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PubMed1998

Recombinant swinepox virus expressing beta-galactosidase: investigation of viral host range and gene expression levels in cell culture.

Swinepox virus (SPV) has been proposed as a potential vector for generating recombinant vaccines for swine. However, little is known about important aspects of SPV biology, such as the functionality of SPV promoters or the host range of SPV. Using a transient expression assay, well-characterized vaccinia virus promoters were shown to be active in cells infected with SPV. A recombinant SPV expressing beta-galactosidase (beta-gal) was constructed and characterized. The E. coli LacZ gene was placed under the control of a strong vaccinia synthetic early/late promoter and was inserted by homologous recombination in a noncoding region of the SPV genome. The recombinant SPV expressing beta-gal was used to characterize the host range of the virus by measuring protein expression and virus production in different cell lines. In general, SPV expressed more protein and grew more efficiently than vaccinia virus in porcine cell lines. Surprisingly, the recombinant SPV was able to infect and replicate in several cell lines of nonswine origin. The virus directed regulated early and late gene expression of beta-gal in those cells and formed blue plaques in cell monolayers in the presence of X-gal. Upon infection with the recombinant SPV, there was a significant level of viral replication, and the virus can be serially passaged in some nonswine cell lines. The data presented suggest that despite the strict host tropism of SPV, the virus exhibits a relatively broad host range in cell culture.

Animals↗

PubMed1964

HOST RANGE OF CERTAIN VIRULENT AND TEMPERATE BACTERIOPHAGES ATTACKING GROUP D STREPTOCOCCI.

Brock, Thomas D. (Indiana University, Bloomington). Host range of certain virulent and temperate bacteriophages attacking group D streptococci. J. Bacteriol. 88:165-171. 1964.-Host range studies with five bacteriophages isolated from sewage showed that a high degree of strain specificity occurred among the species Streptococcus faecalis, S. faecalis var. zymogenes, and S. faecalis var. liquefaciens. Certain phages were able to attack various members of these subgroups but not other members. A high degree of strain specificity was also found with respect to eight phages attacking strains of S. faecium. Many strains of this latter group were resistant to all the phages available. The only firm conclusion was that no phage attacked both S. faecium and S. faecalis strains. In several selected strains of the S. faecalis-liquefaciens-zymogenes group, resistance to several virulent phages seemed to be related to the presence of certain prophages in these strains. A search for transduction with the temperate phages was unsuccessful. Mutants of S. faecium 8043 resistant to certain growth-factor analogues showed unpredictable changes in resistance and sensitivity to two virulent phages.

Bacteriophage Typing↗

PubMed1991

Structural, molecular, and genetic analysis of the kilA operon of broad-host-range plasmid RK2.

The kil loci (kilA, kilB, kilC, and kilE) of incompatibility group P (IncP), broad-host-range plasmid RK2 were originally detected by their potential lethality to Escherichia coli host cells. Expression of the kil determinants is controlled by different combinations of kor functions (korA, korB, korC, and korE). This system of regulated genes, known as the kil-kor regulon, includes trfA, which encodes the RK2 replication initiator. The functions of the kil loci are unknown, but their coregulation with an essential replication function suggests that they have a role in the maintenance or host range of RK2. In this study, we have determined the nucleotide sequence of a 3-kb segment of RK2 that encodes the entire kilA locus. The region encodes three genes, designated klaA, klaB, and klaC. The phage T7 RNA polymerase-dependent expression system was use to identify three polypeptide products. The estimated masses of klaA and klaB products were in reasonable agreement with the calculated molecular masses of 28,407 and 42,156 Da, respectively. The klaC product is calculated to be 32,380 Da, but the observed polypeptide exhibited an apparent mass of 28 kDa on sodium dodecyl sulfate-polyacrylamide gels. Mutants of klaC were used to confirm that initiation of translation of the observed product occurs at the first ATG in the klaC open reading frame. Hydrophobicity analysis indicated that the KlaA and KlaB polypeptides are likely to be soluble, whereas the KlaC polypeptide was predicted to have four potential membrane-spanning domains. The only recognizable promoter sequences in the kilA region were those of the kilA promoter located upstream of klaA and the promoter for the korA-korB operon located just downstream of a rho-independent terminatorlike sequence following klaC. The transcriptional start sites for these promoters were determined by primer extension. Using isogenic sets of plasmids with nonpolar mutations, we found that klaA, klaB, and klaC are each able to express a host-lethal (Kil+) phenotype in the absence of kor functions. Inactivation of the kilA promoter causes loss of the lethal phenotype, demonstrating that all three genes are expressed from the kilA promoter as a multicistronic operon. We investigated two other phenotypes that have been mapped to the kilA region of RK2 or the closely related IncP plasmids RP1 and RP4: inhibition of conjugal transfer of IncW plasmids (fwB) and resistance to potassium tellurite. The cloned kilA operon was found to express both phenotypes, even in the presence of korA and korB, whose functions are known to regulate the kilA promoter. In addition, mutant and complementation analyses showed that the kilA promoter and the products of all three kla genes are necessary for expression of both phenotypes. Therefore, host lethality, fertility inhibition, and tellurite resistance are all properties of the kilA operon. We discuss the possible role of the kilA operon for RK2.

