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PubMed1983

Host range transforming gene of polyoma virus plays a role in virus assembly.

Polyoma virus host range transforming (hr-t) mutants are blocked in virion assembly. In normal 3T3 cells, a nonpermissive host, these mutants synthesize 30-40% as much viral DNA and 80-100% as much capsid proteins as does wild-type virus and yet produce only 1-2% as much infectious virus. Intermediates in virion assembly have been followed by [3H]thymidine incorporation. hr-t mutants synthesize 95S replicating minichromosomes, which accumulate as 75S forms. However, the latter fail to undergo efficient transition to 240S virion structures. This block in encapsidation is overcome in permissive hosts such as primary baby mouse kidney (BMK) epithelial cells. The block in assembly of 240S particles is accompanied by a failure to induce a series of acidic isoelectric forms of the major capsid protein, VP1. Multiple species of post-translationally modified VP1 are seen by two-dimensional gel electrophoresis in wild-type virus-infected cells. These acidic VP1 subspecies are decreased 6- to 10-fold in hr-t mutant-infected 3T3 cells but are produced in normal amounts when the same mutants infect BMK cells. When 3T3 cells are coinfected with hr-t mutant and wild-type viruses, normal amounts of the VP1 subspecies are present, and hr-t mutant viral DNA is efficiently packaged into virions. These studies demonstrate an important role of the hr-t gene of polyoma virus in virus assembly. Specifically, we propose that VP1 is a target for hr-t gene-controlled modification and that modified forms of VP1 are essential for encapsidation of viral minichromosomes.

Animals↗
PubMed2001

Diseases of humans and their domestic mammals: pathogen characteristics, host range and the risk of emergence.

Pathogens that can be transmitted between different host species are of fundamental interest and importance from public health, conservation and economic perspectives, yet systematic quantification of these pathogens is lacking. Here, pathogen characteristics, host range and risk factors determining disease emergence were analysed by constructing a database of disease-causing pathogens of humans and domestic mammals. The database consisted of 1415 pathogens causing disease in humans, 616 in livestock and 374 in domestic carnivores. Multihost pathogens were very prevalent among human pathogens (61.6%) and even more so among domestic mammal pathogens (livestock 77.3%, carnivores 90.0%). Pathogens able to infect human, domestic and wildlife hosts contained a similar proportion of disease-causing pathogens for all three host groups. One hundred and ninety-six pathogens were associated with emerging diseases, 175 in humans, 29 in livestock and 12 in domestic carnivores. Across all these groups, helminths and fungi were relatively unlikely to emerge whereas viruses, particularly RNA viruses, were highly likely to emerge. The ability of a pathogen to infect multiple hosts, particularly hosts in other taxonomic orders or wildlife, were also risk factors for emergence in human and livestock pathogens. There is clearly a need to understand the dynamics of infectious diseases in complex multihost communities in order to mitigate disease threats to public health, livestock economies and wildlife.

Animal Diseases↗
PubMed1989

Serologic evidence of hantaviral infections within small mammal communities of Baltimore, Maryland: spatial and temporal patterns and host range.

Serologic evidence was used to investigate the spatial and temporal distribution and host range of hantaviruses in small mammal communities in Baltimore, MD. Immunofluorescent antibody (IFA) reactive to a Baltimore rat isolate of Seoul virus was detected in 44% of 404 Norway rats captured at 4 park or residential locations; 21% of 121 meadow voles captured at 4 park locations possessed significant IFA titers to Prospect Hill virus. Evidence from plaque neutralization assay of rodent sera suggested that Seoul virus and Prospect Hill virus circulated concurrently in voles and rats, respectively, at 1 park. No cross infection of virus between these primary reservoir species was observed, as determined by higher specific neutralizing antibody titers to the characteristic virus for each host species. Only 4% of 449 house mice and 1% of 94 white-footed mice captured at the same sites as the primary host species were seropositive to hantaviruses, despite extensive demonstrated overlap in time and space with the primary host species.

