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PubMed2002

Broad-host-range cre-lox system for antibiotic marker recycling in gram-negative bacteria.

Complete genome sequences are now available for many bacterial species that lack sophisticated genetic tools. We describe the development of a broad-host-range cre-lox system that allows antibiotic marker recycling in a variety of gram-negative bacteria. This system consists of an allelic exchange vector bearing a kanamycin cassette flanked by loxP sites and a tetracycline-resistant IncP plasmid that provides expression of the Cre recombinase. We demonstrate this system by generating unmarked deletions of genes in two different bacteria, Methylobacterium extorquens AM1 and Burkholderia fungorum LB400. This new antibiotic marker recycling system offers the possibility of creating unmarked mutants in a wide variety of gram-negative bacteria. Furthermore, marker recycling allows the generation of strains bearing multiple genetic manipulations in organisms for which few antibiotic markers are currently available.

Alleles↗
PubMed2000

A broad-host-range plasmid for isolating mobile genetic elements in gram-negative bacteria.

Plasmid pGBG1 was constructed to isolate mobile genetic elements in a wide variety of gram-negative bacteria. The mutation target, carried on a broad-host-range vector, allows positive selection for tetracycline resistance. In tests using several gram-negative bacteria we could detect transposition events of either insertion sequences or transposons. A new insertion sequence (IS) element was identified in Ralstonia eutropha.

Agrobacterium tumefaciens↗
PubMed1993

Use of helper cells with two host ranges to generate high-titer retroviral vectors.

We have recently described Avian Leukosis Virus (ALV)-based packaging cell lines that can produce helper-free ALV-based retrovirus vectors with A, B, C, and E envelope host ranges. Here, we report that lacZ retroviral vectors of subgroup C or E can infect helper cells of subgroup A (Isolde) which are then able to produce high titers of lacZ recombinant viruses of subgroup A. Superinfection of helper cells by lacZ recombinant virus was performed by cocultivating packaging cells with two subgroup specificities (A and E), but this did not result in increased recombinant virus titers. This "ping-pong" process caused the emergence of replication-competent (RC) viruses which are shown to result from recombination between the viral sequences of the helper cell lines and the cis-acting sequences of the lacZ recombinant virus.

Avian Leukosis Virus↗
PubMed2001

Construction and Use of Broad Host Range Mercury and Arsenite Sensor Plasmids in the Soil Bacterium Pseudomonas fluorescens OS8.

We have generated new sensors for the specific detection and studies of bioavailability of metals by engineering Pseudomonas fluorescens with reporter gene systems. One broad host range mercury (pTPT11) and two arsenite (pTPT21 and pTPT31) sensor plasmids that express metal presence by luminescence phenotype were constructed and transferred into Escherichia coli DH5a and Pseudomonas fluorescens OS8. The maximal induction was reached after 2 h of incubation in metal solutions at room temperature (22 degrees C). In optimized conditions the half maximal velocity of reaction was achieved at acidic pH using a d-luciferin substrate concentration that was nearly sixfold lower for P. fluorescens OS8 than for E. coli DH5a. When using a luciferin concentration (150 mM) that was optimal for E. coli the luminescence declined rapidly in the case of Pseudomonas, for which the substrate level 25 mM gave a stable reading between about 20 min and 3 h. The ability of the strain OS8 to quantitatively detect specific heavy metals in spiked soil and soil extracts is as good, or even better in being a real-time reporter system, than that of a traditional chemical analysis. The Pseudomonas strain used is an isolate from pine rhizosphere in oil and heavy metal contaminated soil. It is also a good humus soil colonizer and is therefore a good candidate for measuring soil heavy metal bioavailability.

Journal Article↗
PubMed1992

The transformation-defective adenovirus 12 host range mutant CS-1 lacks the E1a-specific 9.5S mRNA and contains a second deletion in E1b.

We have further analyzed structure and expression of the E1 region of the transformation-defective adenovirus 12 (Ad12) host range mutant CS-1. Using cDNA polymerase chain reaction analysis, the E1a region was found to give raise to five transcripts analogous to the Ad12wt 13S, 12S, 11S, 10S species and the normal 9S mRNA. Due to loss of a splice acceptor site at position 852, the Ad12wt-specific 9.5S transcript cannot be synthesized by the mutant CS-1. The fact that the virus is, however, completely viable indicates that this mRNA is dispensable for lytic growth of Ad12. Besides the deletions in E1a described previously, a second deletion of 31 base pairs was found in the E1b gene. It affects the start site of the E1b-specific mRNAs and destroys or eliminates the AUG sequence for the E1b 19-kD protein. As shown earlier, normal E1b mRNA expression is found in mutant-infected cells, but no 19-kD E1b protein was immunoprecipitated by an E1-specific rat tumor serum.

