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Purification and characterization of hyperthermotolerant leucine aminopeptidase from Geobacillus thermoleovorans 47b.

A thermophilic bacterium, which we designated as Geobacillus thermoleovorans 47b was isolated from a hot spring in Beppu, Oita Prefecture, Japan, on the basis of its ability to grow on bitter peptides as a sole carbon and nitrogen source. The cell-free extract from G. thermoleovorans 47b contained leucine aminopeptidase (LAP; EC 3.4.11.10), which was purified 164-fold to homogeneity in seven steps, using ammonium sulfate fractionation followed by the column chromatography using DEAE-Toyopearl, hydroxyapatite, MonoQ and Superdex 200 PC gel filtration, followed again by MonoQ and hydroxyapatite. The enzyme was a single polypeptide with a molecular mass of 42,977.2 Da, as determined by matrix-assisted laser desorption ionization and time-of-flight mass spectrometry, and was found to be thermostable at 90 degrees C for up to 1 h. Its optimal pH and temperature were observed to be 7.6-7.8 and 60 degrees C, respectively, and it had high activity towards the substrates Leu-p-nitroanilide (p-NA)(100%), Arg-p-NA (56.3%) and LeuGlyGly (486%). The K(m) and V(max) values for Leu-p-NA and LeuGlyGly were 0.658 mM and 25.0 mM and 236.2 micromol min(-1) mg(-1) protein and 1,149 micromol min(-1) mg(-1) protein, respectively. The turnover rate (k(cat)) and catalytic efficiency (k(cat)/ K(m)) for Leu-p-NA and LeuGlyGly were 10,179 s(-1) and 49,543 s(-1) and 15,470 mM(-1 ) s(-1) and 1981.7 mM(-1 ) s(-1), respectively. The enzyme was strongly inhibited by EDTA, 1,10-phenanthroline, dithiothreitol, beta-mercaptoethanol, iodoacetate and bestatin; and its apoenzyme was found to be reactivated by Co(2+) .

Bacillaceae↗

Increase in D-tagatose production rate by site-directed mutagenesis of L-arabinose isomerase from Geobacillus thermodenitrificans.

Among single-site mutations of L-arabinose isomerase derived from Geobacillus thermodenitrificans, two mutants were produced having the lowest and highest activities of D-tagatose production. Site-directed mutagenesis at these sites showed that the aromatic ring at amino acid 164 and the size of amino acid 475 were important for D-tagatose production. Among double-site mutations, one mutant converted D-galactose into D-tagatose with a yield of 58% whereas the wild type gave 46% D-tagatose conversion after 300 min at 65 degrees C.

Aldose-Ketose Isomerases↗

Pro-antibiotic substrates for the identification of enantioselective hydrolases.

A new growth-based screening method for the identification of enantioselective hydrolases, such as lipases and esterases, using pro-antibiotic substrates was devised. An enantioselective hydrolase could be identified by measuring growth rates of cells in liquid media containing (R)- or (S)-2-phenylbutyric chloramphenicol esters. This method can be applied to the screening of novel enantioselective microbes and to the high-throughput screening for the directed evolution of enantioselective hydrolytic enzymes.

Anti-Bacterial Agents↗

Biodegradation of crude oil by thermophilic bacteria isolated from a volcano island.

One-hundred and fifty different thermophilic bacteria isolated from a volcanic island were screened for detection of an alkane hydroxylase gene using degenerated primers developed to amplify genes related to the Pseudomonas putida and Pseudomonas oleovorans alkane hydroxylases. Ten isolates carrying the alkJ gene were further characterized by 16s rDNA gene sequencing. Nine out of ten isolates were phylogenetically affiliated with Geobacillus species and one isolate with Bacillus species. These isolates were able to grow in liquid cultures with crude oil as the sole carbon source and were found to degrade long chain crude oil alkanes in a range between 46.64% and 87.68%. Results indicated that indigenous thermophilic hydrocarbon degraders of Bacillus and Geobacillus species are of special significance as they could be efficiently used for bioremediation of oil-polluted soil and composting processes.

Bacillaceae↗

Conjugated methyl sulfide and phenyl sulfide derivatives of oxidosqualene as inhibitors of oxidosqualene and squalene-hopene cyclases.