Animals↗
PubMed1991

Nucleotide sequence and characterization of the broad-host-range lactococcal plasmid pWVO1.

The nucleotide sequence of the Lactococcus lactis broad-host-range plasmid pWVO1, replicating in both gram-positive and gram-negative bacteria, was determined. This analysis revealed four open reading frames (ORFs). ORF A appeared to encode a trans-acting 26.8-kDa protein (RepA), necessary for replication. The ORF C product was assumed to play a regulatory role in replication. Both RepA and the ORF C product showed substantial sequence similarity with the Rep proteins of the streptococcal plasmid pLS1. In addition, the plus origin of replication was identified on the basis of strong similarity with the plus origin of pLS1. Derivatives of pWVO1 produced single-stranded (ss) DNA in Bacillus subtilis and L. lactis, suggesting that this plasmid uses the rolling-circle mode of replication. In B. subtilis, but not in L. lactis, the addition of rifampicin resulted in increased levels of ssDNA, indicating that in the former organism the host-encoded RNA polymerase is involved in the conversion of the ssDNA to double-stranded plasmid DNA (dsDNA). Apparently, in L. lactis the conversion of ss to ds pWVO1 DNA occurs by a mechanism which does not require the host RNA polymerase.

Amino Acid Sequence↗
PubMed1992

Host range mutants of v-src: alterations in kinase activity and substrate interactions.

Host range mutants of Schmidt-Ruppin v-src that transform chicken embryo fibroblasts (CEF) but not Rat-2 cells were generated previously by linker insertion-deletion mutagenesis (J. E. DeClue and G. S. Martin, J. Virol. 63:542-554, 1989). One of these mutants, SRX5, in which Tyr-416 is substituted by the sequence Ser Arg Asp, retained high levels of kinase activity in vitro and in vivo, both in CEF and in Rat-2 cells. Phosphorylation of p36 (the calpactin I heavy chain) was drastically reduced in cells expressing SRX5 src, suggesting that the phenotype of SRX5 results from an alteration in substrate recognition by the src kinase. Three mutants, SPX1, SHX13, and XD6, containing linker insertions or small deletions within the src homology 2 (SH2) region, induced reduced levels of kinase activity in both CEF and Rat-2 cells. However, the residual levels of kinase activity in Rat-2 cells were above the threshold at which wild-type pp60v-src transforms Rat-2 cells, indicating that the reduction in kinase activity was not sufficient to account for the failure to transform. Cells infected by these mutants exhibited reduced levels of phosphorylation of 120- and 62-kDa proteins. We have reported elsewhere (M. F. Moran, C. A. Koch, D. Anderson, C. Ellis, L. England, G. S. Martin, and T. Pawson, Proc. Natl. Acad. Sci. USA 87:8622-8626, 1990) that ras GTPase-activating protein GAP and associated protein p62 are not tyrosine phosphorylated in Rat-2 cells expressing SHX13 or XD6. The transformation defect in Rat-2 cells may result from the failure to phosphorylate those proteins. The fifth mutant, XD4, contains a deletion which removes all of the src homology 3 (SH3) and most of the SH2 sequences of src. The protein encoded by XD4 is active as a kinase when expressed in CEF, indicating that in CEF the SH2 and SH3 regions of v-src are not necessary for kinase activity and transformation. The XD4 src product is not tyrosine phosphorylated and is inactive as a kinase when expressed in Rat-2 cells. Thus, host cell factors can affect the tyrosine phosphorylation and activity of the v-src kinase in the absence of the SH2 and SH3 regions. These results indicate that the host-dependent transformation phenotype results from alterations in src kinase activity and substrate specificity.

