Primary structure of the chromosomal protein MC1 from the archaebacterium Methanosarcina sp. CHTI 55.

The DNA of the thermophilic archaebacterium Methanosarcina sp. CHTI 55 has been shown to be associated with two proteins called MC1 and MC2, of molecular mass 11 kDa and 17 kDa (Chartier et al. (1988) Biochim. Biophys. Acta 951, 149-156). The most abundant of these proteins, protein MC1, can protect DNA against thermal denaturation. In the present paper we report the covalent structure of protein MC1 and its effect on transcription of DNA in vitro. The covalent structure was determined from automated sequence analysis of the protein and from structural data provided by peptides derived from cleavage of the protein at aspartic acid and arginine residues. The amino-acid sequence of protein MC1 from Methanosarcina sp. CHTI 55 is closely related to that of the protein MC1 (previously called HMb) isolated from Methanosarcina barkeri strain MS: among the nine substitutions observed between the two proteins seven are conservative. Transcription of DNA in vitro is stimulated by protein MC1 at low protein-to-DNA ratio but is inhibited at a ratio higher than 0.1 (w/w), which is the one determined in the bacterial deoxyribonucleoprotein complex.

Crystallisation of the glycyl-tRNA synthetase from Thermus thermophilus and initial crystallographic data.

The glycyl-tRNA synthetase from Thermus thermophilus is a dimer of molecular mass 115 kDa, which has been crystallised using the vapour diffusion method from 5 to 7% polyethylene glycol 6000, 0.8 to 1.4 M NaCl at protein concentrations of 2 to 8 mg/ml. Nucleation is carried out at 4 degrees C and crystals are subsequently transferred to 15 degrees C to maximise growth. Crystals are truncated rhombohedra measuring on average 0.4 mm x 0.4 mm x 0.2 mm, which appear within a few days and reach full size in one to two months. GlyRS crystallises in two closely related space groups, P2(1)2(1)2(1) and C2,2,2(1), both with the same cell a = 125 A, b = 254 A, c = 104 A. Crystal packing in P2(1)2(1)2(1) is strongly C-centred. The crystals have VM = 3.6 A3/Da and a solvent content of 61%, with one dimer in the asymmetric unit in C2,2,2(1) and two dimers in P2(1)2(1)2(1). The best native data extend to 2.9 A in C2,2,2(1) and are 90.6% complete with an R-factor between symmetry-related reflections of 10.0%. The structure has been solved by multiple isomorphous replacement and model building is in progress.

Molecular characterization of the glutathione peroxidase gene of the human malaria parasite Plasmodium falciparum.

In this paper we report the isolation and the characterization of a gene encoding the antioxidant enzyme glutathione peroxidase from the human malaria parasite Plasmodium falciparum. This gene contains two introns of 208 and 168 bp and is present in a single copy on chromosome 13. The open reading frame encodes a protein with a predicted length of 205 amino acids, which possesses a potential cleavage site between residues 21 and 22 after a hydrophobic region with the characteristics of a signal sequence. Therefore, the mature protein is predicted to be 184 residues long with a molecular mass of 21404 Da. In comparison with other known glutathione peroxidases many amino acid residues implicated in catalysis are conserved in the malarial enzyme. Phylogenetic analysis indicates that the deduced protein sequence is more closely related to plant glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase. A 1.5-kb transcript was identified in asynchronous erythrocytic stages.

Characterization of iron-dependent endogenous superoxide dismutase of Plasmodium falciparum.

Two main superoxide dismutase activities at isoelectric points (pI) 6.2 and 6.8 and two minor at pI 5.6 and 6.4 were found in crude extracts of Plasmodium falciparum. These activities were cyanide-resistant and hydrogen peroxide-sensitive and represented 20-30% of the total SOD activity found in the crude extract. A fragment of 424 bp, amplified from genomic DNA from P. falciparum, was cloned and sequenced. The deduced amino acid sequence identified this fragment as a coding region of an SOD gene. A cDNA corresponding to SOD was then isolated from a P. falciparum cDNA library and sequenced. The deduced amino acid sequence of SOD (197 aa) was compared with 32 known Feor Mn-SODs by the 'DARWIN' system. This analysis showed that the parasitic enzyme was related to typical Fe-SODs. The SOD subunit was purified and the N-terminal sequence, determined up to 29 residues, corresponded to that of cDNA isolated. The iron-dependent SOD activity found in Plasmodium falciparum represents the first level of the antioxidant defence system of the parasite. It is also the first SOD characterized in the parasitic Apicomplexa phylum whose sequence can be compared to equivalent iron-dependent enzymes known in other protozoa and bacteria.

High level production of a cellulase-free xylanase in glucose-limited fed batch cultures of a thermophilic Bacillus strain.