Amino Acid Sequence↗

PubMed1983

Replication of derivatives of the broad host range plasmid RK2 in two distantly related bacteria.

A 0.7-kb segment of the broad host range plasmid RK2 containing the replication origin of this plasmid will replicate in Escherichia coli and Pseudomonas putida when this segment is joined to a 1.8-kb region of RK2 designated traA*. The presence of another region of RK2, designated trfB, that previously was implicated in RK2 replication had no effect on the maintenance of the RK2 trfA*-oriV replicon in these two organisms. These observations indicate a requirement for a minimal account of information for replication of this broad host range plasmid in two distantly related bacteria.

DNA Replication↗

PubMed1986

Specific sequences of the env gene determine the host range of two XC-negative viruses of the Rauscher virus complex.

Two viruses which do not give rise to XC plaques in the standard XC assay (XC-negative) have been isolated from the Rauscher virus (RV) complex. These viruses differ in their host range. One, R-MCF-1, is dualtropic and will therefore infect both murine and non-murine cells. However, unlike other mink cell focus-inducing (MCF) viruses, it cannot infect NIH 3T3 cells. The other, R-XC-, is ecotropic. It will infect murine cells, including NIH 3T3 cells, but does not infect mink lung cells. Analysis of hybrid viruses, in which homologous regions of the genomes of R-MCF-1 and R-XC- virus were exchanged, indicated that the NH2-terminal portion of the gp70 is responsible for the particular host ranges of these viruses. The nucleotide sequence of the env gene of R-XC- virus was therefore determined and compared with the known env sequences of ecotropic MLVs and dualtropic MCF viruses of the Rauscher and Friend virus complexes. R-XC- virus was found to be a recombinant virus. Its env gene contained sequences derived from an endogenous env gene which were closely related to those of the MCF viruses but differed from any previously described sequences. The particular properties of R-MCF-1 and R-XC- virus suggest that the two viruses arose by recombination between R-MLV and two endogenous env sequences which differ from those of the known MCF viruses. If so, this suggests that the mouse genome contains at least five env sequences which can give rise to MCF-like viruses. In addition, since the host range and interference properties of R-XC- virus are very similar to those of the previously described ecotropic recombinant viruses, it may be that the ecotropic recombinant viruses arose by recombination with the same endogenous env sequences as did R-XC- virus.

Animals↗

PubMed1984

Involvement of kil and kor genes in the phenotype of a host-range mutant of RP4.

Plasmid pRP761 is a derivative of the promiscuous plasmid RP4, which has a Tn76 insert 1.8 kb from its EcoRI site within the trfB region (Barth 1979). This mutation was pleiotropic, having three effects: the plasmid is unstably maintained in E. coli, it reduces the growth rate of its host and it has suffered a reduction in host-range. We show that pRP761 has reduced expression from both its korA and korB genes and that Tn76 has inserted between them. Fragment exchange experiments showed that this is the only mutant region in pRP761 and is therefore solely responsible for the pleiotropic effects. A spontaneous deletion derivative pRP761-6 has lost Tn76 and its adjacent kilA and korA genes: it has reacquired stability, does not inhibit host growth but is still reduced in its host-range. The provision of cloned korA+ in trans complements the first two phenotypic effects in pRP761 to a large extent, but neither korA+ alone nor korA+ with korB+ complements the host-range reduction in pRP761 or pRP761-6. A possible explanation for these results is that there is a site between korA and korB, affected by the Tn76 insert, that is essential to stable replication of these plasmids in some of their bacterial hosts.

