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PubMed2004

Plasmid evolution and interaction between the plasmid addiction stability systems of two related broad-host-range IncQ-like plasmids.

Plasmid pTC-F14 contains a plasmid stability system called pas (plasmid addiction system), which consists of two proteins, a PasA antitoxin and a PasB toxin. This system is closely related to the pas of plasmid pTF-FC2 (81 and 72% amino acid identity for PasA and PasB, respectively) except that the pas of pTF-FC2 contains a third protein, PasC. As both pTC-F14 and pTF-FC2 are highly promiscuous broad-host-range plasmids isolated from bacteria that share a similar ecological niche, the plasmids are likely to encounter each other. We investigated the relative efficiencies of the two stability systems and whether they had evolved apart sufficiently for each pas to stabilize a plasmid in the presence of the other. The three-component pTF-FC2 pas was more efficient at stabilization of a heterologous tester plasmid than the two component pas of pTC-F14 in Escherichia coli host cells (+/- 92% and +/- 60% after 100 generations, respectively). The PasA antidote of each pas was unable to neutralize the PasB toxin of the other plasmid. The pas proteins of each plasmid autoregulated their own expression as well as that of the pas of the other plasmid. The pas of pTF-FC2 was more effective at repressing the pas operon of pTC-F14 than the pas of pTC-F14 was able to repress itself or the pas of pTF-FC2. This increased efficiency was not due to the PasC of pTF-FC2. The effect of this stronger repression was that pTF-FC2 displaced pTC-F14 when the two plasmids were coresident in the same E. coli host cell. Plasmid curing resulted in the arrest of cell growth but did not cause cell death, and plasmid stability was not influenced by the E. coli mazEF genes.

Antidotes↗
PubMed1995

Host growth temperature and a conservative amino acid substitution in the replication protein of pPS10 influence plasmid host range.

pPS10 is a replicon isolated from Pseudomonas syringe pv. savastanoi that can be established at 37 degrees C efficiently in Pseudomonas aeruginosa but very inefficiently in Escherichia coli. The establishment of the wild-type pPS10 replicon in E. coli is favored at low temperatures (30 degrees C or below). RepA protein of pPS10 promotes in vitro plasmid replication in extracts from E. coli, and this replication depends on host proteins DnaA, DnaB, DnaG, and SSB. Mutant plasmids able to efficiently replicate in E. coli at 37 degrees C were obtained. Three of four mutants whose mutations were mapped show a conservative Ala-->Val change in the amino-terminal region of the replication protein RepA. Plasmids carrying this mutation maintain the capacity to replicate in P. aeruginosa and have a fourfold increase in copy number in this host. The mutation does not substantially alter the autoregulation mediated by RepA. These results show that the physiological conditions of the host as well as subtle changes in the plasmid replication protein can modulate the host range of the pPS10 replicon.

Amino Acid Sequence↗
PubMed2004

Amino acid substitutions and an insertion in the spike glycoprotein extend the host range of the murine coronavirus MHV-A59.

The murine coronavirus [murine hepatitis virus (MHV)] is limited to infection of susceptible mice and murine cell lines by the specificity of the spike glycoprotein (S) for its receptor, murine carcinoembryonic antigen cell adhesion molecule 1a (mCEACAM1a). We have recently shown that 21 aa substitutions and a 7-aa insert in the N-terminal region of S are associated with the extended host range of a virus variant derived from murine cells persistently infected with the A59 strain of MHV (MHV-A59). We used targeted RNA recombination (TRR) to generate isogenic viruses that differ from MHV-A59 by the 21 aa substitutions or the 7-aa insert in S. Only viruses with both the 21 aa substitutions and the 7-aa insert in S infected hamster, feline, and monkey cells. These viruses also infected murine cells in the presence of blocking anti-mCEACAM1a antibodies. Thus, relatively few changes in the N-terminal region of S1 are sufficient to permit MHV-A59 to interact with alternative receptors on murine and non-murine cells.

Amino Acid Substitution↗
PubMed1985

Analysis of wild-derived mice for Fv-1 and Fv-2 murine leukemia virus restriction loci: a novel wild mouse Fv-1 allele responsible for lack of host range restriction.

