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At least 505 records · Page 28Linked to original sources
PubMed1993

The opsX locus of Xanthomonas campestris affects host range and biosynthesis of lipopolysaccharide and extracellular polysaccharide.

Xanthomonas campestris pv. citrumelo strain 3048 is the causal agent of citrus bacterial leaf spot disease and has a wide host range that includes rutaceous and leguminous plants. A spontaneous prototrophic mutant of strain 3048 (strain M28) that had lost virulence on citrus but retained virulence on bean plants was recovered. Growth studies in planta showed that M28 cells died rapidly in citrus leaves but grew normally in bean leaves. In addition to the loss of citrus-specific virulence, M28 displayed the following mutant phenotypes in culture: decreased growth rate, reduction of the amount of exopolysaccharide (to ca. 25% of the amount in 3048), loss of capsules, and significant alterations of the two 3048 lipopolysaccharide (LPS) bands visualized by silver stain on polyacrylamide gels, consistent with a defect(s) in LPS assembly. A 38-kb DNA fragment from a 3048 total DNA library that complemented the mutant phenotypes of M28 was identified. The 38-kb fragment did not hybridize to two similarly sized fragments carrying different hrp (hypersensitive response and pathogenicity) genes cloned from 3048. Subcloning, DNA sequence analyses, and gene disruption experiments were used to identify a single gene, opsX (for outer-membrane polysaccharide), responsible for the mutant phenotypes of M28. At least one other gene downstream from opsX also affected the same phenotypes and may be part of a gene cluster. We report here the DNA sequence and transcriptional start site of opsX. A search of protein sequence data bases with the predicted 31.3-kDa OpsX sequence found strong similarity to Lsi-1 of Neisseria gonorrhoeae and RfaQ of Escherichia coli (both are involved in LPS core assembly). The host-specific virulence function of opsX appears to involve biosynthesis of the extracellular polysaccharide and a complete LPS. Both may be needed in normal amounts for protection from citrus, but not bean, defense compounds.

Amino Acid Sequence↗
PubMedDate not supplied

A broad-host-range cloning vector transposable to various replicons.

A system is described for the stable insertion of cloned DNA sequences into the chromosomes of Gram-negative bacteria. Two broad-host-range plasmids form the basis of the system: one (the "carrier") contains a transposable DNA sequence into which foreign DNA can be cloned; the second (the "helper") provides transposition functions in trans. Both plasmids can be readily transferred between Gram-negative bacteria by conjugation. Instability of the carrier allows enrichment for the products of transposition to the chromosome of the new host, following which the insertion can be stabilised by elimination of the helper. The system was successfully tested in Escherichia coli, Methylophilus methylotrophus and Pseudomonas aeruginosa, and the insertions were stable in each case (less than 0.02% loss per generation).

Chromosomes, Bacterial↗
PubMed1995

The host range of RK2 minimal replicon copy-up mutants is limited by species-specific differences in the maximum tolerable copy number.

The minimal replicon of the broad-host-range plasmid RK2 consists of a gene, trfA (trans-acting replication), encoding a protein required for initiation of plasmid replication. The TrfA protein binds to iterons in the cis-acting origin of vegetative replication (oriV), but the exact mechanism by which TrfA-mediated replication initiation takes place is not known. We report here the isolation and characterization of five mini RK2 trfA mutant plasmids with an elevated plasmid copy number, four in Pseudomonas aeruginosa and one in Azotobacter vinelandii. The mutations are localized between or downstream of previously reported Escherichia coli copy-up mutations in trfA, and one of the mutations has been described earlier as an independent copy-up isolate in E. coli. The five mutant plasmids were all moderately copy up in both E. coli and their host of origin, in spite of the use of isolation procedures which were expected to select efficiently in favor of plasmid mutants specifying high copy numbers. In contrast, previously described high copy-up mutants isolated in E. coli could not be established in P. aeruginosa and A. vinelandii. These high copy-up mutants were shown to induce cell killing in E. coli under conditions where the plasmid copy number was increased as a physiological response to reduced growth rate. We propose that the reason for this killing effect is that the copy number under these conditions exceeds an upper tolerance level specific for E. coli. By assuming that the corresponding tolerance level is lower in P. aeruginosa and A. vinelandii than in E. coli, and that the mechanism of copy number regulation is similar, the model can explain the phenotypes of all tested copy up mutants in these two hosts. Analogous studies were also performed in Salmonella typhimurium and Acetobacter xylinum. The data obtained in these studies indicate that the above model is probably generally true for gram-negative bacteria, and the results also indicate that the maximum tolerable copy number is surprisingly low in some hosts.