Amino Acid Sequence↗
PubMed1983

Cold-sensitive expression of transformation by a host range mutant of type 5 adenovirus.

hr1, an E1a (0-4.5 map units) host range mutant of type 5 adenovirus (Ad5), transformed a cloned rat embryo fibroblast (CREF) cell line at about a 5-fold higher frequency than wild-type (wt) Ad5 when cells were cultured at 37 degrees C. However, if the cells were infected with hr1 and maintained at 32 degrees C morphological transformation did not occur. When infected cells were shifted from 32 degrees C to 37 degrees C 2 wk postinfection, the frequency of transformation by 6 wk was identical to that of cells grown continuously at 37 degrees C, whereas cultures shifted from 37 degrees C to 32 degrees C 2 wk postinfection displayed a greater than 96% reduction in morphological transformation. hr1-transformed cells had a fibroblastic morphology as contrasted with the typical epithelioid morphology of wt Ad5-transformed cells, but hr1- and wt Ad5-transformed cells had similar saturation densities, growth rates, and agar cloning efficiencies when assayed at 37 degrees C. However, when cells transformed by hr1 at 37 degrees C were grown at 32 degrees C, they had a saturation density close to that of normal CREF cells and grew at a lower efficiency in agar than wt-transformed cells. DNA transfer/hybridization analysis of two hr1-transformed cloned cell lines, A2 and B3, indicated that A2 cells contained a complete integrated copy of the Ad5 genome, whereas in B3 cells only part of the Ad5 genome was integrated. RNA transfer and RNA/DNA filter hybridization analyses indicated that the types of viral messenger RNAs and the relative amounts of RNA transcribed were similar in the A2 and B3 cell lines when they were grown at 32 degrees C and 37 degrees C. Indirect immunofluorescence, with antisera from hamsters bearing Ad-induced tumors, indicated a temperature dependence in staining--i.e., cells grown at 37 degrees C or shifted from 32 degrees C to 37 degrees C contained intense, particulate staining in the nuclear region, whereas the staining was decreased significantly in cells cultured at 32 degrees C and in cells shifted from 37 degrees C to 32 degrees C. These findings indicate that the gene product affected by the hr1 mutation is cold sensitive and is essential for the expression of the characteristics that identify the transformed cell.

Adenoviridae↗
PubMed1995

Surface polysaccharide mutants of Rhizobium sp. (Acacia) strain GRH2: major requirement of lipopolysaccharide for successful invasion of Acacia nodules and host range determination.

Two transposon Tn5-induced mutants of wild-type broad-host-range Rhizobium sp. GRH2 were isolated and found to harbour different alterations in surface polysaccharides. These mutants, designated GRH2-14 and GRH2-50, induced a few, empty nodules on Acacia and lost the ability to nodulate most host herbaceous legumes. Whereas mutant GRH2-14 produces an acidic exopolysaccharide (EPS) similar to the wild-type, the acidic EPS of mutant GRH2-50 lacks galactose and the pyruvyl and 3-hydroxybutyryl substituents attached to this sugar moiety. In addition, both mutants GRH2-50 and GRH2-14 were altered in smooth lipopolysaccharides (LPS). DNA sequence analyses of the corresponding Tn5 insertions revealed that strain GRH2-50 was mutated in a DNA locus homologous to galE, and in vitro enzyme assays indicated that the UDPglucose 4-epimerase (GalE) activity was missing in this mutant strain. DNA hybridization studies showed that the GRH2-50 mutant DNA has homologous sequences within the different biovars of Rhizobium leguminosarum. However, no DNA homology to GRH2-14 altered DNA was found in those rhizobial strains, indicating that it represents a new chromosomal lps locus in Rhizobium sp. (Acacia) involved in symbiotic development.

Acacia↗
PubMed1990

The kil-kor regulon of broad-host-range plasmid RK2: nucleotide sequence, polypeptide product, and expression of regulatory gene korC.