Adenovirus Early Proteins↗
PubMed2006

Complete genome sequence of the broad host range single-stranded RNA phage PRR1 places it in the Levivirus genus with characteristics shared with Alloleviviruses.

Single-stranded RNA (ssRNA) bacteriophages of the family Leviviridae infect gram-negative bacteria. They are restricted to a single host genus. Phage PRR1 is an exception, having a broad host range due to the promiscuity of the receptor encoded by the IncP plasmid. Here we report the complete genome sequence of PRR1. Three proteins homologous with those of other ssRNA phages, i.e., maturation, coat, and replicase proteins, were identified. A fourth protein has a lysis function. Comparison of PRR1 with other members of the Leviviridae family places PRR1 in the genus Levivirus with some characteristics more similar to those of members of the genus Allolevivirus.

Allolevivirus↗
PubMed1997

The poison-antidote stability system of the broad-host-range Thiobacillus ferrooxidans plasmid pTF-FC2.

In plasmid pTF-FC2, three small open reading frames (ORFs) are situated between the repB (primase) gene and the repA (helicase) gene of its IncQ-type replicon. Disruption of each of the three ORFs followed by tests for plasmid stability and host cell growth indicated that the ORFs encoded a poison-antidote plasmid stability system. The three genes were named pasA, pasB and pasC (plasmid addiction system), in which PasA is the antidote, PasB the toxin and PasC a protein that appears to enhance the ability of the antidote to neutralize the toxin. Disruption of the pasA gene resulted in two different spontaneous deletions, which inactivated the stability system but did not alter the host range or plasmid copy number. This indicated that the three small ORFs were not involved in plasmid replication. When placed behind a tac promoter, induction of pasB was found to be highly lethal to host cells, which suggests that the Pas system acts by killing plasmid-free host cells rather than by retarding the growth of plasmid-free segregants, as occurs in the ParD system of R1. In spite of this, the presence of the Pas poison-antidote system resulted in a relatively modest threefold stabilization of the pTF-FC2 host replicon and a similar increase in the stabilization of an unstable heterologous R1 plasmid replicon. The Pas system is a poison-antidote plasmid stability module, which appears to have become integrated within the pTF-FC2 replicon module.

Amino Acid Sequence↗
PubMed1991

Altered host range of HIV-1 after passage through various human cell types.

HIV-1 strains, including a molecularly cloned isolate, that had been passaged through different cell types adapted to faster growth in the same cell type and displayed a different host cell tropism. The only change in viral proteins revealed by immunoblot analyses was the molecular size of the envelope glycoprotein gp120 that varied for viruses recovered from the different infected cells. The alteration in size was most likely the result of modification of gp120. Host range differences were also observed for a molecularly cloned HIV-1 strain when passed through the peripheral white blood cells from different individuals. Thus, this phenomenon could have clinical relevance in HIV pathogenesis.

Blotting, Southern↗
PubMed1988

Isolation and characterization of adenovirus type 12 E1 host-range mutants defective for growth in nontransformed human cells.

In order to define the functions of human adenovirus type 12 (Ad12) early region 1 (E1) products in lytic infection and oncogenic transformation we have isolated and phenotypically characterized a set of host-range (hr) mutants of this serotype. These mutants grow efficiently upon HER3 cells, which contain and express type 12 E1 genes, but are restricted for growth upon A549 carcinoma and HeLa cells. Inter- and intratypic complementation analysis, marker-rescue mapping, and DNA sequence analysis have assigned some of the mutations to E1A sequence, and some to the reading frame encoding the E1B 54-kDa (482R) protein. Phenotypic analysis of the E1B mutants in particular has revealed some interesting, and in some cases surprising, findings relating to the roles of that protein in virus-cell interactions. This Ad12 gene product is required, either directly or indirectly, for efficient viral DNA replication in A549 and HeLa cells, unlike its counterpart in type 5 virus. Surprisingly, however, despite the severe defect in viral DNA replication, the synthesis of a few species of viral late proteins continues in cells infected by some of the E1B mutants. In contrast, none of these mutants brings about the inhibition of host-cell protein synthesis characteristic of wild-type virus infection, and with some E1B mutants no viral late proteins are made. Further, in a separate study reported elsewhere, we have demonstrated that the E1B 54-kDa product may also be involved, either directly or indirectly, in positive regulation of both E1A and E1B 19-kDa (163R) gene expression. The molecular and/or physiological bases for these various effects remain to be determined, but our initial results suggest that the E1B 54-kDa protein may carry out multiple regulatory functions during the viral life cycle.

Adenoviruses, Human↗
PubMed1993

Host range selection of vaccinia recombinants containing insertions of foreign genes into non-coding sequences.

