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At least 955 records · Page 53Linked to original sources
PubMed1993

Construction and use of a new broad-host-range lacZ transcriptional fusion vector, pHRP309, for gram- bacteria.

A new lacZ transcriptional fusion vector, pHRP309, based on the IncQ plasmid RSF1010, was constructed and shown to be easily mobilized into a variety of Gram- eubacteria. We also developed a two-step cloning procedure to facilitate the cloning of small promoter fragments into the fusion vector. A set of 'cohort' vectors was constructed which allowed directed cloning of fragments downstream from an omega streptomycin/spectinomycin-resistance cassette while maintaining multiple flanking restriction sites. The omega cassette provides a selectable antibiotic-resistance marker for cloning promoters into the fusion vector and makes mapping to determine fragment orientation unnecessary. The presence of the omega cassette also decreases background beta-galactosidase activity by decreasing readthrough transcription from plasmid sequences. The fusion vector carries a gentamicin-resistance-encoding gene as the selectable marker and can therefore be used in Tn5 (kanamycin-resistant) and Tn10 (tetracycline-resistant) mutant strains. Since pHRP309 is a member of the IncQ incompatibility group, it is compatible with IncP cloning vectors and can be used in strains carrying cloned regulatory genes. Using this system, we cloned the positively regulated Pseudomonas putida pcaI promoter and studied its regulation.

Base Sequence↗
PubMed1992

An attempt to define the host range for African horse sickness virus (Orbivirus, Reoviridae) in east Africa, by a serological survey in some Equidae, Camelidae, Loxodontidae and Carnivore.

A survey was carried out in horse, zebra, elephant, camel, sheep and goat and wild carnivore sera for virus-serum neutralising antibody to the nine type strains of African horse sickness virus. Antibody was found amongst the horse, zebra and elephant sera to all nine different strains. No antibody was detected in any sera from camels, sheep and goats. None was found in sera from hyaena and jackals in this series but had been detected earlier.

African Horse Sickness↗
PubMed1991

Characterization of gram-positive broad host-range plasmids carrying a thermophilic replicon.

The cryptic plasmid pBC1 (1.6 kb) isolated from Bacillus coagulans Zu1961 was genetically marked with the genes for chloramphenicol and ampicillin resistance (CmR and ApR) from the Escherichia coli plasmid pJH101. The recombinant vector obtained (pCP49, 7.0 kb) replicated and expressed CmR in B. subtilis and CmR and ApR in E. coli. Different shuttle vectors for Gram+ bacteria were also constructed by inserting pBC1 into the Staphylococcus aureus plasmid pC194. The smallest of these, pLM6 (2.8 kb), containing essentially pBC1 and the chloramphenicol acetyl transferase gene from pC194, replicated in B. subtilis at a copy number of 60. By electroporation, these plasmids were introduced and stably maintained in B. subtilis, B. amyloliquefaciens, S. aureus, S. carnosus and Lactobacillus reuteri.

Bacillus subtilis↗
PubMed2005

Nucleotide sequence, structural organization and host range of pRS4, a small cryptic Pediococcus pentosaceus plasmid that contains two cassettes commonly found in other lactic acid bacteria.

The complete nucleotide sequence of the cryptic plasmid pRS4 (3550 bp) from Pediococcus pentosaceus RS4 was determined. Sequence analysis revealed the presence of three open reading frames (ORFs). The putative protein coded by ORF 1 showed 93% identity with the mobilization protein of Lactobacillus casei plasmid pLC88 and 94% identity with a sequenced fragment of the mobilization protein of P. damnosus plasmid pF8801, suggesting a common origin for these three mobilization proteins. The putative protein coded by ORF 2 showed 92% identity with the replication protein of L. plantarum plasmid pWCFS101, a plasmid that replicates via the rolling circle (RC) mechanism, suggesting a similar replication mechanism for pRS4. Supporting this hypothesis, a putative double strand origin (dso) and a region with palindromic sequences that could function as single strand origin (sso), were detected in pRS4. A function could not be assigned to ORF 3. Since ORF 1 exhibits high identity with L. casei plasmid pLC88 but lower identity (58%) with other Lactobacillus plasmids, and ORF 2 exhibits high identity with the L. plantarum plasmid pWCFS101 but lower identity (55-58%) with other Lactobacillus plasmids (including pLC88), two independent cassettes, from different sources, seem to be involved in the structure of pRS4. Plasmids derived from pRS4 containing the chloramphenicol resistance gene were successfully electrotransformed in L. plantarum, L. casei, P. pentosaceus, and Pediococcus acidilactici, suggesting that pRS4 could be used as a cloning vector for lactic acid bacteria. To our knowledge pRS4 is the first RC-replicating plasmid of P. pentosaceus that has been completely sequenced and used as cloning vector.

Base Sequence↗
PubMed2003

Roles of outer capsid proteins as determinants of pathogenicity and host range restriction of avian rotaviruses in a suckling mouse model.