Broad-host-range plasmid RK2 encodes several kil operons (kilA, kilB, kilC, kilE) whose expression is potentially lethal to Escherichia coli host cells. The kil operons and the RK2 replication initiator gene (trfA) are coregulated by various combinations of kor genes (korA, korB, korC, korE). This regulatory network is called the kil-kor regulon. Presented here are studies on the structure, product, and expression of korC. Genetic mapping revealed the precise location of korC in a region near transposon Tn1. We determined the nucleotide sequence of this region and identified the korC structural gene by analysis of korC mutants. Sequence analysis predicts the korC product to be a polypeptide of 85 amino acids with a molecular mass of 9,150 daltons. The KorC polypeptide was identified in vivo by expressing wild-type and mutant korC alleles from a bacteriophage T7 RNA polymerase-dependent promoter. The predicted structure of KorC polypeptide has a net positive charge and a helix-turn-helix region similar to those of known DNA-binding proteins. These properties are consistent with the repressorlike function of KorC protein, and we discuss the evidence that KorA and KorC proteins act as corepressors in the control of the kilC and kilE operons. Finally, we show that korC is expressed from the bla promoters within the upstream transposon Tn1, suggesting that insertion of Tn1 interrupted a plasmid operon that may have originally included korC and kilC.

Bacterial Proteins↗
PubMed1989

Host-specific effects of the korA-korB operon and oriT region on the maintenance of miniplasmid derivatives of broad host-range plasmid RK2.

Two genetic determinants are sufficient for small derivatives of broad host-range plasmid RK2 to replicate in different Gram-negative bacteria: trfA, which encodes a replication initiator, and oriV, the origin of replication. In this study, nonessential RK2 determinants in the region encoding oriT, the origin of conjugative transfer, and the korA-korB operon, whose products regulate trfA expression, were tested for their effects on the stability of mini-RK2 plasmids in eight different hosts. We found that determinants of both regions can substantially alter plasmid stability, but the effects are not uniform in all hosts. The results also indicate that the effects of the korA-korB operon extend beyond that of the regulation of trfA transcription. This study further illustrates the different requirements for stable plasmid maintenance in diverse bacteria and the ability of wild-type RK2 to adapt to a variety of intracellular environments. The data also provide further evidence for the involvement of different regions of RK2 for stable maintenance in various hosts.

Conjugation, Genetic↗
PubMed1986

Evidence for the involvement of the incC locus of broad host range plasmid RK2 in plasmid maintenance.

Plasmid pRK2501 is a deletion derivative of broad host range plasmid RK2 and encodes two trfB-regulated operons: the trfA operon which codes for both kilD, which interferes with plasmid maintenance if unregulated, and trfA whose protein product(s) is essential for replication from oriVRK2; and part of the trfB operon, containing both trfB/korA/korD, whose product negatively regulates transcription of both trfA and trfB operons, and incC, the product of which interferes in trans with inheritance of RK2 and certain of its derivatives. Plasmid pRK2501ts3 is a derivative with a point mutation in trfB, rendering plasmid maintenance temperature sensitive. Transcriptional fusions of the trfB operon and the galK gene demonstrate that this mutation derepresses trfB operon transcription at both 30 and 42 degrees C. The trfA operon is also derepressed by this mutation. Since the trfB gene product appears to be defective at both permissive and non-permissive temperatures the temperature sensitivity of pRK2501ts3 must be due to a secondary effect. In fact, it appears to arise from the inhibitory behavior of derepressed incC at the non-permissive temperature since a major class of "revertant" of pRK2501ts3 contains deletions inactivating incC and a reconstruction experiment demonstrates that such a deletion is sufficient for "reversion." Maxicell experiments show that at the non-permissive temperature the trfA operon polypeptide products are produced at much lower levels, an effect partly reversed by a deletion affecting incC. It is proposed that incC normally plays a role in maintenance of IncP plasmids by modulation of trfA operon expression.

Bacterial Proteins↗
PubMed1987

A recombinant human interferon-alpha B/D hybrid with a broad host-range.

