- Volume 136, Issue 10, 1990

Two carboxymethylcellulases (CMCase, 1,4-1,4-β-d-glucan glucanohydrolase, EC 3.2.1.4), designated E-H and E-L, were purified to homogeneity from a culture filtrate of the alkalophilic Bacillus sp. KSM-635, by chromatography on DEAE-Toyopearl 650S and gel filtration on Bio-Gel A-0·5m. The purified CMCases both contained approximately 2–3% (w/w) glucosamine. Molecular masses deduced from SDS-PAGE were 130 kDa for E-H and 103 kDa for E-L. The pH optima of the enzymes were both about 9·5, and their optimum temperatures were around 40 °C. Activities of both enzymes were inhibited by Hg2+, Cu2+, Fe2+ and Fe3+, but sulphydryl inhibitors, such as N-ethylmaleimide, monoiodoacetate and 4-chloromercuribenzoate, had either no effect or a slightly inhibitory effect. N-Bromosuccinimide was strongly inhibitory, suggesting that a tryptophan residue is essential for the activity of the CMCases from Bacillus. In addition, the activities of both E-H and E-L were stimulated by Co2+, and they required Mg2+, Ca2+, Mn2+ or Co2+ for stabilization. Both enzymes efficiently hydrolysed carboxymethylcellulose (β-1,4-linkage) and lichenan (β-1,3; 1,4-linkage), but crystalline cellulosic substrates, curdlan (β-1,3-linkage), laminarin (β-1,3; 1,6-linkage) and 4-nitrophenyl-β-d-glucopyranoside were hydrolysed very little, if at all. 4-Nitrophenyl-β-d-cellobioside was hydrolysed by both enzymes to liberate 4-nitrophenol, and their hydrolysis rates were higher at neutral pH than at alkaline pH.

The rumen entodiniomorphid ciliate protozoon Polyplastron multivesiculatum was shown, by biochemical and electron microscopic techniques, to possess hydrogenosomes. After differential centrifugation of whole cell homogenates the hydrogenosomal marker enzymes pyruvate: ferredoxin oxidoreductase and hydrogenase were recovered predominantly (61% and 70% of activity respectively) in the large granular fractions that were sedimented by centrifugation for 104 g-min (fraction P1) and 105 g-min (fraction P2). These subcellular fractions contained membrane-bound organelles that were approximately 0·4–0·6 μm in diameter and which had a mean equilibrium density of 1·22–1·24 g ml−1 after isopycnic centrifugation in sucrose gradients. Malate dehydrogenase (decarboxylating) activity, however, was predominantly non-sedimentable after centrifugation for 6 × 106 g-min. Numerous hydrogenosome-like organelles were present in the ectoplasm and endoplasm of the cell. Hydrogenase activity was demonstrated and localized in the protozoan cell using a novel staining procedure with distyryl nitroblue tetrazolium chloride (DSNBT).

The proposal that nitrite exerts its inhibitory effect on anaerobic bacteria by direct interaction with the iron-sulphur proteins of the phosphoroclastic system was investigated. The effects of nitrate, nitrite with or without ascorbate, and nitric oxide on the growth of Clostridium sporogenes in liquid cultures at pH 7·4, on the rates of hydrogen production, and on the activities of the enzymes pyruvate-ferredoxin oxidoreductase and hydrogenase, and of ferredoxin were investigated. In agreement with previous studies, nitrate was the least effective inhibitor of cell growth, and nitric oxide the most effective. Nitrite reductase activity was very low in C. sporogenes, indicating that the presence of external reducing agents would be necessary for the reduction of nitrite to nitric oxide. Inhibition by nitrite was enhanced by ascorbate; 0·5 mm-nitrite with 10 mm-ascorbate stopped growth completely. In partially-purified preparations 4·1 mm-NaNO2 and equimolar ascorbate caused complete inactivation of hydrogenase activity but only partial (up to 78 %) inactivation of pyruvate-ferredoxin oxidoreductase. This agreed with the loss of hydrogen production observed with nitrite in vivo. Inhibition occurred within 5 min, and was irreversible in each case. Electron paramagnetic resonance (EPR) spectroscopy showed that paramagnetic [Fe(NO)2(SR)2] species were formed during growth in the presence of nitrite, and were associated with cells. However, the intensity of these EPR signals did not correlate with the inhibition of cell growth. The [4Fe-4S] clusters in ferredoxin were shown by EPR spectroscopy to be resistant to treatment with 3·6 mm-NaNO2 and 3·6 mm-ascorbate. It is concluded that the effects of nitrite on pre-formed ironsulphur proteins are not convincing as a basis for the lethal effects on bacterial cells.