We previously demonstrated that a pigeon rotavirus, PO-13, but not turkey strains Ty-3 and Ty-1 and a chicken strain, Ch-1, induced diarrhea in heterologous suckling mice. In this study, it was suggested that these avirulent strains, but not PO-13, were inactivated immediately in gastrointestinal tracts of suckling mice when they were orally inoculated. To determine which viral proteins contribute to the differences between the pathogenicitiy and the inactivation of PO-13 and Ty-3 in suckling mice, six PO-13 x Ty-3 reassortant strains that had the genes of the outer capsid proteins, VP4 and VP7, derived from the opposite strain were prepared and were orally inoculated to suckling mice. A single strain that had both PO-13 VP4 and VP7 with the genetic background of Ty-3 had an intermediate virulence for suckling mice. Three strains with Ty-3 VP7, regardless of the origin of VP4, rapidly disappeared from gastrointestinal tracts of suckling mice. These results indicated that the difference between the pathogenicity of PO-13 and that of Ty-3 was mainly dependent on both their VP4 and VP7. In particular, VP7 was found to be related to the inactivation of Ty-3 in gastrointestinal tracts of suckling mice.

Animals↗
PubMed2005

Functional analysis of the baculovirus host range gene, hrf-1.

The hrf-1 gene from Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV) prevents translation arrest and promotes Autographa californica multiple nucleopolyhedrovirus (AcMNPV) replication in IPLB-Ld652Y cells (Ld652Y), a non-permissive L. dispar cell line. There are no motifs in the predicted protein sequence to suggest how it might function and the only homolog identified is encoded by another baculovirus, Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV). In this study, we report a functional analysis of the hrf-1 protein. AcMNPV bearing carboxy- or amino-terminally truncation hrf-1, and hrf-1 mutated by two-amino acid insertions did not replicate Ld652Y cells. Neither OpMNPV hrf-1 nor an OpMNPV/LdMNPV chimeric hrf-1 supported AcMNPV replication. Mutations in a highly acidic domain of hrf-1, in which aspartic acid residues were replaced with alanine, had varied effects on hrf-1 function. They had no effect, abolished hrf-1 function completely, or partially supported protein synthesis in infected Ld652Y cells. A slight increase in protein synthesis was achieved by increasing the expression of hrf-1 acidic domain mutant proteins. Together, these results indicate a critical role for hrf-1 structure and suggest a functional role for the acidic domain.

Animals↗
PubMed2005

The infectivity and host range of the ecotropic porcine endogenous retrovirus, PERV-C, is modulated by residues in the C-terminal region of its surface envelope protein.

Endogenous retroviral genetic material serves as a reservoir for the generation of retroviral pathogens by recombination between activated endogenous or exogenous infectious agents. Some porcine tissues actively express infectious porcine endogenous retroviruses (PERVs). Of the three classes of PERV characterized to date, two, PERV-A and B, are capable of infecting human cells in vitro, whereas PERV-C cannot. Here, we demonstrate that the PERV-C envelope surface protein (SU) when disassociated from its C-terminus binds human cells. Further, we show that PERV-C binding to human cells is not inhibited in 293 cells productively infected with PERV-A, confirming that the molecule PERV-C interacts with on human cells is distinct from that used by PERV-A. Moreover, we demonstrate that the envelope region encompassing the proline-rich region is required for binding to cells in addition to the putative variable region A (VRA) and B (VRB). The region in the C-terminus of the SU that alters the binding and infectivity properties of PERV-C differs by only nine residues from the analogous region of PERV-A. Caution may be warranted even when a xenotransplantation product is from source pigs that do not express human-tropic viruses, as minimal mutations within PERV-C combined with selection in a human recipient could render PERV-C infectious in humans.

Animals↗
PubMed2006

Host range and receptor utilization of canine distemper virus analyzed by recombinant viruses: Involvement of heparin-like molecule in CDV infection.

We constructed recombinant viruses expressing enhanced green fluorescent protein (EGFP) or firefly luciferase from cDNA clones of the canine distemper virus (CDV) (a Japanese field isolate, Yanaka strain). Using these viruses, we examined susceptibilities of different cell lines to CDV infection. The results revealed that the recombinant CDVs can infect a broad range of cell lines. Infectivity inhibition assay using a monoclonal antibody specific to the human SLAM molecule indicated that the infection of B95a cells with these recombinant CDVs is mainly mediated by SLAM but the infection of 293 cell lines with CDV is not, implying the presence of one or more alternative receptors for CDV in non-lymphoid tissue. Infection of 293 cells with the recombinant CDV was inhibited by soluble heparin, and the recombinant virus bound to immobilized heparin. Both F and H proteins of CDV could bind to immobilized heparin. These results suggest that heparin-like molecules are involved in CDV infection.

Animals↗
PubMed2001

Stability and DNA-binding properties of the omega regulator protein from the broad-host range Streptococcus pyogenes plasmid pSM19035.