A recombinant interferon (IFN) hybrid has been found to have a broad host-range of activity in an antiviral assay (plaque reduction of vesicular stomatitis virus) and also high efficacy as an antiviral agent in at least 12 different animal cell species. The IFN hybrid consists of amino acids 1 to 60 from HuIFN-alpha B and amino acids 61 to 166 from HuIFN-alpha D. The profile of cross-species activity of the IFN-alpha B/D hybrid has been compared with that of HuIFN-alpha F, and of the parents HuIFN-alpha B and -alpha D. When both IFN-alpha B and -alpha D were active in a cell species, the hybrid IFN had comparable or better activity than the more active parental IFN. The hybrid shared a broad cross-species activity with IFN-alpha D. However, the IFN-alpha B/D hybrid was 10-fold more active on human cells, 30-fold more active on rabbit cells, and 50-fold more active on mouse cells than IFN-alpha D.

Animals↗
PubMed1992

Transcription and autoregulation of the stabilizing functions of broad-host-range plasmid RK2 in Escherichia coli, Agrobacterium tumefaciens and Pseudomonas aeruginosa.

The broad-host-range plasmid RK2 has been shown to encode several proteins important for its maintenance within bacterial populations of a number of Gram-negative bacteria. Their genes are organized into two operons: parCBA and parD. These operons have been proposed to be transcribed from two divergent promoters, p-parCBA and p-parD, located within a sequence of approximately 150 bases. In this report we identify and characterize the sequences required for regulated transcription from these promoters in Escherichia coli, Agrobacterium tumefaciens and Pseudomonas aeruginosa. Both of these promoters are repressed by their own gene products in the same manner in all three bacteria tested, with ParA functioning as the primary repressor of p-parCBA and ParD functioning as the repressor of p-parD. The binding regions of these proteins were determined through deletion analyses, DNA mobility shift assays, and an examination of the effect of mutations in this region. Based on these observations, the ParA protein appears to bind to either two inverted repeat or two direct repeat sequences, one downstream from the transcriptional initiation site and the other upstream of the p-parCBA -35 box. The ParD protein appears to bind to one inverted repeat sequence, located between the -35 and -10 boxes of p-parD.

Agrobacterium tumefaciens↗
PubMed2007

A type IV-secretion-like system is required for conjugative DNA transport of broad-host-range plasmid pIP501 in gram-positive bacteria.

Plasmid pIP501 has a very broad host range for conjugative transfer among a wide variety of gram-positive bacteria and gram-negative Escherichia coli. Functionality of the pIP501 transfer (tra) genes in E. coli was proven by pIP501 retrotransfer to Enterococcus faecalis (B. Kurenbach, C. Bohn, J. Prabhu, M. Abudukerim, U. Szewzyk, and E. Grohmann, Plasmid 50:86-93, 2003). The 15 pIP501 tra genes are organized in a single operon (B. Kurenbach, J. Kopeć, M. Mägdefrau, K. Andreas, W. Keller, C. Bohn, M. Y. Abajy, and E. Grohmann, Microbiology 152:637-645, 2006). The pIP501 tra operon is negatively autoregulated at the transcriptional level by the conjugative DNA relaxase TraA. Three of the 15 pIP501-encoded Tra proteins show significant sequence similarity to the Agrobacterium type IV secretion system proteins VirB1, VirB4, and VirD4. Here we report a comprehensive protein-protein interaction map of all of the pIP501-encoded Tra proteins determined by the yeast two-hybrid assay. Most of the interactions were verified in vitro by isolation of the protein complexes with pull-down assays. In conjunction with known or postulated functions of the pIP501-encoded Tra proteins and computer-assisted prediction of their cellular location, we propose a model for the first type IV-secretion-like system encoded by a conjugative plasmid from gram-positive bacteria.

Bacterial Proteins↗
PubMed1991

Isolation and characterization of insertion sequence elements from gram-negative bacteria by using new broad-host-range, positive selection vectors.

