. frontalis produce the same range of enzymes, with similar properties, able to degrade cellulose and hemicellulose.

The kinetics of levansucrase secretion were examined in a strain of Bacillus subtilis which overproduces the enzyme (sacU h). Pulse-labelling experiments indicated that the second step of the levansucrase secretion process has the properties of a membrane active-transport system. This event appears to be directly linked to the influx of iron into the bacteria. The response of B. subtilis to the inducer of levansucrase synthesis was modulated by ferrichelators and the extent of the response varied with the nature of the ferrichelator. Ferridihydroxybenzoate markedly shortened the induction lag period. It is inferred from these data that iron occurs as a cofactor for components of the membrane sites of synthesis/secretion of B. subtilis levansucrase.

Conidia of Trichoderma reesei QM 9414 are able to degrade crystalline cellulose to glucose in the absence of protein synthesis, indicating the presence of a cellulase enzyme system. Measurement of enzyme activities revealed the presence of several glycanases and glycosidases. The cellulase enzyme system appeared irrespective of the conditions applied to induce conidiation. Removal of the endo-1,4-β-glucanase from the cellulase system could be achieved by treatment of conidia with β-octylglucoside or Triton X-100. Conidia treated in this way grew poorly on cellulose but well on glycerol. The endo-1,4-β-glucanase released from conidia by β-octylglucoside appeared as a single enzyme upon SDS-PAGE/immunoblotting with an apparent molecular mass of 68 kDa, an isoelectric point of 4·8–5·3, and a pH optimum between pH 4–6. These properties were similar to endo-1,4-β-glucanase I purified from culture filtrates of cellulose-grown T. reesei. Thermostability of the released enzyme was, however, lower than that of endo-1,4-β-glucanase I. Germination of T. reesei conidia in a medium containing glycerol as a carbon source led to a loss of endo-1,4-β-glucanase activity which paralleled the decrease in the number of ungerminated conidia.

Alkylsulphatases have been localized in cells of two bacterial isolates using transmission electron microscopy. Cells were incubated with the appropriate alkyl sulphate ester in the presence of Ba2+ ions. Inorganic sulphate liberated by alkylsulphatases was precipitated at the site of liberation as BaSO4. Electron microscopy of thin sections was used to locate electron-dense grains which were identified by energy dispersive analysis of X-rays (EDAX) as BaSO4. The long-chain primary alkylsulphatases of the detergent-degrading bacterium Pseudomonas C12B, active on C6-C14 primary alkyl sulphates, were located on the outer cell-wall. There was no activity inside the cells. In contrast, the short-chain (C3-C7) alkylsulphatase in a coryneform isolated for its ability to grow on but-1-yl sulphate was located entirely in the cytoplasm. The butylsulphatase was apparently associated with granules of poly-β-hydroxybutyric acid. The different locations for the long- and short-chain alkylsulphatases may be related to the relative potential toxicities of their ester substrates.

Toxic effects of both main colicin types, i.e. of porin and nuclease types, involve the direct contact of their molecules with the plasma membrane of sensitive cells. In the present study, it was tested whether this contact provokes a lateral or vertical movement of intramembrane protein particles (IMP) or a direct cleavage of the proteins. IMP were visualized by freeze-fracturing and electron microscopy on the protoplasmic fracture face (PF) of colicin-treated cells of Escherichia coli. Possible changes in distribution and in density of IMP due to treatment with colicins E1-E7 and K were followed. As a control, the bacteria were equilibrated at 0C before quenching, which caused a reversible formation of smooth areas and a decrease in the mean density of IMP on the PF. Colicins E1-E7 had no clear-cut effect on the disposition of IMP. Only colicin K decreased the IMP density, by 10% in E. coli strain 58–161 and by 17% in strain C6; the distribution of IMP remained homogeneous. Trypsin reactivation of colicin-K-inactivated bacteria was not reflected by restoration of the original density of IMP; on the contrary, it led to a further decrease, of 1–13%, in IMP density, presumably by proteolytic cleavage. Varying densities of IMP in different strains of the same bacterial species (under standard conditions) were confirmed.