Certain reagents, such as ascorbate or iron salts and thiols, enhance the bacteriostatic action of nitrite on food-spoilage bacteria. This may be due to the formation of nitric oxide and iron-thiol-nitrosyl ([Fe-S-NO]) complexes. The minimum concentrations of these reagents required to inhibit growth of Clostridium sporogenes were investigated. A mixture of nitrite (0·72 mm) with iron (1·44 mm) and cysteine (2·16 mm) was found to be extremely inhibitory when autoclaved and diluted into the culture medium. This mixture caused rapid cessation of growth and loss of cell viability at a final concentration corresponding to 40 µm-nitrite. If added to the initial culture medium, it prevented growth at 5 µm-nitrite. The mixture was more inhibitory, on the basis of the nitrite concentration used, than the ‘Perigo factor’, obtained by autoclaving nitrite in growth medium. [Fe-S-NO] compounds of known chemical structure were tested to determine if they were responsible for this effect. Total inhibition of cell growth was observed with the tetranuclear clusters [Fe4S3(NO)7] (Roussin’s black salt), [Fe4S4(NO)4] or [Fe4Se3(NO)7], added at concentrations equivalent to 10 µm-nitrite, or with [Fe2(SMe)2(NO)4] (methyl ester of Roussin's red salt), equivalent to 200 µm-nitrite. The rate of hydrogen production in growing cell cultures was inhibited by [Fe4S3(NO)7] at levels equivalent to 2·5 µm-nitrite. EPR spectra of the inhibited cells showed features with g-values of 2·03, characteristic of mononuclear iron-nitrosyl species, and, under non-reducing conditions, an unusual signal at g = 1·65. There was no correlation between growth inhibition and the g = 2·03 signal, though there was a better correlation between inhibition and the g = 1·65 signal. The direct effects of the compounds were tested on the iron-sulphur proteins of the phosphoroclastic system, namely ferredoxin, pyruvate-ferredoxin oxidoreductase and hydrogenase. EPR spectra and enzyme assays showed that these proteins were not destroyed by [Fe4S3(NO)7], [Fe4S4(NO)4], [Fe2(SMe)2(NO)4], [Fe(SPh)2(NO)2], or M2 (an autoclaved mixture of 66 mm-cysteine, 3·6 mm-FeSO4 and 0·72 mm-NaNO2) at concentrations higher than those that caused total inhibition of cell growth. Inhibition of cells by [Fe-S-NO] compounds is unlikely to be due to interaction with the preformed enzymes. The possible formation of iron-nitrosyl complexes in vivo, and their inhibitory actions, are discussed.

Genomic DNA from Butyrivibrio fibrisolvens strain A46 was digested with EcoRI and ligated into λgt11. Two recombinant phages isolated from the gene bank hydrolysed carboxymethylcellulose and were shown to contain the same 2·3 kb EcoRI restriction fragment, which was cloned into pUC12 to generate pBA46. Escherichia coli JM83 harbouring pBA46 expressed an endoglucanase (EGA) which hydrolysed a range of other substrates including barley-glucan, Avicel, filter paper and p-nitrophenyl-d-cellobioside. Nucleotide sequencing of the B. fibrisolvens strain A46 DNA cloned in pBA46 revealed a single open reading frame (ORF) of 1296 bp, encoding a protein of 48863 Da. Confirmation that the ORF coded for EGA was obtained by comparing the N-terminal sequence of the purified endoglucanase with that deduced from the nucleotide sequence. EGA contains a typical prokaryotic signal peptide at its N-terminus and shows some homology with the Bacillus family of cellulases. The enzyme does not contain distinct functional domains, which are prevalent in cellulases from Pseudomonas fluorescens subsp. cellulosa and Cellulomonas fimi.