Bacillus sp. strain XE and its mutant derivative strain D3 produce a thermostable xylanase which is suitable for enzymatic bleaching of kraft pulp. Xylanase synthesis was shown to be induced by the soluble products of xylan hydrolysis (xylooligosaccharide) and equally, catabolically-repressed when these oligosaccharides accumulate in the medium. An optimal balance between these two antagonistic effects was obtained in a carbon-limited, fed-batch culture with continuous xylooligosaccharide feeding. To reduce substrate cost, the xylooligosaccharide could be 90% substituted by glucose without reduction of the xylanase production rate. Xylanase production ceased when the activity reached approximately 380 U ml-1 due to an amino acid shortage. A continuous supply of exogenous amino acids allowed the production to continue to more than 1000 U ml-1.

Purification, characterization and amino terminal sequence of the superoxide dismutase from Babesia hylomysci.

Babesia hylomysci was found to contain two superoxide dismutase (SOD) isoenzymes with isoelectric points (pI) of 4.9 and 5.2. The two isoenzymes (45 and 47 kDa) were composed of two subunits of 22 kDa. An unique amino terminal sequence was determined up to 34 residues from the pooled isoenzymes and was identified as a sequence of SOD. The comparison of this N-terminal sequence of B. hylomysci SOD with 29 known Fe- or Mn-SODs showed more homologies with Fe-SODs.

Thermobacillus xylanilyticus gen. nov., sp. nov., a new aerobic thermophilic xylan-degrading bacterium isolated from farm soil.

An aerobic, thermophilic, xylanolytic, spore-forming bacterium, XETP (T = type strain; P = patent strain), has been isolated from farm soil situated underneath a manure heap in northern France. Strain XETP, which stained negative in the Gram test, occurs as short rods which sometimes form chains. Its spores are ellipsoidal, central to subterminal and occur in swollen sporangia. It grows at temperatures up to 63 degrees C and in the pH range 6.5-8.5. When grown on glucose in optimal conditions, its doubling time was found to be 33 min. CO2 was observed to have a growth-stimulating effect at the start of the culture. In addition to glucose, the isolate utilizes xylose, arabinose, mannose, cellobiose, galactose, maltose, sucrose, xylan and starch. Growth is inhibited by 5% NaCl. The G+C content of strain XETP is 57.5 mol%. The 16S rDNA sequence analysis indicated that strain XETP falls into the radiation of the Bacillus-Lactobacillus-Streptococcus subdivision of the Gram-positive phylum. Its three closest phylogenetic relatives are 'Bacillus viscosus', Paenibacillus curdlanolyticus and Bacillus popilliae with identity values of 91.15, 90.94 and 90.92%, respectively. The major cellular fatty acids are 14-methyl pentadecanoic acid (16:0 iso), hexadecanoic acid (16:0) and 14-methyl hexadecanoic acid (17:0 anteiso). On the basis of 16S rRNA sequence and chemotaxonomic characteristics, the isolate is different enough for it to be considered as a member of a new genus. It is therefore proposed that this isolate represents a new genus and species: Thermobacillus xylanilyticus. Strain XETP, the type strain of Thermobacillus xylanilyticus, has been deposited in the Collection Nationale de Cultures Microbiennes (CNCM I-1017) as a patent strain.

Methanosarcina mazei JC2, a new methanogenic strain isolated from lake sediments, that does not use H2/CO2.

A new mesophilic methanogenic strain, which produced methane from acetate, methanol, and methylamines, was isolated from lake sediments obtained from the lake Banyoles, near Girona (Spain). The cells were irregular in shape, from 1 to 3 micron in diameter, aggregated in masses of a few to several hundred units. Colonies were about 1-2 mm and irregularly shaped. Their color was yellow or white. Growth occurred throughout the pH range of 5 to 9 with optimal growth around pH 7. The optimal growth temperature was 37 degrees C. The molar deoxiribonucleic acid base composition was 37.2% (G + C). Studies of DNA homologies showed that this isolated was a strain of Methanosarcina mazei, but it differs from other reported strains, in that was not able to use H2/CO2 for growth or methane production.

Preferential binding of the archaebacterial histone-like MC1 protein to negatively supercoiled DNA minicircles.

The interaction of the archaebacterial MC1 protein with 207 bp negatively supercoiled DNA minicircles has been examined by gel retardation assays and compared to that observed with the relaxed DNA minicircle. MC1 binding induces a drastic DNA conformational change of each minicircle, leading to an increase of the electrophoretic mobility of the DNA. A slight increase in salt concentration enhances the amount of bound MC1, and high NaCl concentrations are required to dissociate the complexes. Furthermore, the salt effect on binding depends on the supercoiling state of the DNA. The dissociation rates decrease with increasing linking difference of the minicircles relative to their relaxed configuration to reach a maximum at -2 turns. In addition, differences between the topoisomers are also observed in terms of stoichiometry of the strongest complexes. So with the -2 topoisomer the complex with two MC1 molecules is the most stable, while with the -1 and -3 topoisomers, the strongest ones are those with one MC1 molecule per DNA ring.