Ampicillin↗
PubMed1997

Improved broad-host-range RK2 vectors useful for high and low regulated gene expression levels in gram-negative bacteria.

This report describes the construction and use of improved broad-host-range expression vectors based on the previously constructed pJB137 and pJB653 plasmids (Blatny et al., 1997). These vectors contain the minimal replicon of RK2 and the inducible Pu or Pm promoters together with their regulatory xylR or xylS genes, respectively, from the Pseudomonas putida TOL plasmid pWWO. A set of ATG vectors were derived from pJB653, and these vectors are characterized by the relatively small size, the presence of multiple cloning sites downstream of Pm, the establishment of their nucleotide sequence, the presence of RK2 oriT, and different antibiotic selection markers. The copy numbers of all the vectors can easily be modified by using copy-up mutations of the trfA gene, required for initiation of replication of RK2 replicons. The vectors were used to study the expression levels of the Acetobacter xylinum phosphoglucomutase gene celB and the two commonly used reporter genes luc and cat in Escherichia coli, Pseudomonas aeruginosa, and Xanthomonas campestris. Good induction properties and tight regulation of Pm were achieved in all three species tested, and higher gene expression levels were obtained by using the ATG vectors compared to pJB653. By introducing different trfA copy-up mutations into the vectors, a wide range of gene expression levels from Pu and Pm were obtained in E. coli. Induced expression levels of luc, cat, and celB from Pm were found to be comparable to or higher than those from the Ptrc and PT7 promoters located on high copy number plasmids. The induced levels of Luc activity were higher in P. aeruginosa than in E. coli, indicating that these vectors may be useful for maximization of gene expression in strains other than E. coli. We believe that the well-characterized vectors described here are useful for gene expression studies and routine cloning experiments in many Gram-negative bacteria.

Acetobacter↗
PubMed1997

Characterization of host range factor 1 (hrf-1) expression in Lymantria dispar M nucleopolyhedrovirus- and recombinant Autographa californica M nucleopolyhedrovirus-infected IPLB-Ld652Y cells.

We previously identified a gene, host range factor 1 (hrf-1), in Lymantria dispar M nucleopolyhedrovirus (LdMNPV) which promoted Autographa californica M nucleopolyhedrovirus (AcMNPV) replication in a nonpermissive cell line IPLB-Ld652Y (Ld652Y). A recombinant AcMNPV, vAcLdPS, that bore hrf-1 controlled by two synthetic baculovirus late promoters and that replicated in Ld652Y cells was constructed. In this study, we constructed a new recombinant AcMNPV, vAcLdPD, bearing only hrf-1 controlled by its own promoter. vAcLdPD replicated in Ld652Y cells in the same manner as vAcLdPS, confirming that hrf-1 alone was sufficient to promote AcMNPV replication in Ld652Y cells. hrf-1 was transcribed as a delayed early gene in LdMNPV but as an immediate early gene in both recombinant AcMNPVs. Primer extension analysis showed that the initiator sequence TCAGT was used as the transcription start site in both LdMNPV and recombinant AcMNPVs. Additional sequencing revealed several regulatory motifs in the hrf-1 upstream region. hrf-1 transcripts in LdMNPV- and vAcLdPS-infected Ld652Y cells terminated near the polyadenylation signal at the end of hrf-1 ORF while in vAcLdPD, the hrf-1 transcripts terminated at a downstream polyadenylation signal at the end of ORF 603. Using Western blot analysis, we detected HRF-1 expression in both recombinant AcMNPV-infected Ld652Y cells but not in LdMNPV-infected Ld652Y cells.

Animals↗
PubMed2001

Novel virulence and host range genes of African swine fever virus.

Current work is beginning to reveal the complex mechanisms by which African swine fever virus interacts with its swine and tick hosts. This work includes the identification of novel viral genes that mediate virulence and host range, and influence important cellular regulatory pathways.

African Swine Fever↗
PubMed1995

Cloning and sequence analysis of Vibrio parahaemolyticus ompK gene encoding a 26-kDa outer membrane protein, OmpK, that serves as receptor for a broad-host-range vibriophage, KVP40.

The ompK gene of Vibrio parahaemolyticus 1010 (RIMD 2210001) encoding an outer membrane protein (OMP), OmpK, which serves as the receptor for a broad-host-range vibriophage, KVP40, was cloned and sequenced. The gene consisted of 789 nucleotides encoding 263 amino acids. Since the first 20 amino acids most likely constitute the signal peptide, mature OmpK would consist of 243 amino acids with a calculated molecular mass of 27458 Da. Sequence comparisons indicate that OmpK is unique among Vibrio OMPs so far sequenced, but may be distantly related to Tsx of enteric bacteria and is homologous to an Aeromonas hydrophila OMP, protein IV.

