Detection and identification of the fire blight pathogen, Erwinia amylovora, can be accurately done by polymerase chain reaction (PCR) analysis in less than 6 h. Two oligomers derived from a 29-kb plasmid which is common to all strains of E. amylovora were used to amplify a 0.9-kb fragment of the plasmid. By separation of the PCR products on agarose gel, this fragment was specifically detected when E. amylovora DNA was present in the amplification assay. It was not found when DNA from other plant-pathogenic bacteria was used for the assay. A visible band specific to the 0.9-kb fragment was produced with DNA from fewer than 100 E. amylovora cells. A signal of similar strength was also obtained from E. amylovora cell lysates in the presence of the mild detergent Tween 20. Signals were weaker when bacteria were added to the PCR mixture without the detergent. As with results obtained from hybridization experiments using pEA29 DNA, the PCR signal was obtained with E. amylovora isolates from various geographic regions. This technique could also be used for detection of the fire blight pathogen in extracts of tissue obtained from infected plant material.

The human spuma retrovirus or foamy virus integrase (HFV IN) is an enzymatically active protein consisting of domains similar to other retroviral integrases: an amino-terminal HH-CC finger, a centrally located region with the conserved D, D-35-E protein motif required for catalytic activity and oligomerization, and at least one DNA binding domain implicated in the 3' DNA processing activity and integrase. Recombinant, purified HFV IN protein carrying 10 histidine residues displays a site-specific endonuclease, an integrase, and a disintegrase activity with oligonucleotide substrates that mimic the viral long terminal repeat (LTR) ends. Site-directed mutagenesis of conserved HFV IN residues of the catalytic domain had increased endonuclease and disintegrase activities. Deletion mutants at both ends of the HFV IN protein were generated, purified, and characterized. Unexpectedly, it was found that the HFV integrase and disintegrase activities require an intact NH2-terminal sequence and that COOH-terminal deletions led to an increase in disintegrase activity. The HH-CC finger of HFV IN was exchanged with that of the human immunodeficiency virus-1 (HIV-1) IN protein. The resulting chimeric IN had a 3' processing activity that utilized the HFV LTR instead of the HIV LTR, indicating that the central domain is crucial for substrate recognition. Functional complementation of the amino-terminal deletion mutant of HFV IN was achieved by a carboxyl-terminal deletion mutant of the chimeric IN, resulting in high levels of integrase activity.

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The bacterial expression plasmids, pET3b and pET16b, that contain the integrase domain of the human foamy virus (HFV) reverse transcriptase were constructed and expressed in Escherichia coli. The histidinetagged HFV IN protein was purified to near homogeneity by single-step Ni2+ chelate affinity chromatography.