A simple yet powerful selection system was developed for the insertion of foreign genes in vaccinia virus. The selection system utilizes the vaccinia virus K1L (29K) host range gene which is located in HindIII M. This gene is necessary for growth in RK-13 cells but not in BSC40 or CV-1 cells. A vaccinia mutant (vAbT33) unable to grow on RK-13 cells was constructed having sequences at the 3' end of the K1L gene and the adjacent M2L gene deleted and replaced with the beta-galactosidase gene regulated by the BamHI F (F7L) promoter. A recombination plasmid containing the hepatitis B surface (HBs) antigen gene regulated by the M2L promoter and the complete sequence of the K1L gene was used to insert the HBs gene into vAbT33. The M2L negative K1L positive recombinant was easily isolated in two rounds of plaque purification by plating the virus on RK-13 cell monolayers. The K1L gene selection system allows the isolation of recombinants arising at frequencies as low as 1/100,000. It was noted that recombinants containing vaccinia sequence duplications (promoters) resulted in intragenomic recombinations that eliminated all sequences between the duplications. A second recombination plasmid was constructed that allowed insertion into the vaccinia genome without the loss of vaccinia coding sequences. This was achieved by insertion of the pseudorabies virus GIII gene regulated by the vaccinia H5R (40K) promoter between the translation and transcription stop signals at the 3' end of the K1L gene. The K1L gene transcription stop signal thus became the stop signal for the inserted GIII gene and an upstream transcription stop signal present in the H5R promoter fragment provided the stop signal for the K1L gene. This manipulation of the vaccinia genome had no effect on the accumulation or 5' end of the M2L gene transcripts. Although the insertion lengthened the 3' end and lowered the accumulation of K1L transcripts it altered neither the virulence nor the immunogenicity of the recombinant.

Base Sequence↗
PubMed1985

Mutations near the carboxyl terminus of the simian virus 40 large tumor antigen alter viral host range.

We report the characterization of three mutants of simian virus 40 with mutations that delete sequences near the 3' end of the gene encoding large tumor antigen (T antigen). Two of these mutants, dl1066 and dl1140, exhibit an altered viral host range. Wild-type simian virus 40 is capable of undergoing a complete productive infection on several types of established African green monkey kidney lines, including BSC40 and CV1P. dl1066 and dl1140 grow on BSC40 cells at 37 degrees C. However, both mutants fail to form plaques on BSC40 cells at 32 degrees C or on CV1P cells at any temperature. These mutants are capable of replicating viral DNA in the nonpermissive cell type, indicating a defect in an activity of T antigen not related to its replication function. Furthermore this defect can be complemented in trans by the wild type or by a variety of DNA replication-negative T antigen mutants, so long as they produce a normal carboxyl-terminal region of the molecule. Our data are consistent with the hypothesis that the C-terminal region of T antigen constitutes a functional domain. We propose that this domain encodes an activity that is required for simian virus 40 productive infection on the CV1P cell line, but not on BSC40.

Animals↗
PubMed2000

Host range variability in the transfer of antibiotic resistance determinants from Pseudomonas aeruginosa strains.

Twelve multiply drug-resistant strains of Pseudomonas aeruginosa, isolated in Frankfurt University Clinics during the year 1998, transferred determinants of antibiotic resistance with remarkable variability in the host range of transferred plasmids. As a new phenomenon, two of them transferred resistance genes to both groups of recipients, i.e. to Enterobacteriaceae as well as to a strain of P. aeruginosa. Six strains had more restrictive activity and transferred resistance determinants to Enterobacteriaceae only, while four additional strains demonstrated transferability only to P. aeruginosa. Two isolates transferred imipenem resistance and one cefepime resistance. Differences in selection pressure occurring during therapy of individual patients seems to be one of the possible factors influencing the spectrum of antibiotic resistance genes present in P. aeruginosa and their transfer to susceptible strains.

Drug Resistance, Multiple↗
PubMed1991

Probing the activation of the replicative origin of broad host-range plasmid R1162 with Tus, the E.coli anti-helicase protein.

The E.coli Tus protein is an anti-helicase involved in the termination of chromosome replication. The binding site for this protein, ter, was cloned into derivatives of the broad host-range plasmid R1162. The ter site caused the orientation-specific termination of plasmid replication fork movement in cell extracts containing Tus. Plasmids were constructed so that two sites for initiation of R1162 replication flanked the iteron-containing domain of the origin. In these plasmids, the site next to the AT-rich region within the iteron-containing domain was more active. In addition, when ter was placed between the more active site and the iterons, initiation of replication from this site was specifically inhibited. The data support a model for entry of the essential, plasmid-encoded helicase at one side of the direct repeats, and for its movement primarily in one direction away from these repeats to activate the initiation sites for DNA replication.

Bacterial Proteins↗
PubMed2003

Analysis of the mobilization region of the broad-host-range IncQ-like plasmid pTC-F14 and its ability to interact with a related plasmid, pTF-FC2.