Wild-derived mice originally obtained from Asia, Africa, North America, and Europe were typed for in vitro sensitivity to ecotropic murine leukemia viruses and for susceptibility to Friend virus-induced disease. Cell cultures established from some wild mouse populations were generally less sensitive to exogenous virus than were cell cultures from laboratory mice. Wild mice also differed from inbred strains in their in vitro sensitivity to the host range subgroups defined by restriction at the Fv-1 locus. None of the wild mice showed the Fv-1n or Fv-1b restriction patterns characteristic of most inbred strains, several mice resembled the few inbred strains carrying Fv-1nr, and most differed from laboratory mice in that they did not restrict either N- or B-tropic murine leukemia viruses. Analysis of genetic crosses of Mus spretus and Mus musculus praetextus demonstrated that the nonrestrictive phenotype is controlled by a novel allele at the Fv-1 locus, designated Fv-10. The wild mice were also tested for sensitivity to Friend virus complex-induced erythroblastosis to type for Fv-2. Only M. spretus was resistant to virus-induced splenomegaly and did not restrict replication of Friend virus helper murine leukemia virus. Genetic studies confirmed that this mouse carries the resistance allele at Fv-2.

Alleles↗
PubMed1984

Nucleotide sequence and functional properties of DNA encoding incompatibility in the broad host-range plasmid R1162.

A 370 base pair (bp) fragment of R1162 DNA encoding the incompatibility determinant has been cloned and sequenced. The DNA is located between 6.1 and 6.5 on the R1162 map, near the origin of replication. The sequence contains three perfectly conserved 20 bp direct repeats, with 11 bp of this sequence repeated a fourth time. The direct repeat unit shows some homology with that of another, unrelated broad host-range plasmid, RK2. The cloned DNA has two other properties: it lowers the copy number of R1162 when cloned into this plasmid, and it is required in cis for replication of R1162 satellite plasmids.

Base Sequence↗
PubMed2006

Functional analysis of replication and stability regions of broad-host-range conjugative plasmid CTX-M3 from the IncL/M incompatibility group.

Plasmid CTX-M3 (89 kb) isolated from Citrobacter freundii from a Warsaw hospital is a mosaic plasmid with replication functions 100% identical with those of pMU407.1 of the IncL/M group, conjugative operons with up to 60% homology to ColIb-P9 (IncI) and stability functions originating either from NR1(R100) (IncFII) or ColIb-P9 /R1/NR1 plasmids. We established the broad-host-range for pCTX-M3 and defined its minireplicon in Escherichia coli. We analyzed the role of stability cassettes and showed that the par operon consists of three orfs parA (stbA), parB (stbB) and nuc with a centromere-like region located upstream of the operon. Deletion of the par operon strongly destabilized pCTX-M3 despite the presence of the pemIK toxin-antidote system identical to that on NR1(R100) plasmids. Deletion of the pemIK operon had no effect on plasmid stability.

Amino Acid Sequence↗
PubMed1984

High-efficiency gene transfer into mammalian cells: generation of helper-free recombinant retrovirus with broad mammalian host range.

We have constructed a chimeric retrovirus genome containing ecotropic gag-pol sequences from Moloney murine leukemia virus and envelope sequences derived from the amphotropic virus 4070A. This reconstructed genome, termed pMAV-psi-, lacks the psi site required for encapsidation of the viral genome. NIH 3T3 cells transfected with pMAV-psi-, called psi-AM lines, are capable of producing high titer stocks of helper-free recombinant retrovirus with amphotropic host range after transfection with recombinant retroviral vectors carrying the neomycin phosphotransferase gene. Most transfected psi-AM cells remain helper-free, even after months in culture. psi-AM virus stocks infect nearly all human and murine cell lines tested thus far, as assayed by resistance to the neomycin analogue G418. Southern and RNA blot analyses of psi-AM-infected human cells show that recombinant murine retroviruses integrate randomly into genomic DNA as normal proviruses and express high levels of the subgenomic and genomic viral messages in the expected stoichiometry of 1:1.

Animals↗
PubMed1999

Rhizobium sp. strain NGR234 and R. fredii USDA257 share exceptionally broad, nested host ranges.