Various (1E,3E)- and (1Z,3E)-conjugated methylthio derivatives of oxidosqualene (OS) and conjugated and non-conjugated phenylthio derivatives of OS were obtained. These compounds, designed as inhibitors of pig liver and Saccharomyces cerevisiae 2,3-oxidosqualene-lanosterol cyclases (OSC) (EC 5.4.99.7) and of Alicyclobacillus acidocaldarius squalene-hopene cyclase (SHC) (EC 5.4.99.-), contain the reactive function adjacent to carbons involved in the formation of the third and the fourth cycle during OS cyclization. All the new compounds are inhibitors of OSC and SHC, with various degrees of selectivity. The conjugated methylthio derivatives behaved as potent inhibitors of S. cerevisiae OSC, whereas most of the phenylthio derivatives were especially active toward SHC.

Animals↗

Intestinal perforations in a premature infant caused by Bacillus cereus.

Although Bacillus cereus is a ubiquitous bacterium, the incidence of neonatal infections is very low with only a few cases of B. cereus infections in neonates reported in the literature. We report the case of a premature infant with multiple intestinal perforations and an abdominal B. cereus infection. The initial course was characterized by severe cardiovascular shock, anemia, thrombocytopenia and disseminated intravascular coagulation, leading to periventricular leukomalacia, alopecia capitis and toxic epidermal necrolysis. The possible role of B. cereus-associated enterotoxins for the clinical manifestations are discussed. Our case confirms previous reports of severe clinical symptoms in B. cereus infection in premature neonates. We speculate that the systemic complications of B. cereus infection are at least partly related to the effect of B. cereus-associated enterotoxins.

Abnormalities, Multiple↗

Necrotizing gastritis due to Bacillus cereus in an immunocompromised patient.

Bacillus cereus is increasingly being acknowledged as a serious bacterial pathogen in immunocompromised patients. We present a case of acute necrotizing gastritis caused by B. cereus in a 37-year-old woman with acute myeloblastic leukemia, who recovered following total parenteral nutrition and treatment with imipenem and vancomycin. B. cereus was isolated from gastric mucosa and blood cultures. Up to now, no case of acute necrotizing gastritis due to this organism has been reported.

Adult↗

Fibronectin type III-like sequences and a new domain type in prokaryotic depolymerases with insoluble substrates.

Fibronectin type III-like sequences are present in many proteins from higher eukaryotes and are involved in protein-protein interactions, heparin binding and cell adhesion. A nine-member family of bacterial sequences is shown to be significantly homologous to the type III-like sequences. All the sequences are contained in secreted depolymerases acting on complex, energy-rich insoluble substrates, in which they apparently do not participate in catalysis or substrate-binding, their exact function remaining unclear. Furthermore, a new family of sequences, present in some cellulases, is presented.

Actinomycetales↗

Bacillus cereus meningitis in two neurosurgical patients: an investigation into the source of the organism.

Two patients developed Bacillus cereus meningitis following neurosurgery. During the subsequent investigation into the source of the organism, linen was discovered to be heavily contaminated with B. cereus. No other prolific source of the organism was found. It seems probable that lint from contaminated fabric was the vehicle of transmission of the organism during extended surgery. Linen should be considered as a possible source of B. cereus infection.

Adult↗

Physiology of Sporolactobacillus strains isolated from different habitats and the indication of in vitro antagonism against Bacillus species.

In an ecological study only low numbers of Sporolactobacillus were found in habitats such as the faeces of herbivores, the rumen of cattle and the final waste water of an abattoir. Their presence in the final waste water of an abattoir indicates their possible association with food, and, more specifically, with meat. Differences were found in some physiological characteristics. One isolate (L2404) differed from the authentic Sporolactobacillus ATCC 15538 by its inability to ferment inulin, its growth in presence of 6.5% NaCl and in 0.2% tellurite, by the isomer(s) of lactic acid produced and the mol% G + G in the DNA. One Sporolactobacillus isolate (L2407) showed antagonism against Bacillus cereus, Bacillus cereus var, mycoides, Bacillus megaterium and Bacillus subtilis.

Animals↗

Resistance of vegetative cells and endospores of Sporolactobacillus to gamma-irradiation.