DNA Replication↗

PubMed1985

Extension of the host range of Escherichia coli vectors by incorporation of RSF1010 replication and mobilization functions.

The broad-host-range vectors pSUP104, pSUP106, pSUP204, pSUP304, and pSUP404 are based on conventional Escherichia coli vectors (such as pBR325 and pACYC184) which have been modified to include the mobilization and broad-host-range replication functions of the IncQ plasmid RSF1010. These vector plasmids now can be maintained in a wide range of bacterial genera including Rhizobium, Agrobacterium, and Pseudomonas. They are efficiently mobilized by RP4 and thus are of particular interest for bacteria refractory to transformation. They offer the selection markers and cloning sites characteristic of the basic E. coli vectors. Therefore, they can be applied and adapted to a variety of cloning strategies. However, the cloning of very large fragments (e.g., in cosmid hybrids of pSUP106) was found to affect the stability of the recombinant molecules in a Rec+ background. This instability was not observed with smaller inserts of about 5 kilobases.

Cloning, Molecular↗

PubMed1987

Nucleotide sequence of korB, a replication control gene of broad host-range plasmid RK2.

The korB gene is a major regulatory element in the replication and maintenance of broad host-range plasmid RK2. It negatively controls the replication gene trfA, the host-lethal determinants kilA and kilB, and the korA-korB operon. Here, we present the nucleotide sequence of an 1167 base-pair region that encodes korB. Using sequence data from korB mutants, we identified the korB structural gene. The predicted polypeptide product is negatively charged and has a molecular weight of 39,015, which is considerably less than that estimated by its electrophoretic mobility in SDS/polyacrylamide gels. Secondary-structure predictions of korB polypeptide revealed three closely spaced helix-turn-helix regions with significant homology to similar structures in known DNA-binding proteins. The korB gene, like all other sequenced RK2 genes, shows a strong preference for codons ending in a G or C residue. This is similar to codon usage by genes of Klebsiella and Pseudomonas, the original hosts for RK2 and some closely related plasmids. We also sequenced the site of transposon Tn76 insertion in the host-range mutant pRP761 and found it to be located immediately upstream from korB in the incC gene. Finally, we report the presence of sequences resembling a replication origin within the korB structural gene: a cluster of four 19 base-pair direct repeats and a nearby potential binding site for Escherichia coli dna A replication protein.

Base Composition↗

PubMed1987

Functional analysis of a retroviral host-range mutant: altered long terminal repeat sequences allow expression in embryonal carcinoma cells.

A retroviral host-range neomycin-resistant myeloproliferative sarcoma virus mutant, which is expressed in the embryonal carcinoma cell lines F9 and PCC4aza1R, was molecularly cloned and analyzed. This mutant virus, PCMV, differs from myeloproliferative sarcoma virus by two major deletions, one of which spans exactly a 75-base-pair repeat of the long terminal repeat. Functional analysis of recombinant viruses shows that the host-range expansion of PCMV is a property of nucleotide changes within the U3 region of the long terminal repeat. Furthermore, expression assays of chimeric long terminal repeats show that the enhancer region of PCMV joined to the promoter region of Moloney murine leukemia virus is sufficient to direct the synthesis of chloramphenicol acetyltransferase in F9 and PCC4 cells.

Animals↗

PubMed1991

Host range determinants located on the interior of the poliovirus capsid.

The inability of certain poliovirus strains to infect mice can be overcome by the expression of human poliovirus receptors in mice or by the presence of a particular amino acid sequence of the B-C loop of the viral capsid protein VP1. We have identified changes in an additional capsid structure that permit host-restricted poliovirus strains to infect mice. Variants of the mouse-virulent P2/Lansing strain were constructed containing amino acid changes, deletions and insertions in the B-C loop of VP1. These variants were attenuated in mice, demonstrating the importance of the B-C loop sequence in host range. Passage of two of the B-C loop variants in mice led to the selection of viruses that were substantially more virulent. The increased neurovirulence of these strains was mapped to two different suppressor mutations in the N-terminus of VP1. Whereas the B-C loop of VP1 is highly exposed on the surface of the capsid, near the five-fold axis of symmetry, the suppressor mutations are in the interior of the virion, near the three-fold axis. Introduction of the suppressor mutations into the genome of the mouse-avirulent P1/Mahoney strain resulted in neurovirulent viruses, demonstrating that the P2/Lansing B-C loop sequence is not required to infect mice. Because the internal host range determinants are in a structure known to be important in conformational transitions of the virion, the host range of poliovirus may be determined by the ability of virions to undergo transitions catalyzed by cell receptors.