Uninucleate amoebae of Physarum polycephalum strain CL undergo apogamic development to form multinucleate plasmodia via an intermediate stage of large, uninucleate cells irreversibly committed to plasmodial development. This amoebal-plasmodial transition involves major changes in tubulin gene expression and the organization of microtubular structures. We analysed the expression of the betC locus, which encodes the plasmodial-specific β2-tubulin, during plasmodial development. A key question addressed was the timing of expression of betC in relation to the last open mitosis of the amoeba and the first closed mitosis of the plasmodium during the transition. Culture conditions were improved to yield partly synchronous differentiating cultures containing 50–60% committed cells, in order to facilitate biochemical analysis of development. Northern blotting indicated that betC RNA was virtually absent from amoebae and from early differentiating cultures. However, betC transcripts could already be detected in differentiating cultures containing only 0·1% of committed cells; the relative amount of betC transcripts increased as the percentage of committed cells in differentiating cultures increased. In fully developed plasmodia, there was at least a 330-fold increase in the betC transcript level compared to that in amoebae. We conclude that betC is activated during the amoebal-plasmodial transition immediately before or during the commitment event. Small amounts of β2-tubulin polypeptide could first be detected by Western blotting around the stage of the first closed mitosis. Thus β2-tubulin may participate in the first closed mitosis that committed cells undergo during their development into plasmodia.

When sodium chloride crystallizes in an evaporitic environment, living halobacteria are entrapped within the fluid inclusions which form as the crystals develop. Trapped cells have been observed in natural salts from a marine saltern and from Lake Magadi. Entrapment occurs under both neutral and alkaline conditions. Salt crystals have been grown under controlled conditions from solutions containing pure culture suspensions of halobacteria at densities comparable to those reported from natural evaporitic habitats. Crystals formed in solutions heavily loaded with bacterial cells contained more and larger inclusions than crystals formed from sterile solutions, and thus bacterial entrapment may affect the physical characteristics of the product. Representative strains of each major grouping within the Halobacteriaceae except the genus Halococcus exhibited entrapment and survival within salt crystals. Continued motility has been demonstrated for up to three weeks after entrapment. All strains tested to date retained viability for a minimum of six months. Non-motile and non-viable cells were also entrapped.

The complete nucleotide sequence of the LPD1 gene, which encodes the lipoamide dehydrogenase component (E3) of the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multienzyme complexes of Saccharomyces cerevisiae, has been established. The flanking region 5′ to the LPD1 gene contains DNA sequences which show homology to known control sites found upstream of other yeast genes. The primary structure of the protein, determined from the DNA sequence, shows strong homology to a group of flavoproteins including Escherichia coli lipoamide dehydrogenase and pig heart lipoamide dehydrogenase. The amino acid sequence also reveals the presence of a potential targeting sequence at its N-terminus which may facilitate transport to and entry into mitochondria.

A recombinant plasmid, pRSR100, containing the functional analogue of the Escherichia coli recA gene was isolated from a genomic library of Thiobacillus ferrooxidans ATCC 33020. The plasmid complemented defects in DNA repair and homologous recombination in E. coli recA mutant strains. Antiserum raised against E. coli RecA protein reacted with the native but defective E. coli HB101 RecA protein; it did not react with protein extracts from the recA deletion mutant E. coli JK696, but it reacted with two protein bands in extracts of E. coli JK696(pRSR100). A single band with an apparent M r equal to the higher-M r band in E. coli JK696(pRSR100) was detected in T. ferrooxidans cell extracts with the E. coli RecA antiserum.

Twelve mycobacillin-negative (My−) mutants of Bacillus subtilis B3 were isolated from an auxotrophically tagged mycobacillin producer organism. In whole-cell fermentations of some of these My− mutants a penta- and a nonapeptide accumulated; these peptides were also obtained in a cell-free system in which a new tripeptide was also detected. The amino acid composition, N- and C-terminal residues and amino acid sequence of these peptides agreed with those of equivalent segments of the mycobacillin molecule. The mycobacillin-synthesizing enzyme can be divided into three fractions that catalyse different steps in biosynthesis, and the defective enzyme fractions in the various mutant strains were identified by reconstitution experiments in vitro. The defects were further pin-pointed in mutant enzyme fractions by an ATP⇌Pi exchange reaction and also by cell-free synthesis involving the use of membrane-bound enzyme. The defects so identified indicated the formation of tri-, penta- and nonapeptides as intermediates in the mycobacillin biosynthetic pathway.

The presence of origin-region DNA in preparations containing bacterial cell wall and cytoplasmic membrane is well established, but little is known about the relationship between this association and events of the cell cycle. We have observed, during renewed growth of stationary-phase cultures of Bacillus subtilis, an association of DNA, including newly synthesized regions, with a specific region of the plasma membrane. Attachment was transitory, occurring once per replication cycle, and was prevented by inhibitors of cell wall synthesis.