Genetically, Rhizobium sp. strain NGR234 and R. fredii USDA257 are closely related. Small differences in their nodulation genes result in NGR234 secreting larger amounts of more diverse lipo-oligosaccharidic Nod factors than USDA257. What effects these differences have on nodulation were analyzed by inoculating 452 species of legumes, representing all three subfamilies of the Leguminosae, as well as the nonlegume Parasponia andersonii, with both strains. The two bacteria nodulated P. andersonii, induced ineffective outgrowths on Delonix regia, and nodulated Chamaecrista fasciculata, a member of the only nodulating genus of the Caesalpinieae tested. Both strains nodulated a range of mimosoid legumes, especially the Australian species of Acacia, and the tribe Ingeae. Highest compatibilities were found with the papilionoid tribes Phaseoleae and Desmodieae. On Vigna spp. (Phaseoleae), both bacteria formed more effective symbioses than rhizobia of the "cowpea" (V. unguiculata) miscellany. USDA257 nodulated an exact subset (79 genera) of the NGR234 hosts (112 genera). If only one of the bacteria formed effective, nitrogen-fixing nodules it was usually NGR234. The only exceptions were with Apios americana, Glycine max, and G. soja. Few correlations can be drawn between Nod-factor substituents and the ability to nodulate specific legumes. Relationships between the ability to nodulate and the origin of the host were not apparent. As both P. andersonii and NGR234 originate from Indonesia/Malaysia/Papua New Guinea, and NGR234's preferred hosts (Desmodiinae/Phaseoleae) are largely Asian, we suggest that broad host range originated in Southeast Asia and spread outward.

Carbohydrate Sequence↗
PubMed1985

Adenovirus 5 early region 1A host range mutants hr3, hr4, and hr5 contain point mutations which generate single amino acid substitutions.

The early region 1A (E1A) gene of adenovirus 5 encodes two proteins, 289AA and 243AA, which are translated from mRNAs of 13S and 12S, respectively. These two E1A proteins are identical except for an internal stretch of 46 amino acids unique to the larger protein. The 289AA protein activates transcription from promoters of other early adenoviral genes. The adenovirus type 5 host range mutants hr3, hr4, and hr5 are unable to activate transcription of these early viral genes. We show here that hr3, hr4, and hr5 each contain a distinct missense mutation in the E1A gene. We first localized the mutations in a series of constructed wild-type-hr hybrid E1A genes by using a biological assay which can discriminate between functional and nonfunctional E1A proteins. We then identified the mutations by DNA sequencing. In hr3 lysine replaced methionine at position 176, and in hr4 phenylalanine replaced leucine at position 173; both substitutions occurred in the region unique to the 289AA protein. In hr5, due to the splicing patterns of the two mRNAs, asparagine replaced serine as the last amino acid in the unique region of the 289AA protein at position 185, while aspartic acid replaced glycine at position 139 in the 243AA protein, which is the last amino acid common to both proteins before the unique region. These results substantiate the role of the 289AA protein in transcriptional activation and underscore the importance of the unique region as the basis of the functional difference between the two E1A proteins. Implications as to how these mutations affect the structure and function of the E1A proteins in transcriptional activation and transformation are discussed.

Acetyltransferases↗
PubMed1992

Human immunodeficiency virus (HIV) type 1 can superinfect HIV-2-infected cells: pseudotype virions produced with expanded cellular host range.

In studies on viral interference, cloned T-cell lines chronically infected with human immunodeficiency virus (HIV) type 1 or HIV-2 were inoculated with several strains of these two AIDS retrovirus subtypes. HIV-2UC1-infected cells, which still express the CD4 receptor, could be superinfected with a variety of HIV-1 and HIV-2 strains. This event was accompanied by cytopathic effects in the cells and production of pseudotype virions with an expanded cellular host range. HIV-1- or HIV-2-infected clonal cell lines, which did not express CD4, could not be superinfected by any HIV strains but were coinfected after transfection of molecular clones into the persistently infected cells. These observations indicate that viral interference with HIV occurs at the cell surface and involves a down-modulation of the CD4 molecule. If the CD4 protein is expressed, superinfection can take place, and phenotypically mixed virus particles are produced. Since HIV-1 and HIV-2 dually infected individuals have been detected, these in vitro observations may have relevance to the in vivo state.

B-Lymphocytes↗
PubMed1982

Replication of the broad host range plasmid RSF1010 in cell-free extracts of Escherichia coli and Pseudomonas aeruginosa.

Replication of exogenous RSF1010 DNA can be carried out by soluble enzyme systems from Escherichia coli and Pseudomonas aeruginosa. It requires the function of RSF1010-encoded replication protein(s), which is not expressed in extracts from plasmid-free bacteria. In contrast to previously described in vitro systems for plasmid replication, initiation of RSF1010 DNA synthesis is independent of transcription catalyzed by host RNA polymerase. This is indicated by the insensitivity of RSF1010 replication to rifampicin as well as to RNA polymerase antibodies. It is proposed that a host RNA polymerase transcription-independent initiation mechanism might be a general property of broad host range plasmids.