Alleles↗

PubMed2005

Foot-and-mouth disease: host range and pathogenesis.

In this chapter the host range of foot-and-mouth disease (FMD) under natural and experimental conditions is reviewed. The routes and sites of infection, incubation periods and clinical and pathological findings are described and highlighted in relation to progress in understanding the pathogenesis of FMD.

Animals↗

PubMed1984

Genetic evidence for the direction of transcription of the trfA gene of broad host range plasmid RK2.

For replication of broad host range plasmid RK2 in Escherichia coli two regions of the plasmid genome are essential, oriV RK2 between 12.0 and 12.7 kb on the genome (defined clockwise from the unique EcoRI site) and trfA, located between 16.0 and 17.4 kb, which provides a trans-acting product necessary for oriV RK2 function. The properties of an insertion mutant of a mini-RK2/ColE1 hybrid plasmid suggest that the trfA promoter lies clockwise from the 17.4 kb RK2 coordinate. Fusion of the trfA gene lacking its normal promoter to the E. coli trpE gene in a hybrid plasmid confirmed that trfA is transcribed anticlockwise, towards the Tcr gene of RK2. Repression of trpE promoter activity in these fusions showed that replication of an RK2 replicon can be regulated by varying the level of trfA gene expression. The results also indicate the presence of a transcription unit running anticlockwise through the trfB region located between 54.0 and 56.0 kb on the RK2 genome.

Escherichia coli↗

PubMed1989

Identification and sequence of the basic replication region of a broad-host-range plasmid isolated from Thiobacillus ferrooxidans.

The minimum region required for replication of the broad-host-range Thiobacillus ferrooxidans plasmid pTF-FC2 in Escherichia coli was shown to be contained on a 2.05-kilobase fragment of DNA. A 184-base-pair fragment that was required in cis for plasmid replication was identified. This region was also involved in plasmid incompatibility. Nucleotide sequencing of this region revealed three perfectly conserved 22-base-pair tandemly repeated sequences. A comparison of this region with the equivalent region of the broad-host-range plasmid R1162 showed that the repeated sequences had 60% nucleotide homology. The 106-base-pair region immediately adjacent to the repeated sequences was 75% homologous. These plasmids were compatible.

Base Sequence↗

PubMed1999

A comparative analysis of the phosphorylation and biochemical properties of wild type and host range variant DNA binding proteins of human adenovirus 5.

Specific mutation of the DNA binding protein (DBP) of human adenovirus types 2 and 5 can extend the host range of these viruses to simian cells. These mutations replace histidine at position 130 in the highly phosphorylated N-terminal domain of DBP with a potentially phophorylatable tyrosine residue. To investigate the possibility that alternative phosphorylation might contribute to the functional differences between wild type (wt) and host range (hr) DBP molecules, radiolabeled proteins were compared by partial proteolysis and tyrosine phosphorylation was analyzed. These studies confirmed the previous tentative assignment of a chymotrypsin-sensitive site at position 121 of DBP. No host range-specific tyrosine phosphorylation was detected, and no gross difference in the extent of phosphorylation between wt and hr DBP was observed. However, the cleaved N-terminal domains of wt and hr DBP exhibited different sensitivities to further chymotryptic digestion in vitro and different fragmentation patterns, suggesting that they might have different conformations. Such a difference could underlie the differing ability of these proteins to support Ad replication in simian cells.

Adenoviruses, Human↗

PubMed1992

Isolation and molecular characterization of a novel broad-host-range plasmid from Bordetella bronchiseptica with sequence similarities to plasmids from gram-positive organisms.

