An improved transposon for the halophilic archaeon Haloarcula hispanica

An improved transposon (ThD73) for Haloarcula hispanica is described. Based on the halobacterial insertion sequence ISH28, it showed little target sequence specificity but was biased toward a lower G+C content. Twenty randomly selected ThD73 mutants were analyzed, and the DNA flanking their insertions revealed several recognizable sequences, including two (unrelated) ISH elements.     

Characterization of 13 newly isolated strains of anaerobic, cellulolytic, thermophilic bacteria

Characteristics of 13 newly isolated thermophilic, anaerobic, and cellulolytic strains were compared with previously described strains of Clostridium thermocellum: ATCC 27405 and JW20 (ATCC 31549). Colony morphology, antibiotic sensitivity, fermentation end-products, and cellulose degradation were documented. All 13 strains were sensitive to erythromycin (5 μg/ml) and chloramphenicol (25 μg/ml), and all strains but one were sensitive to kanamycin (20 μg/ml). Polymerase chain reaction (PCR) amplification using primers based on gene sequences from C. thermocellum ATCC 27405 was successful for all 13 strains in the case of the hydrogenase gene and 11 strains in the case of phosphotransacetylase/acetate kinase genes. Ten strains amplified a product of the expected size with primers developed to be specific for C. thermocellum 16SrRNA primers. Two of the 13 strains did not amplify any product with the PCR primers designed for the phosphotransacetylase/acetate kinase and 16SrRNA primers. A MboI-like GATC- recognizing restriction activity was present in all of the five strains examined. The results of this study have several positive implications with respect to future development of a transformation system for cellulolytic thermophiles. Journal of Industrial Microbiology & Biotechnology (2001) 27, 275–280. Received 12 September 2000/ Accepted in revised form 20 November 2000     

A31P NMR study of the interaction of amphibian antimicrobial peptides with the membranes of live bacteria

Amphibian skin is a rich source of peptides that are specificto pathogens and act by disrupting bacterial membranes. Threeantimicrobial peptides were isolated from the skin glands ofAustralian tree frogs, Litoria caerulea and Litoriagenimaculata. NMR spectroscopy was used to observe changesinduced by these peptides in the ³¹P resonances of bacterialmembranes in vivo. Caerin 1.1 and maculatin 1.1, both wide-spectrum antibiotics, disrupted the membranes ofBacillus cereus and Staphylococcus epidermidis (Gram-positive), leadingto an increase in the isotropic ³¹P NMR signal. Caerin 4.1, anarrow-spectrum antibiotic, however, did not affect the ³¹Pspectra of these organisms. The results demonstrate the use of³¹P NMR to study the effects of membrane-disrupting agents onthe membranes of live bacteria.     

BioGraph: unsupervised biomedical knowledge discovery via automated hypothesis generation

We present BioGraph, a data integration and data mining platform for the exploration and discovery of biomedical information. The platform offers prioritizations of putative disease genes, supported by functional hypotheses. We show that BioGraph can retrospectively confirm recently discovered disease genes and identify potential susceptibility genes, outperforming existing technologies, without requiring prior domain knowledge. Additionally, BioGraph allows for generic biomedical applications beyond gene discovery. BioGraph is accessible at .     

Demonstration of an immunodominant neutralization site by analysis of antigenic variants of SA11 rotavirus

Serotype-specific monoclonal antibodies were used to select mutants of SA11 rotavirus that were resistant to neutralization. The antigenic characteristics of these mutants were studied with with a panel of monoclonal antibodies. We isolated one type of mutant which showed a dramatic increase (greater than 10-fold) in resistance to neutralization by hyperimmune antiserum, and this together with other data indicates the presence on the rotavirus major outer shell glycoprotein of an immunodominant antigenic site involved in virus neutralization. The mutants were also useful in classifying neutralizing monoclonal antibodies.     

Serotypic analysis of VP3 and VP7 neutralization escape mutants of rhesus rotavirus.

Neutralization escape mutants of simian rotaviruses (rhesus rotavirus and SA11) were tested in hemagglutination inhibition and neutralization assays against hyperimmune and infection sera to determine if mutation in an immunodominant epitope could enable neutralization escape. An SA11 mutant with a new glycosylation site at amino acid 211 of VP7 was shown to escape neutralization by hyperimmune but not infection sera.     

Cytochemical demonstration of hydrogen peroxide in polymorphonuclear leukocyte phagosomes

Phagocytosis by polymorphonuclear leukocytes (PMN) is accompanied by specific morphological and metabolic events which may result in the killing of internalized micro-organism. Hydrogen peroxide is produced in increased amounts during phagocytosis (17) and in combination with myeloperoxidase and halide ions constitute a potent, microbicidal mechanism (8,9,11). There can be direct iodination of micro-organisms (10), or alternatively, other intermediate reaction products, i.e. chloramines and aldehydes (21), can exert a microbicidal effect. The H2O2-peroxidase-halide system is presumed to operate within the phagocytic vacuole (12,18). Myeloperoxidase, present in the primary granules of PMN, enters the phagocytic vacuole during degranulation (1,4,7), and halide ions are probably derived from the extracellular medium or are present in the PMN (see 11, 18). For the operation of this system in intact cells, the presence of H2O2 in the phagocytic vacuole is necessary, and indeed this has been suggested by the work of several investigators (12, 18, 21). In the present investigation, the diaminobenzidine reaction of Graham and Karnovsky (5), modified to utilize endogenous myeloperoxidase and hydrogen peroxide, has been applied to actively phagocytizing PMN to demonstrate cytochemically the presence of H2O2 in the phagocytic vacuole.     

Comparisons of rotavirus polypeptides by limited proteolysis: close similarity of certain polypeptides of different strains

The polypeptides of SA11 rotavirus produced in virus-infected cells were analyzed by limited proteolysis, using Staphylococcus aureus V8 protease. This clearly distinguished between all the known primary gene products of the virus and allowed relationships between other infected-cell proteins, and between infected-cell and virus structural proteins, to be ascertained. A comparison of the proteolysis cleavage patterns between SA11 rotavirus and the human rotavirus Wa was also performed which demonstrated a marked conservation in the digestion patterns among nonstructural and inner-shell structural proteins, but a marked variation in the digestion patterns among outer-shell structural proteins.     

Halophage HF2: genome organization and replication strategy.

Halophage HF2 is a lytic, broad-host-range bacteriophage of the extremely halophilic domain Archaea. It has a 79.7-kb double-stranded DNA genome which is linear, contains no modified nucleotides, and is not susceptible to cleavage by many type II restriction endonucleases. This insensitivity is attributed to selection against palindromic restriction sites, a commonly observed feature of broad-host-range phages. Interestingly, enzymes that did cut the genome recognized AT-rich sites, and five such enzymes, DraI, AseI, HpaI, HindIII, and SspI, were used to construct a physical map of the genome. Southern hybridization experiments used to order fragments on the map indicated homologies between the phage termini, and subsequent sequence analysis showed that HF2 possessed 306-bp direct terminal repeats. The presence of such repeats suggested replication through concatameric intermediates, and this was confirmed by analysis of the state of the phage genome in infected cells. This is a replication strategy adopted by many well-studied bacterial phages, for example T3 and T7. Other similarities between the terminal repeats of T3 or T7 and HF2 include a putative nick site at the repeat border and a series of short imperfect repeats. These observations suggest a long evolutionary history for concatamer-based strategies of phage replication, possibly predating the divergence of Archaea/Eucarya and Bacteria, or alternatively, indicate possible lateral transfer of phage genes or modules between the domains Archaea and Bacteria.