Anti-Bacterial Agents↗
PubMed1992

Foreign gene expression by a baculovirus vector with an expanded host range.

A nuclear polyhedrosis virus (NPV) (Baculoviridae)-based gene expression system was improved by DNA recombination. The BmN cell line established from Bombyx mori and the Sf21 cell line established from Spodoptera frugiperda are non-permissive for Autographa californica multicapsid NPV (AcMNPV) and B. mori NPV (BmNPV) replication, respectively. After cotransfection of AcMNPV DNA and BamHI-digested BmNPV DNA into Sf21 cells, progeny viruses were isolated by plaque purification on BmN cell monolayers and the host specificity of one viral isolate was analysed. The virus had a wider host range, and replicated and produced polyhedra in Sf21 cells, BmN cells and larvae of the silkworm, B. mori. DNA restriction endonuclease analysis showed that the isolate was a hybrid of AcMNPV and BmNPV. Using the AcMNPV transfer vector pAcYM1 a portion of the polyhedrin gene of the hybrid virus was replaced with the coding region of the firefly luciferase gene, producing a recombinant virus. The latter expressed firefly luciferase in both cell lines and in silkworm larvae under the control of the polyhedrin promoter.

Baculoviridae↗
PubMed1986

Mutation in the VP-1 gene is responsible for the extended host range of a monkey B-lymphotropic papovavirus mutant capable of growing in T-lymphoblastoid cells.

Monkey B-lymphotropic papovavirus (LPV) replicates only in B-lymphoblastoid cells, whereas the LPV mutant 76 (LPV-76) can grow in either B- or T-lymphoblastoid cells. The nucleotide sequence of the wild-type LPV PstI B segment was compared with that of LPV-76 PstI-B, within which the mutation responsible for the extended host range had been located. The VP-1 coding region of LPV-76 was found to have mutations, single-base substitutions causing three amino acid substitutions.

Animals↗
PubMed2003

The transferrin receptor genes of Trypanosoma equiperdum are less diverse in their transferrin binding site than those of the broad-host range Trypanosoma brucei.

Trypanosoma brucei and T. equiperdum infect the mammalian bloodstream and tissues. T. brucei is transmitted by tsetse flies between an extremely large range of mammals in sub-Saharan Africa. In contrast, T. equiperdum is restricted to equines, where it is transmitted as a venereal disease. Both species evade immune destruction by changing their variant surface glycoprotein (VSG), encoded in a telomeric VSG expression site. T. brucei has about 20 VSG expression sites, and it has been proposed that their genetic diversity plays a role in host adaptation. Two expression site-associated genes ESAG6 and ESAG7, encode variable transferrin receptor subunits allowing trypanosomes to internalize polymorphic transferrin molecules from different mammals. We investigated if there was a correlation between the size of the trypanosome host range and the degree of ESAG6 genetic diversity. Both T. equiperdum and T. brucei appear to have approximately similar numbers of ESAG6, however, the genetic diversity of the ESAG6 family varies in the two species. We sequenced 114 T. equiperdum ESAG6 genomic clones, resulting in the isolation of 10 T. equiperdum ESAG6 variants. The T. equiperdum ESAG6 genes were less genetically diverse than those of T. brucei in regions known to play a role in transferrin binding. This indicates that ESAG6 genetic diversity playing a role in host adaptation could have been lost in the absence of selection pressure. There was also evidence of positive selection ( d(N) /d(S) = approximately 5) acting on other ESAG6 regions not involved in transferrin binding, perhaps due to antigenic variation of these surface molecules.

Amino Acid Sequence↗
PubMedDate not supplied

Evaluation of acidic heteropolysaccharide structures in Rhizobium leguminosarum biovars altered in nodulation genes and host range.

1H-NMR spectroscopy showed that the extracellular heterpolysaccharides (EPS) from derivatives of Rhizobium leguminosarum bv. trifolii ANU843 altered in pSym nod composition or function (transposon insertions, deletion of pSym, induction by flavone, and introduction of cloned pSym nod regions from ANU843 and R. l. bv. viciae 248 on recombinant plasmids into the pSym-cured background of ANU843) differed only in 3-hydroxybutyrate stoichiometry per octaglycosyl unit. This change in EPS was likely to be an indirect effect of altered growth during expression of pSym nod genes in the presence of the flavone. No modifications were found in EPS made by R. l. bv. phaseoli 8002 when its resident pSym was deleted or replaced with pSym from R. l. bv. viciae 248, or with a derivative of this pSym lacking the host-specific nodulation genes nodFELMNTO. Thus, although certain O-acyl noncarbohydrate substitutions in EPS are affected by pSym nod genes (including the ones that determine host range) in certain backgrounds of R. leguminosarum, this change does not occur universally among all strains of R. leguminosarum. We conclude that the structure of the acidic EPS does not control host-specific nodulation of white clover, hairy vetch, and beans for the strains of R. leguminosarum tested here.