The correlation between the melting temperature of intracellular DNA, determined by differential scanning calorimetry (DSC) of whole bacteria, and its guanine + cytosine (G + C) content, was examined for 58 species of bacteria. Samples of vegetative cells were heated in a Perkin-Elmer DSC-2C at 10 °C min-1 from 5 to 130 °C, cooled to 5 °C and then re-heated as before. Literature values for the mole fraction of G + C, X GC, were linearly related to the temperature, T max, at which the reversible peak, p r, observed on the second heating run was at a maximum, via the equation X GC = (T max − 73·8)/41·0. This equation accounted for 91·9% of the variance in X GC with 95% confidence limits of ±7·3%, approximately 1·6 times the corresponding uncertainty (±4·5%) quoted by De Ley (Journal of Bacteriology 101, 738–754, 1970) for estimates based on the spectroscopically determined melting temperature of purified DNA. Random errors of measurement of T max did not greatly limit the precision of the prediction and it was concluded that factors additional to base composition affected the temperature of DNA melting within the bacterial cell. Displacement of T max values from the fitted line was particularly noticeable in Campylobacter, Corynebacterium and Bacterionema species and part of the residual variation appeared to be species specific, possibly caused by differences in intracellular solute concentration.

A series of step-level penicillin-resistant derivatives of Streptococcus sanguis V288 (Challis) were obtained through successive genetic transformations. The DNA donor used was a laboratory-derived, penicillin-resistant multistep mutant of the recipient strain. Detection of the penicillin-binding proteins (PBPs) of wild-type and transformants revealed five major PBPs. While it was found that S. sanguis can acquire intrinsic resistance in a stepwise manner and the mechanism was similar to those of some other organisms (changes in penicillin-binding protein affinity and/or in extent of penicillin binding), multiple-PBP changes accompanied a single step-level of resistance. All of the PBPs showed varying degrees of decreased affinity for [3H]benzylpenicillin with increasing penicillin resistance. Of these, the consistent, dramatic and progressive decrease of PBP 4 binding was most notable. After an initial decrease at the first step-level of resistance, PBP 5 was restored to wild-type levels, indicating a possible important role in survival. Genetic linkage of the first two step-levels of resistance was demonstrated by examination of transformation frequencies and by hit-kinetics experiments. A convenient method is described for the quantitative comparison of fluorographs containing PBPs with a wide range of affinities for penicillin.

A mutant of Escherichia coli dependent on erythromycin for growth spontaneously gives erythromycin-independent strains with altered or missing ribosomal proteins. Strains with defects in ribosome assembly were sought and obtained from among these revertants. Two organisms in which ribosomal protein L19 is altered and absent respectively have 70S ribosomes whose dissociation into sub-units is particularly sensitive to pressures generated during centrifuging. The mutant that lacks protein L19 also accumulates ribosome precursor particles during exponential growth as do others including mutants that lack proteins S20 or L1. These strains also show unbalanced synthesis of RNA and so will be useful in investigating both the pathways and the regulation of ribosome assembly.

Bacteriophage B278 has been characterized and compared with Mu, the only phage known to produce random mutations in E. coli. Although both phages are morphologically indistinguishable and have a similar host range, they clearly differ at both the protein and the DNA level. B278 apparently possesses a DNA protection mechanism that is different from the mom system described for Mu.

Fifty-five lytic bacteriophages isolated from water and soil samples were active on many strains of the genus Hyphomicrobium. The optimal isolation procedure was an adsorption method in which samples from a habitat similar to that of the respective host bacterium were used as the phage inoculum. According to the morphology and nucleic acid type these bacteriophages belonged to different families: Myoviridae (type A1: five phages); Styloviridae (type B1: 33 phages; type B2: eight phages) and Podoviridae (type C1: nine phages). The Styloviridae (type B1) appeared in two morphological variants (tails flexible or rigid). All phages investigated were specific for the genus Hyphomicrobium and were unable to lyse members of other genera of hyphal, budding bacteria (e.g. Hyphomonas, Pedomicrobium, genus D, genus T). The host specificity of 42 phages was tested with 156 Hyphomicrobium strains: 122 strains were lysed by at least one of these phages, but 34 Hyphomicrobium strains were not susceptible. Morphotype B1 phages with identical morphology could be distinguished according to their host-range properties on prophage-containing Hyphomicrobium strains. With regard to differences in morphology and host range, 25 phages were selected for more detailed investigations. From these phages DNA was isolated; the melting transition midpoints (T m) ranged from 67 to 93 C. The upper and higher values suggested the presence of DNA modifications. Six different adsorption patterns could be distinguished among the Hyphomicrobium phages. Preferred attachment sites were the proximal pole of the mother cell, the hyphal tip, the distal pole of the bud, and the distal pole of the swarmer cell.