Aeromonas↗
PubMed1990

Replication of the broad-host-range plasmid RK2: direct measurement of intracellular concentrations of the essential TrfA replication proteins and their effect on plasmid copy number.

The trfA gene of the broad-host-range plasmid RK2 is essential for initiation of plasmid replication. Two related TrfA proteins of 43 and 32 kilodaltons (kDa) are produced by independent translation initiation at two start codons within the trfA open reading frame. These proteins were o overproduced in Escherichia coli and partially purified. Rabbit antisera raised against the 32-kDa TrfA protein (TrfA-32) and cross-reacting with the 43-kDa protein (TrfA-43) were used in Western blotting (immunoblotting) assays to measure intracellular TrfA levels. In logarithmically growing E. coli HB101, RK2 produced 4.6 +/- 0.6 ng of TrfA-32 and 1.8 +/- 0.2 ng of TrfA-43 per unit of optical density at 600 nm (mean +/- standard deviation). On the basis of determinations of the number of cells per unit of optical density at 600 nm, this corresponds to about 220 molecules of TrfA-32 and 80 molecules of TrfA-43 per cell. Dot blot hybridizations showed that plasmid RK2 is present in about 15 copies per E. coli cell under these conditions. Using plasmid constructs that produce different levels of TrfA proteins, the effect of excess TrfA on RK2 replication was tested. A two- to threefold excess of total TrfA increased the copy number of RK2 by about 30%. Additional increases in TrfA protein concentration had no further effect on copy number, even at levels 170-fold above normal. An RK2 minimal origin plasmid showed a similar response to intracellular TrfA concentration. These results demonstrate that TrfA protein concentration is not strictly rate limiting for RK2 replication and that a mechanism that is independent of TrfA concentration functions to limit RK2 copy number in the presence of excess TrfA.

Bacterial Proteins↗
PubMed1992

Sequence analysis and characterization of the mobilization region of a broad-host-range plasmid, pTF-FC2, isolated from Thiobacillus ferrooxidans.

The nucleotide sequence of a 5,317-bp fragment which includes the region required for mobilization of broad-host-range plasmid pTF-FC2 was determined. A region of approximately 3.5 kb was required for plasmid mobilization, and oriT was localized on a 138-bp fragment. Polypeptides which corresponded in size and location to several of the open reading frames were detected in an in vitro transcription-translation system. Three open reading frames essential for plasmid mobilization and two which affect the mobilization frequency were identified. There was a distinct similarity in the sizes, amino acid sequences, and locations of the proteins from the mobilization region of pTF-FC2 and the Tra1 region of IncP plasmid RP4. Similarity in the structures and sequences of the oriT regions was also apparent. A sequence with 37-of-38-bp homology to the inverted repeated sequences of Tn21 and an open reading frame with strong homology to the MerR regulatory protein was identified outside of the region required for mobilization.

Amino Acid Sequence↗
PubMed2002

Host range and receptor binding properties of vectors bearing feline leukemia virus subgroup B envelopes can be modulated by envelope sequences outside of the receptor binding domain.

To evaluate host range differences between two different strains of feline leukemia virus subgroup B (FeLV-B), we compared the binding and infectivity patterns of retrovirus vectors bearing either FeLV-B-90Z or FeLV-B-GA envelopes. We report here that the ability of these envelopes to utilize different Pit1 orthologs is mediated primarily by the receptor binding domain; however, in the case of FeLV-B-90Z, the C terminus also contributes to the recognition of certain Pit1 orthologs.

Animals↗
PubMed2004

Host range factor 1 from Lymantria dispar Nucleopolyhedrovirus (NPV) is an essential viral factor required for productive infection of NPVs in IPLB-Ld652Y cells derived from L. dispar.

Host range factor 1 (HRF-1) of Lymantria dispar multinucleocapsid nucleopolyhedrovirus promotes Autographa californica MNPV replication in nonpermissive Ld652Y cells derived from L. dispar. Here we demonstrate that restricted Hyphantria cunea NPV replication in Ld652Y cells was not due to apoptosis but was likely due to global protein synthesis arrest that could be restored by HRF-1. Our data also showed that HRF-1 promoted the production of progeny virions for two other baculoviruses, Bombyx mori NPV and Spodoptera exigua MNPV, whose replication in Ld652Y cells is limited to replication of viral DNA without successful production of infectious progeny virions. Thus, HRF-1 is an essential viral factor required for productive infection of NPVs in Ld652Y cells.