DNA relatedness among some thermophilic members of the genus Methanobacterium: emendation of the species Methanobacterium thermoautotrophicum and rejection of Methanobacterium thermoformicicum as a synonym of Methanobacterium thermoautotrophicum.

DNA reassociation was used to determine levels of relatedness among four thermophilic Methanobacterium strains that are able to use formate and between these organisms and two representative strains of Methanobacterium thermoautotrophicum, strain delta HT (= DSM 1053T = ATCC 29096T) (T = type strain) and strain Marburg (= DSM 2133). Three homology groups were delineated, and these groups coincided with the clusters identified by antigenic fingerprinting. The first group, which had levels of cross hybridization that ranged from 73 to 99%, included M. thermoautotrophicum delta HT, Methanobacterium thermoformicicum Z-245, Methanobacterium sp. strain THF, and Methanobacterium sp. strain FTF. The second and third groups were each represented by only one strain, Methanobacterium sp. strain CB-12 and M. thermoautotrophicum Marburg, respectively (cross-hybridization levels, 13 to 30 and 29 to 33%, respectively). Our results indicate that the name M. thermoformicicum should be rejected as it is a synonym of M. thermoautotrophicum. The taxonomic positions of strains Marburg and CB-12 need further investigation.

Clostridium neopropionicum sp. nov., a strict anaerobic bacterium fermenting ethanol to propionate through acrylate pathway.

Strain X4 was isolated several years ago from an anaerobic mesophilic plant treating vegetable cannery waste waters. It was the first example of propionic fermentation from ethanol. Morphologic and physiologic characterizations of the strain are presented here. This strain is described as type strain of a new species, Clostridium neopropionicum sp. nov. Whole cells of strain X4 ferment [1-13C] ethanol and CO2 to [2-13C] propionate, [1-13C] acetate and [2-13C] propanol, suggesting the absence of a randomizing pathway during the propionate formation. Enzymes involved in this fermentation were assayed in cell-free extracts of cells grown with ethanol as sole substrate. Alcohol dehydrogenase, aldehyde dehydrogenase, phosphate acetyl transferase, acetate kinase, pyruvate synthase, lactate dehydrogenases, and the enzymes of the acrylate pathway were detected at activities sufficient to be involved in ethanol fermentation. The same pathway may be used for the degradation of lactate or acrylate to acetate.

Trophic relationships between Saccharomyces cerevisiae and Lactobacillus plantarum and their metabolism of glucose and citrate.

Glucose and citrate are two major carbon sources in fruits or fruit juices such as orange juice. Their metabolism and the microorganisms involved in their degradation were studied by inoculating with an aliquot of fermented orange juice a synthetic model medium containing glucose and citrate. At pH 3.6, their degradation led, first, to the formation of ethanol due to the activity of yeasts fermenting glucose and, eventually, to the formation of acetate resulting from the activity of lactobacilli. The yeast population always outcompeted the lactobacilli even when the fermented orange juice used as inoculum was mixed with fermented beet leaves containing a wider variety of lactic acid bacteria. The evolution of the medium remained similar between pH 3.3 and 5.0. At pH 3.0 or below, the fermentation of citrate was totally inhibited. Saccharomyces cerevisiae and Lactobacillus plantarum were identified as the only dominant microorganisms. The evolution of the model medium with the complex microbial community was successfully reconstituted with a defined coculture of S. cerevisiae and L. plantarum. The study of the fermentation of the defined model medium with a reconstituted microbial community allows us to better understand the behavior not only of fermented orange juice but also of many other fruit fermentations utilized for the production of alcoholic beverages.

Structural basis of the properties of an industrially relevant thermophilic xylanase.

A thermophilic xylanase from Bacillus strain D3 suitable for use as a bleach booster in the paper pulping industry has been identified and characterized. The enzyme is suited to the high temperature and alkaline conditions needed for using xylanases in the pulp industry. The xylanase is stable at 60 degrees C and relatively stable at high temperatures, with a temperature optimum of 75 degrees C. The pH optimum is 6, but the enzyme is active over a broad pH range. The xylanase has been cloned and sequenced, and the crystal structure has been determined. The structure of Bacillus D3 xylanase reveals an unusual feature of surface aromatic residues, which form clusters or "sticky patches" between pairs of molecules. These "sticky patches" on the surface of the enzyme are responsible for the tendency of the protein to aggregate at high concentrations in the absence of reagents such as ethylene glycol. The formation of dimers and higher order polymers via these hydrophobic contacts may also contribute to the thermostability of this xylanase.