Modulation of lambda plasmid and phage DNA replication by Escherichia coli SeqA protein.
SeqA protein, a main negative regulator of the replication initiation of the Escherichia coli chromosome, also has several other functions which are still poorly understood. It was demonstrated previously that in seqA mutants the copy number of another replicon, the lambda plasmid, is decreased, and that the activity of the lambda p(R) promoter (whose function is required for stimulation of ori lambda) is lower than that in the wild-type host. Here, SeqA-mediated regulation of lambda phage and plasmid replicons was investigated in more detail. No significant influence of SeqA on ori lambda-dependent DNA replication in vitro was observed, indicating that a direct regulation of lambda DNA replication by this protein is unlikely. On the other hand, density-shift experiments, in which the fate of labelled lambda DNA was monitored after phage infection of host cells, strongly suggested the early appearance of sigma replication intermediates and preferential rolling-circle replication of phage DNA in seqA mutants. The directionality of lambda plasmid replication in such mutants was, however, only slightly affected. The stability of the heritable lambda replication complex was decreased in the seqA mutant relative to the wild-type host, but a stable fraction of the lambda O protein was easily detectable, indicating that such a heritable complex can function in the mutant. To investigate the influence of seqA gene function on heritable complex- and transcription-dependent lambda DNA replication, the efficiency of lambda plasmid replication in amino acid-starved relA seqA mutants was measured. Under these conditions, seqA dysfunction resulted in impairment of lambda plasmid replication. These results indicate that unlike oriC, SeqA modulates lambda DNA replication indirectly, most probably by influencing the stability of the lambda replication complex and the transcriptional activation of ori lambda.
Narajczyk
Magdalena
M
Department of Molecular Biology, University of Gdańsk, 80-822 Gdańsk, Poland.
Barańska
Sylwia
S
Department of Molecular Biology, University of Gdańsk, 80-822 Gdańsk, Poland.
Szambowska
Anna
A
Department of Molecular Biology, University of Gdańsk, 80-822 Gdańsk, Poland.
Glinkowska
Monika
M
Department of Molecular Biology, University of Gdańsk, 80-822 Gdańsk, Poland.
Węgrzyn
Alicja
A
Laboratory of Molecular Biology (affiliated with University of Gdańsk), Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 80-822 Gdańsk, Poland.
Węgrzyn
Grzegorz
G
Department of Molecular Biology, University of Gdańsk, 80-822 Gdańsk, Poland.
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000502:physiology*; D010582:Bacteriophage lambda / Q000235:genetics*; D004261:DNA Replication / Q000502:physiology*; D004279:DNA, Viral / Q000096:biosynthesis*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology*; D004926:Escherichia coli / Q000235:genetics / Q000502:physiology* / Q000821:virology; D029968:Escherichia coli Proteins / Q000235:genetics / Q000502:physiology*; D009154:Mutation; D010957:Plasmids / Q000235:genetics*; D018741:Replication Origin; D014764:Viral Proteins / Q000032:analysis
D001425:Bacterial Outer Membrane Proteins; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C027812:O protein, Bacteriophage lambda; C089746:SeqA protein, E coli; D014764:Viral Proteins