The fepA gene of Escherichia coli encodes the outer-membrane receptor protein for ferrienterobactin. Previous genetic studies indicated that fepA mutations occur frequently and suggested that most of the mutations were deletions. In this work seven spontaneous fepA mutations were analysed by enzyme assay (enterobactin synthase and enterobactin esterase) and by DNA hybridization studies. In two strains, UT500 and UT700, the mutations were confined to the fepA gene. In the remaining mutants, the mutations were large deletions; in several cases, 27 kb or more of DNA had been lost. The deletions, all of which eliminated approximately the left half of the enterobactin gene cluster, extended from the vicinity of the fepC gene counterclockwise into the chromosome. A minimum of three clockwise endpoints were identified and at least two counterclockwise endpoints were detected. The variation in endpoints among the deletions argues against the involvement of a normal transposon in their formation. Also, unexpected homology was found between enterobactin gene cluster DNA and lacPOZ and pSC101.

Streptococcus mutans serotype g secretes at least three kinds of glucosyltransferase with different enzymological and immunological properties. One of them is a primer-independent enzyme and seems to be the source of primer for the others, both of which are primer-dependent enzymes. Recently, we purified the primer-independent enzyme, the third glucosyltransferase in this group from S. mutans strain AHT-k serotype g. In the present study, we examined the specificity of the antiserum against the primer-independent glucosyltransferase using extracellular culture-conditioned fluids of many strains of the various serotypes of S. mutans. The antiserum cross-reacted with the extracellular culture fluids from strains of serotypes d and a, in addition to serotype g, but not with those of other serotypes, indicating that the primer-independent glucosyltransferase is secreted by the S. sobrinus and S. cricetus, but not by S. mutans and S. rattus. The antiserum did not completely inhibit the activity of the enzyme, even at more than twofold antibody excess, determined by indirect precipitation with immobilized staphylococcal protein A.

Cell wall components from Candida albicans were compared to intact cells for their ability to induce natural cytotoxic immunoeffectors in the peritoneal cavity of mice. A soluble mannoprotein extract (MP) and an insoluble glucan fraction (GG) strongly stimulated the generation of peritoneal effectors capable of lysing YAC-1 and P-815 tumour cell lines in vitro. The anti-YAC-1 effectors were characterized as natural killer (NK) lymphocytes while the anti-P-815 effectors appeared to be activated macrophages. The activity of each fraction was typically dose-dependent and both fractions differed from whole cells in the kinetics of induction of cytotoxicity. However, the NK and macrophage effectors generated by these materials had similar functional and phenotypic properties, irrespective of the material used as inducer. No mannoprotein was detected in the insoluble glucan fraction GG. Hence, the immunoenhancing activity of GG could not be attributed to the presence of some MP or MP-like component. Mannan-rich fractions with low (<3%) protein content (M) or extracted by hot alkaline reagent (M-alk) were inactive as NK and macrophage inducers. Thus, the cell wall of C. albicans contains at least two distinct macromolecular complexes which mediate the induction in murine peritoneal exudates of cytotoxic effectors active against tumour cell lines.

Protein kinases I (PK I) and II (PK II) were purified 253- and 13·5-fold, respectively, from an extract of sonically disrupted cells of Legionella micdadei by ion-exchange chromatography on QAE-Sephadex, by histone affinity chromatography, and by HPLC-gel filtration chromatography. Both enzymes catalysed the phosphorylation of calf thymus histones, with a K m of 2·7 mg ml−1 for PK I and 2·9 mg ml−1 for PK II. Histone H2b was the best protein kinase substrate for both PK I and PK II. The pH optima were 6·8 and 7·0 for PK I and PK II respectively. The K m for ATP was 0·29 mm for PK I and 0·33 mm for PK II. PK II activity was stimulated by either cAMP or cGMP, whereas PK I was inhibited by both cyclic nucleotides. The activity of PK I was unaffected by addition of calmodulin, diacylglycerol and mixtures of Ca2+ and acidic phospholipids, but these additions increased PK II activity threefold. The activity of PK II was stimulated by spermine and spermidine, but PK I was inhibited by these compounds. PK I and PK II were both strongly inhibited by heparin.