Vegetative cells of Sporolactobacillus showed average resistance to gamma-irradiation compared to other vegetative bacteria with D10 values ranging from 0.350 to 0.525 kGy. Endospores of Sporolactobacillus showed higher resistance to gamma-irradiation than most Bacillus species but were close to that of Clostridium species. The average D10 value for Sporolactobacillus endospores was 2.5 kGy.

Bacillaceae↗

Application of pyrolysis mass spectrometry to the investigation of outbreaks of food poisoning and non-gastrointestinal infection associated with Bacillus species and Clostridium perfringens.

Eighteen isolates of Bacillus species and 15 of Clostridium perfringens, all of which had been associated with outbreaks of either food poisoning or non-gastrointestinal infection (NGI), were examined for relatedness by pyrolysis mass spectrometry (PyMS). The PyMS-analysis correctly clustered all the groups of epidemiologically related isolates of both genera, and distinguished all the single, epidemiologically unrelated isolates of the same species. PyMS is a simple, rapid and inexpensive technique which can provide useful and accurate inter-strain comparisons within both the Bacillus and Clostridium genera in complete accord with conventional serological typing results.

Bacillaceae Infections↗

Media for the detection and enumeration of Bacillus cereus in foods: a review.

Bacillus cereus is an established cause of food poisoning in addition to being a troublesome and persistent contaminant, responsible for a variety of spoilage defects in processed foods and dairy products. A range of diagnostic and selective media has been developed to facilitate the detection and enumeration of B. cereus in routine surveillance situations and food poisoning investigations. These media are reviewed with respect to the selective and diagnostic systems they employ, their ability to recover and differentiate the target organism, and their advantages and limitations in particular applications.

Animals↗

A survey of bacterial toxins involved in food poisoning: a suggestion for bacterial food poisoning toxin nomenclature.

There is at present no accepted nomenclature for bacterial protein toxins, although there have been several attempts at dividing them into groups by their mode of action. In this paper we will not try to describe all known bacterial protein toxins, but concentrate on the toxins involved in food poisoning. Although most of these toxins are enterotoxins (protein exotoxins with the site of action on the mucosal cells of the intestinal tract) there are also other toxins involved in food poisoning, like the neurotoxins. In Table 1 the most important food pathogens in Europe are listed. For most, but not all, of these food pathogens, toxins are virulence factors. Generally, we divide food poisoning into infections and intoxications, where Salmonella spp. and Shigella spp. are typical examples of infections and Clostridium botulinum and Staphylococcus aureus for intoxications. We consider it better to make four different groups of food pathogenic bacteria, according to Table 2. Today the first three groups are all defined as infections, although for both group 2 and 3 the bacterium itself does not harm the host directly. The bacterium in such locations is like an 'enterotoxin factory'. The bacteria belonging to group 3 do not even interact with the epithelial cells in the intestine, while the bacteria of group 2 must colonise the epithelial cells prior to enterotoxin production.

Bacillaceae↗

Infection due to a contaminated thoracic drainage system.

Infection and colonization of the pleural space in three patients was traced to contaminated thoracic drain equipment. The organisms implicated were Bacillus cereus, Staphylococcus aureus and various non-fermenting Gram-negative bacteria. A careful examination of cleaning and decontamination procedures revealed delay in replacing the drainage bottle jar to be the main factor responsible for the contamination of the drainage unit and the pleural space. Recent technical adaptation of the drainage apparatus had removed the need for daily changing of this jar. These findings confirm that thoracic drainage systems may be a potential source of contamination for the patient. We emphasize that technical improvements to existing operating apparatus should necessitate the adaptation of recommendations governing their use in accordance with approved hospital hygiene standards.

Aged↗

Phylogenetic analysis of the genera Planococcus, Marinococcus and Sporosarcina and their relationships to members of the genus Bacillus.

A phylogenetic analysis based on 16S rRNA was performed on the genera Planococcus, Marinococcus, Sporosarcina and endospore-forming rods. In agreement with earlier 16S rRNA cataloguing data, Planococcus citreus and Sporosarcina ureae clustered with Bacillus pasteurii and other bacilli containing lysine in their cell walls. Sporosarcina halophila was shown to be genetically distinct from S. ureae and formed a loose association with the main Bacillus subtilis grouping. Marinococcus halophilus (formerly Planococcus halophilus) exhibited low levels of relatedness to all reference species examined and formed a distinct line of descent.

Bacillaceae↗