On the basis of an RSF1010-derived broad-host-range vector, three different systems which enable positive detection and isolation of insertion sequence (IS) elements from gram-negative bacteria were constructed. Vectors pSUP104-pheS, pSUP104-rpsL, and pSUP104-sac were used successfully in a number of Rhizobium strains and in Xanthomonas campestris. More than 20 different IS elements were isolated and characterized. The 16 IS elements from Rhizobium meliloti were further used to characterize various R. meliloti strains by hybridization. The resulting hybridization patterns were different for every strain and gave a clear and definite IS fingerprint of each strain. These IS fingerprints can be used to identify and characterize R. meliloti strains rapidly and unequivocally, as they proved to be relatively stable. Some of the IS elements were found to be identical when the IS fingerprints from a given strain were compared. This method of IS fingerprinting can also establish whether IS elements are the same, related, or different.

Blotting, Southern↗
PubMed1975

In vivo characteristics of temperature-sensitive host range mutants of herpes simplex virus type 2.

The in vivo properties of four host range temperature-sensitive (ts) mutants of herpes simplex virus type 2 (HSV-2) were correlated with known in vitro characteristics. Mutants and parental virus were inoculated into newborn and weanling mice and weanling hamsters by intracranial and subcutaneous routes, and into weanling rabbits by corneal scarification. In all cases the pattern of attenuation of mutants in vivo correlated with their stability in vitro at 39 degrees. The most attenuated mutant (mutant 69) was also the most consistent in its inability to induce cytopathic effects (CPE) in vitro or to replicate under nonpermissive conditions. Conversely, the most virulent mutant (mutant 41) was the least stable under nonpermissive conditions.

Animals↗
PubMed2003

What determines host range in parasitoids? An analysis of a tachinid parasitoid community.

Despite the vast diversity of parasitic insects and their importance in natural and agricultural communities, our knowledge of what determines their patterns of association with hosts remains sparse. Unlike most parasites that tend to be specialized, parasitoid flies in the family Tachinidae exhibit a broad spectrum of host-specificity, with many species attacking a wide range of hosts. This variability in host-specificity makes them a useful model for examining the ecological and historical factors that determine host associations. We analyzed data collected from a 5-year rearing program of Lepidoptera in southern Arizona to investigate the factors that influence tachinid-host associations. After controlling for a strong effect of sample size, a significant portion of the remaining variance in host range was explained by differences among phylogenetic groups of tachinids and/or their correlated reproductive strategies. Relatively specialized tachinids tended to be associated with monophagous or narrowly oligophagous hosts and attacked them at relatively high frequencies, a pattern we suggest is related to host location efficiency. Cluster analysis indicated that host abundance, gregariousness, food-plant type, and morphology are all important determinants of tachinid host use. Little concordance was found between how tachinid species cluster according to characteristics of their hosts and their estimated phylogenetic relationships. Together, the results of this study suggest that ecological factors are important determinants of host use in these parasitoids and although phylogenetic history may influence the range of hosts used, its power to explain the ecological or taxonomic character of hosts used appears limited.

Analysis of Variance↗
PubMed1994

The distribution and host range of species of the blood fluke Sanguinicola in British freshwater fish.

The distribution of host range of Sanguinicola armata, S. inermis and S. volgensis in British freshwater fish are described. S. inermis has been recorded in carp Cyprinus carpio, crucian carp Carassius carassius and tench Tinca tinca in southern, central and eastern England and in koi carp imported from Japan. S. armata has been detected in pike Esox lucius and tench and S. volgensis has been found in chub Leuciscus cephalus, dace Leuciscus leuciscus, perch Perca fluviatilis, roach Rutilus rutilus and pike in southern and eastern England. The first record of the co-occurrence of two species of Sanguinicola (S. inermis and S. volgensis) in a British inland water is reported. The importance of blood flukes and the role of anthropochore movement of fish hosts in dissemination of S. inermis are discussed.

Animals↗