A 2.6 kb plasmid, named pBBR1, was isolated from Bordetella bronchiseptica S87. After insertion of an antibiotic resistance marker, this plasmid could be transferred into Escherichia coli, Bordetella pertussis, B. bronchiseptica, Vibrio cholerae, Rhizobium meliloti, and Pseudomonas putida by transformation or conjugation. Conjugation was possible only when the IncP group transfer functions were provided in trans. As shown by incompatibility testing, pBBR1 does not belong to the broad-host-range IncP, IncQ or IncW groups. DNA sequence analysis revealed two open reading frames: one was called Rep, involved in replication of the plasmid, and the other, called Mob, was involved in mobilization. Both the amino-terminal region of Mob and its promoter region show sequence similarities to Mob/Pre proteins from plasmids of Gram-positive bacteria. In spite of these sequence similarities, pBBR1 does not replicate via the rolling-circle mechanism commonly used by small Gram-positive plasmids. We therefore speculate that pBBR1 may combine a mobilization mechanism of Gram-positive organisms with a replication mechanism of Gram-negative organisms. Determination of the plasmid copy number in E. coli and B. pertussis indicated that pBBR1 has a rather high copy number, which, in conjunction with its small size and broad host range, renders it particularly interesting for studies of broad-host-range replicons and for the development of new cloning vectors for a wide range of Gram-negative bacteria.

Amino Acid Sequence↗

PubMed1992

Promoters of the broad host range plasmid RK2: analysis of transcription (initiation) in five species of gram-negative bacteria.

A broad host range cloning vector was constructed, suitable for monitoring promoter activity in diverse Gram-negative bacteria. This vector, derived from plasmid RSF1010, utilized the firefly luciferase gene as the reporter, since the assay for its bioluminescent product is sensitive, and measurements can be made without background from the host. Twelve DNA fragments with promoter activity were obtained from broad host range plasmid RK2 and inserted into the RSF1010 derived vector. The relative luciferase activities were determined for these fragments in five species of Gram-negative bacteria. In addition, four promoters were analyzed by primer extension to locate transcriptional start sites in each host. The results show that several of the promoters vary substantially in relative strengths or utilize different transcriptional start sites in different bacteria. Other promoters exhibited similar activities and identical start sites in the five hosts examined.

Base Sequence↗

PubMed1990

Risk Assessment Studies: Detailed Host Range Testing of Wild-Type Cabbage Moth, Mamestra brassicae (Lepidoptera: Noctuidae), Nuclear Polyhedrosis Virus.

The host range of a multiply enveloped nuclear polyhedrosis virus (NPV) (Baculoviridae) isolated from the cabbage moth, Mamestra brassicae (Lepidoptera: Noctuidae), was determined by challenging a wide range of insect species with high (10 polyhedral inclusion bodies) and low (10 polyhedral inclusion bodies) doses of the virus. The identity of the progeny virus was confirmed by dot blotting. Analysis of 50% lethal dose was carried out on selected species, and the progeny virus was identified by using restriction enzyme analysis and Southern blotting. Other than the Lepidoptera, none of the species tested was susceptible to M. brassicae NPV. Within the Lepidoptera, M. brassicae NPV was infective to members of four families (Noctuidae, Geometridae, Yponomeutidae, and Nymphalidae). Of 66 lepidopterous species tested, M. brassicae NPV was cross-infective to 32 of them; however, 91% of the susceptible species were in the Noctuidae. The relevance of host range data in risk assessment studies is discussed.

Journal Article↗

PubMed1989

Complete nucleotide sequence and gene organization of the broad-host-range plasmid RSF1010.

We present the complete nucleotide sequence of RSF1010, a naturally occurring broad-host-range plasmid belonging to the Escherichia coli incompatibility group Q and encoding resistance to streptomycin and sulfonamides. A molecule of RSF1010 DNA consists of 8684 bp and has a G + C content of 61%. Analysis of the distribution of translation start and stop codons in the sequence has revealed the existence of more than 40 open reading frames potentially capable of encoding polypeptides of 60 or more amino acids. To date, products of eleven such potential RSF1010 genes have been identified through the application of controlled expression vector systems, and for eight of them, the reading frame has been confirmed by N- and/or C-terminal amino acid sequence determinations on the purified proteins. The sequencing results are discussed in relation to the systems of replication, host range, conjugal mobilization and antibiotic resistance determinants associated with the RSF1010 plasmid.

Amino Acid Sequence↗