In vitro packaging of DNA of the Bacillus subtilis bacteriophage SPP1.
In vitro packaging of bacteriophage SPP1 DNA into procapsids is described and the requirements of this process were determined. Combination of proheads with an extract supplying terminase, DNA and phage tails yielded up to 10(7 )viable phages per milliliter of in vitro reaction under optimized conditions. The presence of neutral polymers and polyamines had a concentration and type dependent effect in the packaging reaction. The terminase donor extract lost rapidly activity at 30 degrees C in contrast to the stability of the prohead donor extract. Maturation to infective virions was observed using both procapsids assembled in SPP1 infected cells and procapsid-like structures assembled in Escherichia coli that overexpressed the SPP1 prohead gene clusters. Neither a majority of aberrant capsid-related structures present in the latter material nor procapsids lacking the portal protein inhibited DNA packaging. Addition of purified portal protein reduced DNA packaging activity in vitro only at concentrations 20-fold higher than those found in the SPP1 infected cell. The SPP1 DNA packaged in vitro originated exclusively from the terminase donor extract. This packaging selectivity was not observed in vivo during mixed infections. The data are compatible with a model for processive headful DNA packaging in which terminase and DNA co-produced in the same cell are tightly associated and can effectively discriminate the portal vertex of DNA packaging-proficient proheads from aberrant structures, from portal-less procapsids, and from isolated portal protein.
Dröge
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A
Max-Planck-Institut für Molekulare Genetik, Ihnestrasse 73, Berlin, D-14195, Germany. droege@molgen.mpg.de
Tavares
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P
D017103:Bacillus Phages / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development* / Q000302:isolation & purification; D001412:Bacillus subtilis / Q000166:cytology / Q000821:virology*; D002213:Capsid / Q000235:genetics / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics; D005814:Genes, Viral / Q000235:genetics / Q000502:physiology; D006023:Glycoproteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008954:Models, Biological; D009154:Mutation / Q000235:genetics; D011073:Polyamines / Q000378:metabolism; D011108:Polymers / Q000378:metabolism; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D014764:Viral Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D019065:Virus Assembly
D004279:DNA, Viral; D006023:Glycoproteins; D011073:Polyamines; D011108:Polymers; D011498:Protein Precursors; D014764:Viral Proteins; C115880:portal protein, Bacteriophage SPP1; D004706:Endodeoxyribonucleases; C030894:terminase