Plasmid pTC-F14 is a 14.2-kb plasmid isolated from Acidithiobacillus caldus that has a replicon that is closely related to the promiscuous, broad-host-range IncQ family of plasmids. The region containing the mobilization genes was sequenced and encoded five Mob proteins that were related to those of the DNA processing (Dtr or Tra1) region of IncP plasmids rather than to the three-Mob-protein system of the IncQ group 1 plasmids (e.g., plasmid RSF1010 or R1162). Plasmid pTC-F14 is the second example of an IncQ family plasmid that has five mob genes, the other being pTF-FC2. The minimal region that was essential for mobilization included the mobA, mobB, and mobC genes, as well as the oriT gene. The mobD and mobE genes were nonessential, but together, they enhanced the mobilization frequency by approximately 300-fold. Mobilization of pTC-F14 between Escherichia coli strains by a chromosomally integrated RP4 plasmid was more than 3,500-fold less efficient than the mobilization of pTF-FC2. When both plasmids were coresident in the same E. coli host, pTC-F14 was mobilized at almost the same frequency as pTF-FC2. This enhanced pTC-F14 mobilization frequency was due to the presence of a combination of the pTF-FC2 mobD and mobE gene products, the functions of which are still unknown. Mob protein interaction at the oriT regions was unidirectionally plasmid specific in that a plasmid with the oriT region of pTC-F14 could be mobilized by pTF-FC2 but not vice versa. No evidence for any negative effect on the transfer of one plasmid by the related, potentially competitive plasmid was obtained.

Bacterial Proteins↗
PubMed2003

Distribution, host range, and climatic constraints on Centistes gasseni (Hymenoptera: Braconidae), a South American parasitoid of cucumber beetles, Diabrotica spp. (Coleoptera: Chrysomelidae).

The genus Diabrotica includes a large number of pest species, including some of the most important crop pests of the Americas. The parasitoid Centistes gasseni Shaw is the first braconid to be described parasitizing Diabrotica in South America, and high natural infestations are reported. Field and experimental observations on the host range, distribution and biology of this parasitoid are described. Centistes gasseniwas collected in southern Brazil, eastern Paraguay and northeastern Argentina, in a region comprising humid lowlands and highlands, and cool temperate to warm subtropical climates, with regular rainfall in excess of 1300 mm. Three Diabroticaspecies, D. limitata (Sahlberg), D. speciosa (Germar) and D. viridula (Fabricius) were found to host the parasitoid, with mean percent parasitism of 5.4, 2.0 and 1.0%, respectively. Diabrotica speciosa and D. viridula are the two most important pest Diabroticaspecies in South America. Laboratory experiments with field-collected beetles and parasitoid cocoons indicated that C. gasseni overwinters in adult host beetles, remaining dormant in its live host below developmental temperatures. A potential distribution of C. gasseni in North America is proposed based on its known climatic range and the distribution of the main pest species of adult overwintering North American Diabrotica.

Animals↗
PubMed1979

Tumor antigens of human Ad5 in transformed cells and in cells infected with transformation-defective host-range mutants.

We have studied the polypeptides associated with the expression of the transforming region of the Ad5 genome by immunoprecipitating antigens (using the double antibody and protein A-Sepharose techniques) from cells infected with wild-type (wt) Ad5 or transformation-defective host range (hr) mutants and from cells transformed by Ad5. Three different antisera were used: P antiserum specific for early viral products (Russell et al., 1967) and two different hamster tumor antisera. Immunoprecipitation of antigens from wt-infected KB cells followed by SDS-polyacrylamide gel electrophoresis of precipitated proteins revealed that a major polypeptide having a molecular weight of approximately 58,000 was detected with all three antisera and with both the double antibody and the protein A-Sepharose techniques, while P antiserum also precipitated polypeptides of molecular weights 72,000, 67,000 and 44,000, which probably represent the DNA binding protein and related polypeptides, respectively. With the double antibody technique, in addition to the proteins mentioned above, P antiserum and the hamster tumor antisera precipitated a 10,500 dalton polypeptide which was not detected when the protein A-Sepharose procedure was used. Using either the double antibody or the protein A-Sepharose technique, we found that hr mutants from complementation group II failed to induce the synthesis of the 58,000 dalton protein, whereas mutants from complementation group I produced normal or near normal amounts. Using the double antibody technique, we found that the 10,500 dalton protein was absent or made in reduced amounts by group I mutants. A 58,000 dalton protein was detected in a number of different Ad5-transformed cell lines, including the 293 human line, the 14b hamster line and several transformed rat cell lines. This observation and the fact that transformation negative group II mutants fail to induce the synthesis of a 58,000 dalton polypeptide suggest that this protein is one of the Ad5-specific products necessary for cell transformation.

Adenoviruses, Human↗