The nucleotide sequence of the Streptococcus mutans GS-5 gtfD gene coding for the glucosyltransferase which synthesizes water-soluble glucan (GTF-S) has been determined. The complete gene contains 4293 base pairs and the unprocessed protein is composed of 1430 amino acids with a molecular mass of 159814 Da. The amino terminus of the unprocessed protein resembles the signal sequences of other extracellular proteins secreted by S. mutans and that of the GTF-I secreted by Streptococcus downei. In addition, the GTF-S protein exhibits high amino acid similarity with the strain GS-5 enzymes responsible for insoluble glucan synthesis (GTF-I, GTF-SI) previously isolated and sequenced in this laboratory. These results indicate that all three gtf genes evolved from a common ancestral gene.

The Escherichia coli–Brevibacterium lactofermentum shuttle vector pBLA was introduced into intact cells of B. lactofermentum by electrotransformation. Several parameters of this procedure such as voltage and cell concentration were analysed. Optimal conditions gave an efficiency of 106 transformants per μg of DNA. Two recalcitrant strains could be electrotransformed when an ampicillin pretreatment step was used. Electrotransformation experiments using DNAase or different structural forms of plasmid DNA showed that the electrotransformation process is quite different from natural transformation involving competence development. Restriction–modification-proficient B. lactofermentum could be efficiently electrotransformed with pBLA DNA isolated from E. coli. This restriction–modification system therefore seems to be overcome by electrotransformation. Thus electrotransformation may efficiently replace the protoplast bacterial transformation method.

Thermotolerance in Escherichia coli is induced by exposing cells to a brief heat shock (42 °C for 15 min). This results in resistance to the lethal effect of exposure to a higher temperature (50 °C). Mutants defective in the recA, uvrA and xthA genes are more sensitive to heat than the wild-type. However, after development of thermotolerance these mutants are like the wild-type in their heat sensitivity. This suggests that thermotolerance is an inducible response capable of protecting cells from the lethal effects of heat, independently of recA, uvrA and xthA. Thermotolerance does not develop in a dnaK mutant. In addition, the dnaK mutant is sensitive to heat and H2O2, but is resistant to UV irradiation. This implies that the E. coli heat-shock response includes a mechanism that protects cells from heat and H2O2, but not from UV.

The urease of Helicobacter pylori (formerly Campylobacter pylori) has been partly purified by fast protein liquid chromatography. This material contained 10 nm doughnut-like structures when examined by electron microscopy and comprised three major polypeptides (61 kDa, 56 kDa and 28 kDa). Only two of these polypetides (61 kDa and 28 kDa) were observed in urese-containing material isolated by preparative non-denatured PAGE. Monoclonal antibodies (mAbs) were produced which were directed against two of these polypeptides (56 kDa and 28 kDa). Only mAbs directed against the 28 kDa polypetptide inhibited or captured urease activity. These results suggest that the 56 kDa polypeptide is not essential for anzyme actiity. Anti-urease mAbs were used in an indirect immunogold technique to localize the enzyme at the ultrastructural level. In both prefixed bacterial and ultrathin cryosectioned bacterial the enzyme was located on the cell surface and in material apparently shed from that surface.

Phenolic glycolipid (PGL-I), an antigen specific to Mycobacterium leprae, was localized subcellularly in M. leprae residing in human skin, in M. leprae isolated from armadillo liver (‘isolated M. leprae’) and outside M. leprae in human lepromatous skin. For a quantitative localization of PGL-I sites, specimens, including skin segments stored for 6 years in glutaraldehyde, were embedded in hydrophilic Lowicryl (K4M) resin for ultrathin sectioning. Ultracryosections and Araldite sections of comparable specimens were used for comparison of localization results. A monoclonal antibody (F 47-21-3) directed to antigenic oligosaccharide of PGL-I was employed as primary antibody in immunogold labelling of ultrathin sections. K4M-immunogold methods gave very satisfactory quantitive gold-labelling of PGL-I. The localization of PGL-I by this method partially corresponded with sites detectable in both ultracryosections and the quantitatively superior Araldite sections, but new sites were also localized. Cell walls in human M. leprae and in isolated M. leprae possessed many PGL-I sites, particularly in dividing organisms. PGL-I or its antigenic oligosaccharide was also found, to a lesser extent, in the bacterial cytoplasm. Capsules discernible around part of isolated M. leprae cells displayed heavy PGL-I labelling, sometimes clearly confined to a zone distant from the cell wall. Extrabacterial PGL-I in M. leprae-infected human skin was encountered (1) in phagolysosomes and cytoplasm proper of dermal macrophages containing M. leprae, and (2) intra- and extracellularly in epidermal areas where basal cells harboured M. leprae in untreated multibacillary patients.