Gene Expression↗
PubMed1987

Genetic analysis of the gentamicin resistance region of pPH1JI and incorporation into a wide host range cloning vehicle.

A region of the IncP plasmid pPH1JI encoding resistance to gentamicin, spectinomycin, and streptomycin was characterized by subcloning, deletion, and insertion mutagenesis. Approximate locations of these resistance determinants were established. A 1.6-kb HindIII-SphI segment of this region expresses gentamicin resistance (Gmr) in Escherichia coli when inserted into various plasmid vectors; this DNA segment encodes a polypeptide of 17.5 kDa. Incorporation of this fragment into an IncP cloning vehicle produced a Gmr wide host range vector, pRAR209, which confers levels of Gmr comparable to those expressed by pPH1JI in E. coli, Agrobacterium tumefaciens, and Rhizobium meliloti.

Cloning, Molecular↗
PubMed1990

A 36 nucleotide deletion mutation in the coding region of the NS1 gene of an influenza A virus RNA segment 8 specifies a temperature-dependent host range phenotype.

Previously a spontaneous 36 nucleotide deletion in the coding region of NS1 was detected in the NS gene of a reassortant virus (CR43-3) recovered from a dual infection by the influenza A/Ann Arbor/6/60 cold-adapted (ca) mutant and wild-type (wt) influenza A/Alaska/6/77 (H3N2). The hemagglutinin, neuraminidase and NS genes were derived from the wild type virus parent while the other 5 genes were derived from the ca parent. The CR43-3 reassortant virus exhibited: (i) a host range (hr) phenotype, i.e. the reassortant replicated efficiently in avian cells in tissue culture but failed to grow in mammalian (MDCK) cell culture and (ii) an attenuation (att) phenotype, i.e., the reassortant was restricted in replication in the upper and lower respiratory tract of ferrets and hamsters. Since the CR43-3 reassortant possessed 5 genes from the ca parent which are each known to contain one or more mutations, it was not possible to assign the hr and att phenotypes solely to the NS deletion mutant gene. In order to determine the phenotype(s) specified solely by the mutant NS gene, it was transferred into a reassortant virus (143-1) which derived its seven other genes from the homologous wild type A/Alaska/6/77 virus. The deletion mutant NS gene specified only a partial hr phenotype manifested by a reduction in plaque size in MDCK tissue, but not a reduction in plaque number. Thus, the complete hr manifested by the CR43-3 parent virus is specified by the mutant NS1 gene acting in concert with one or more genes derived from the ca virus. The clone 143-1 virus exhibited the ts phenotype and was restricted in plaque formation at 37 degrees C in MDCK cells, a level of temperature sensitivity previously shown with other ts mutants to correlate with significant restriction of viral replication in the lower respiratory tract of hamsters. However, the clone 143-1 virus grew almost as well as the wt virus in the upper and lower respiratory tracts of hamsters and chimpanzees and thus did not possess the att phenotype. The finding that the ts phenotype was not manifest in vivo in animals with a 37 degrees C core temperature indicates that the mutated NS1 gene specifies a host dependent ts phenotype with replication restricted in vitro (MDCK tissue culture) at 37 degrees C but not in vivo in the lungs of hamsters and chimpanzees. ts+ virus was readily recovered from infected hamsters and chimpanzees indicating that the ts phenotype specified by the 36-base deletion was not stable following replication in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1988

A broad-host-range shuttle system for gene insertion into the chromosomes of gram-negative bacteria.

A deletion derivative of transposon Tn7 containing the Escherichia coli lacZY genes as a selectable marker for insertion of foreign DNA into the chromosomes of soil bacteria was improved to facilitate the cloning of additional genes and their insertion by this element. This report describes a series of plasmid vectors that enable this cloning to be carried out in small, high-copy, narrow host-range plasmids. The final Tn element can then be easily moved (by transposition) without further use of restriction enzymes, to plasmids suitable for delivering it to the bacterial chromosome. The very high specificity for insertion of Tn7 into single locations in bacterial chromosomes has been exploited in the construction of a shuttle system for delivering these Tn7 elements.

Cloning, Molecular↗