At the transcriptional level, the pSM19035-encoded omega protein coordinates the expression of proteins required for control of copy number and maintenance of plasmids. Using circular dichroism, fluorescence spectroscopy, ultracentrifugation and an electrophoretic mobility shift assay, the wild-type omega protein and a variant with a C-terminal hexa-histidine tag (omega-H(6)) were characterized. The omega protein is mainly alpha-helical (42%), occurs as homodimer in solution, unfolds thermally with half transition temperatures, T(m), between approximately 43 and approximately 78 degrees C depending on the ionic strength of the buffer, and binds PcopS-DNA with high affinity. The omega-H(6) protein has a modified conformation with lower alpha-helix content (29%), lower thermal stability, and strongly reduced affinity to PcopS-DNA.

Bacterial Proteins↗
PubMed1998

Use of a broad-host-range bacteriocin-producing Lactococcus lactis transconjugant as an alternative starter for salami manufacture.

Lactococcus lactis DPC4268 is widely used for Cheddar cheese manufacture in Ireland where it is recognised for its reliable fast acid producing ability in dairy environments. A transconjugant of this strain, L. lactis DPC4275, was generated which produces the broad spectrum, two-component bacteriocin lacticin 3147, which is inhibitory to a variety of undesirable gram-positive bacteria including Clostridium, Bacillus, Enterococcus, Listeria, Staphylococcus and Streptococcus. Both DPC4268 and DPC4275 were used as single strain starters for manufacture of salamis. These products were compared with a salami manufactured with a conventional starter (Lactobacillus sake and Staphylococcus carnosus) in terms of pH development, a(w)-value, weight loss, colour development and sensory characteristics. Salamis produced with either lactococcal culture exhibited pH values below 5.1 and a(w)-values below 0.90 which is favourable for preservation and hygienic stability. In addition, these salamis had good sensory and colorimetric qualities. A minimum lacticin 3147 concentration of 640 AU/g was detected in the salamis which were produced with L. lactis DPC4275.

Animals↗
PubMed2002

Monitoring Azospirillum-wheat interactions using the gfp and gusA genes constitutively expressed from a new broad-host range vector.

To monitor the colonization of wheat roots by Azospirillum brasilense, we constructed several plasmids based on the pBBR1 replicon expressing the gfp and gusA genes constitutively. Both genes were placed under control of the gentamycin resistance gene promoter resulting in high levels of expression in Escherichia coli and A. brasilense. The constructed plasmids were stably maintained in A. brasilense strains even in the absence of selective pressure. The colonization of wheat plants grown under controlled conditions in sterilized vermiculite by A. brasilense strain FP2 (a Sp7-derivative) transconjugants containing these plasmids was monitored. Bacteria expressing GFP were easily observed in fresh plant material by fluorescence microscopy. Cell aggregates and single bacteria were visualized on the surfaces of young root zones, such as roots hairs and lateral roots. Large cellular clumps were observed at the points of lateral root emergence or at intercellular spaces of root epidermal cells 30 days after inoculation. Although we failed to detected bacteria in internal cortical and xylem tissues of wheat roots, the initial stage of endophytic colonization by A. brasilense may involve the sites detected in this work.

Aluminum Silicates↗
PubMed1998

A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants.

An improved method for gene replacement in Pseudomonas aeruginosa was developed. The method employs several new gene replacement vectors that incorporate (1) the counterselectable sacB marker, (2) a lacZ alpha-allele for blue-white screening, (3) the pUC18/19 vectors multiple cloning site with 10 unique restriction sites, (4) an oriT for conjugation-mediated plasmid transfer and (5) carbenicillin, gentamicin (Gm) and tetracycline selectable markers. A cassette was constructed that contains a GmR selectable marker next to the green fluorescent protein structural gene, with both markers being flanked by Flp recombinase target (FRT) sites. The FRT cassette was used to insertionally inactivate the cloned P. aeruginosa pabC gene encoding aminodeoxychorismate lyase. After conjugal transfer into P. aeruginosa, plasmid integrants were selected, and deletion of unwanted DNA sequences was promoted by sucrose counterselection. The FRT cassette was excised with high frequencies (close to 100%) from the chromosome after conjugal transfer of a Flp recombinase-expressing plasmid; this sacB-containing plasmid was subsequently cured by sucrose counterselection, resulting in an unmarked P. aeruginosa delta pabC strain.

Base Sequence↗
PubMedDate not supplied

Glycoprotein-mediated biological properties of a host range mutant of Junin virus.

The biological properties mediated by the main envelope glycoprotein (GP1) of a mouse-attenuated mutant of Junin virus, named Cl67, were investigated in comparison with its parental strain XJCl3. In contrast to the parental strain, this mutant was unable to multiply in primary cultures of mouse embryo fibroblasts (MEFs). Impairment of Cl67 multiplication was associated with a lack of virus binding to MEF, probably due to an altered interaction between GP1 and cellular receptors. Antigenic and immunogenic characterization of GP1, performed by neutralization assays, demonstrated that, under certain conditions, polyclonal and monoclonal antibodies exhibited differential affinity and specificity for each virus. Cl67-infected Vero cells showed a marked pH-dependent fusion capability, suggesting more efficient low pH triggering of fusion by mutant virus GP1 in comparison with the parental strain.

3T3 Cells↗