Animals↗
PubMed1981

Complementation of adenovirus type 5 host range mutants by adenovirus type 12 in coinfected HeLa and BHK-21 cells.

We have studied the ability of adenovirus type 12 (Ad12) to complement the Ad5 transformation-defective host rang (hr) mutants during infection of human cells (HeLa) or hamster cells (BHK-21). The group I mutant hr3 (mapped within 1.3 to 3.7 map units), which is incapable of synthesizing viral DNA, was complemented for both DNA synthesis and infectious virus production in nonpermissive HeLa cells during coinfection with Ad12. Similarly, the group II mutant hr6 (6.1 to 9.4 map units), which does synthesize DNA, was also shown to be complemented for virus production. When the host cells were BHK-21, an established hamster cell line that is permissive for Ad5 but nonpermissive for Ad12 DNA synthesis and virus production, coinfection with Ad5 and Ad12 did not overcome the block to Ad12 DNA synthesis. Coinfection of BHK-21 cells with Ad12 and either hr3 or hr6 leads to the complementation of only the group I mutant (hr3). The inability of Ad12 to complement hr6 in BHK-21 cells may be due to the failure of Ad12 to express an early gene product from the region corresponding to early region 1B (4.5 to 11 map units) Ad5 where hr6 and the other group II mutations are located.

Adenoviruses, Human↗
PubMed1983

Location and nucleotide sequence of the transfer origin of the broad host range plasmid RK2.

The origin of plasmid DNA transfer, oriT, has been localized on RK2, a conjugative drug-resistance plasmid of the IncP group with a very broad host range in gram-negative bacteria. The transfer origin is contained in a 760-base-pair Hae II restriction fragment that maps in the same region as the single-strand nick made by the RK2 relaxation complex. The functional oriT was subcloned as a 112-base-pair Hpa II fragment, and the DNA sequence of this region was determined. The dominant structural feature of the oriT sequence is a 19-base-pair inverted repeat, with 15 of the 19 bases able to form pairs in a hairpin structure. This inverted repeat may be the recognition site for the relaxation complex proteins, which nick the plasmid DNA molecule and initiate the transfer process.

Base Sequence↗
PubMed1992

A region of the broad-host-range plasmid RK2 causes stable in planta inheritance of plasmids in Rhizobium meliloti cells isolated from alfalfa root nodules.

We demonstrate for the first time that the broad-host-range stabilization loci from plasmid RK2 cause total retention of plasmids in cells of Rhizobium meliloti during symbiosis with alfalfa. Two derivatives of plasmid RK2, pRK290 and a 7.3-kb mini-RK2 plasmid, were stabilized in R. meliloti cells isolated from root nodules by the insertion of a 3.2-kb DNA fragment or a smaller 0.8-kb DNA fragment derived from the RK2 stabilization region.

Aerobiosis↗
PubMed1991

High-frequency mobilization of broad-host-range plasmids into Neisseria gonorrhoeae requires methylation in the donor.

Antibiotic resistance in Neisseria gonorrhoeae has been associated with the acquisition of R plasmids from heterologous organisms. The broad-host-range plasmids of incompatibility groups P (IncP) and Q (IncQ) have played a role in this genetic exchange in nature. We have utilized derivatives of RSF1010 (IncQ) and RP1 (IncP) to demonstrate that the plethora of restriction barriers associated with the gonococci markedly reduces mobilization of plasmids from Escherichia coli into strains F62 and PGH 3-2. Partially purified restriction endonucleases from these gonococcal strains can digest RSF1010 in vitro. Protection of RSF1010-km from digestion by gonococcal enzymes purified from strain F62 is observed when the plasmid is isolated from E. coli containing a coresident plasmid, pCAL7. Plasmid pCAL7 produces a 5'-MECG-3' cytosine methylase (M.SssI). The M.SssI methylase only partially protects RSF1010-km from digestion by restriction enzymes from strain PGH 3-2. Total protection of RSF1010-km from PGH 3-2 restriction requires both pCAL7 and a second coresident plasmid, pFnuDI, which produces a 5'-GGMECC-3' cytosine methylase. When both F62 and PGH 3-2 are utilized as recipients in heterospecific matings with E. coli, mobilization of RSF1010 from strains containing the appropriate methylases into the gonococci occurs at frequencies 4 orders of magnitude higher than from strains without the methylases. Thus, protection of RSF1010 from gonococcal restriction enzymes in vitro correlates with an increase in the conjugal frequency. These data indicate that restriction is a major barrier against efficient conjugal transfer between N. gonorrhoeae and heterologous hosts.

Conjugation, Genetic↗