Recombinant fragments of the major outer-membrane protein (MOMP) of Chlamydia trachomatis, expressed at high levels in Escherichia coli, were isolated and purified. Antisera to the recombinant proteins reacted preferentially with overlapping synthetic peptides covering the immunoaccessible variable segments of MOMP. These sera also reacted in a species-specific manner with the surface of intact infectious elementary bodies, and in a Chlamydia genus-specific manner in assays using denatured or bound chlamydial antigens. The ability of recombinant MOMP preparations to elicit antibody to the surface of chlamydial elementary bodies raises the possibility that these proteins may be useful for chlamydial vaccine development.

The pattern of ethanol and lactate formation by continuous cultures of Clostridium thermosaccharolyticum LMG 6564 under glucose limitation is affected by culture conditions such as pH and dilution rate. NADH- and NADPH-mediated lactate dehydrogenase (LDH; EC 1.1.1.27) and alcohol (ethanol) dehydrogenase (ADH; EC 1.1.1.1 and EC 1.1.1.2) activities were measured in cell extracts from continuous cultures grown under different conditions. In conditions of high product formation, the NADH-mediated reaction was higher than the NADPH-mediated reaction for both LDH and ADH. LDH showed an absolute requirement for fructose 1,6-bisphosphate (FBP). Both NADH- and NADPH-linked LDH reactions were cytoplasmic, not sensitive to oxygen, and had a pH optimum of 6·0–6·5; the temperature optimum was 55–60 °C. The reverse reaction (lactate oxidation) could not be demonstrated. ADH activity was found in the particulate fraction of the cell lysate and was sensitive (not completely, but irreversibly) to oxygen. The temperature and pH optima were 43 °C, pH 7·0 and 45 °C, pH 8·8 for the NADH- and NADPH-mediated reactions, respectively. The production of at least two different ADHs is likely. LDH and ADH seemed to be regulated at the level of enzyme synthesis (direct correlation between the in vitro activities and the lactate and ethanol yields in the culture) with a second regulation of LDH by FBP at the reaction level.

Three species of marine, anaerobic free-living ciliates (Metopus contortus, Plagiopyla frontata and Parablepharisma collare) were studied with respect to their relation to O2. Survival was adversely affected at O2 tensions exceeding 1–2% of atmospheric (air) saturation (atm. sat.): Parablepharisma collare survived for about 1 h at 100% atm. sat., while the other two species survived the treatment for up to 2 d. Survival in the presence of O2 depended on the composition of the medium; cells survived longer in clean seawater than in culture medium which, when exposed to O2, became toxic. This is probably related to peroxide production mediated by solutes in the culture medium: addition of catalase prolonged survival. The ciliates did not contain catalase. Two of the species harbour endosymbiotic methanogens; at O2 tensions exceeding 2% atm. sat. the bacteria were inactivated, but they remained viable even after more than 5 h exposure to atmospheric O2 tension. Metopus contortus and Plagiopyla frontata respired O2 at rates similar to those of aerobic species; this O2 uptake is not coupled to energy conservation since the ciliates do not contain cytochromes, and low O2 tensions do not stimulate growth. O2 consumption is probably a detoxification mechanism which can maintain an intracellular anaerobic environment at a low ambient O2 tension. Metopus contortus and Plagiopyla frontata showed chemosensory behaviour in response to O2; this allowed them to find and remain within anaerobic microhabitats.

Mutants of the obligatorily aerobic yeast Rhodotorula glutinis, obtained by treating wild-type cells with N-methyl-N′-nitro-N-nitrosoguanidine, were selected on fructose medium containing 1% 2-deoxy-d-glucose. One mutant, designated R33, displayed a significantly decreased initial rate of uptake of d-glucose, 2-deoxy-d-glucose, d-galactose and d-xylose, and was unable to accumulate the latter three sugars. However, the residual monosaccharide transport was still energy-dependent. The mutant displayed significantly altered transport parameters for d-glucose and d-xylose. These results are evidence for the isolation of a mutant defective in the glucose carrier.