Sequence of Mu-host junctions at 15 insertions recovered in a <i>priA</i> mutant infected with Mu::Cm(<i>Bam</i>1066).

<p>See Methods for sequencing details. Orientation refers to clockwise positions of Mu from <i>oriC</i>, which is at ∼3.92 Mb; <i>ter</i> is at ∼1.59 Mb. The numbers in the Insertion site column refer to nucleotides on the <i>E. coli</i> genome. Black bars, intact Mu with L an R ends indicated; Gray bars, truncated/duplicated Mu with only one end identified; Dotted lines, undetermined host DNA sequence; • a repeated sequence; * insertion of nucleotides not found in the host DNA; l, r, position of insertions in the left and right replicores, respectively.</p

Known steps in replicative and non-replicative (repair) pathways of Mu transposition.

<p>The transposase MuA, in the presence of <i>E. coli</i> protein HU, first introduces single-stranded cleavages at the 3′ ends. With assistance from MuB, the 3′OHs at the cleaved ends are transferred by MuA to phosphodiester bonds spaced 5 bp apart in the target <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002642#pgen.1002642-Chaconas1" target="_blank">[3]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002642#pgen.1002642-Mizuuchi1" target="_blank">[4]</a>. The resultant branched strand transfer intermediate is processed alternately. During the lytic cycle, Mu is inserted in the chromosome, the target is also in the chromosome, so the purple flanking DNA is continuous with the orange target; transposition is intramolecular. The target OHs found in the strand transfer intermediate are used as primers to replicate Mu (green lines). ClpX, IF2-2 and other uncharacterized factors are required for disassembly of the transpososome followed by assembly of the PriA restart primosome on the Mu ends <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002642#pgen.1002642-Nakai1" target="_blank">[5]</a>. During integration of infecting Mu, the purple flanking DNA on the incoming Mu genome is non-covalently joined to itself via phage N protein; transposition into the chromosome target is intermolecular <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002642#pgen.1002642-Harshey2" target="_blank">[9]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002642#pgen.1002642-Puspurs1" target="_blank">[10]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002642#pgen.1002642-Gloor1" target="_blank">[11]</a>. The branched strand transfer intermediate is resolved/repaired by MuA_Nuc-mediated resection of the flap DNA <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002642#pgen.1002642-Choi1" target="_blank">[13]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002642#pgen.1002642-Wu1" target="_blank">[14]</a>. ClpX is required for this reaction. The remaining gaps are thought to be filled by host enzymes.</p

Models for recombinational repair of Mu insertions in the non-replicative pathway.

<p>Both models presented rely on repair of double strand breaks by homologous recombination and replication restart proteins, but differ in the location of the break and the order of the recombination/restart-replication events that follow. In (A), the break is on the chromosomal DNA flanking the Mu insertion. Here, homologous recombination is followed by restart replication. In (B), the break is on the Mu lagging strand. Here, restart replication precedes homologous recombination. Alternate shapes for PriA denote uni- or bi-directional replication. See text for details.</p

Behavior of various <i>priA</i>, <i>dnaT</i>, and <i>rec</i> alleles in a different strain background.

<p>Mu<i>Bam</i>1066ΔSE::Cm was used for infection of indicated strains to assess their role in recovery of Mu insertions. Other descriptions as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002642#pgen-1002642-g004" target="_blank">Figure 4</a>.</p

Identification of <i>E. coli</i> mutants in the Keio library defective in recovery of Mu::Cm insertions.

<p>(A) Functional map of the Mu genome packaged within a phage particle, showing position of inserted Cm^R cassette, and host or flap DNA attached to both ends. The SE (semi-essential) region contains 14 orfs <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002642#pgen.1002642-Morgan1" target="_blank">[27]</a>; only those assigned a phenotype/function are indicated <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002642#pgen.1002642-Symonds1" target="_blank">[2]</a>. (B) Cultures infected with Mu::Cm were spotted on Cm plates as described in Methods. Control panel: Expected results from infection of two different wild type and their derivative mutant strains - <i>clpX</i> and <i>himA</i> - that do not support Mu replication. Bottom four panels: Final set of mutants from the Keio screen showing lower Mu::Cm lysogen recovery compared to the wild type strain, grouped into indicated categories.</p

Mutant screen using replication-defective Mu.

<p>Lysogenization efficiencies (calculated as Cm^R cells/infected cells×100) of the mutant strains infected with either (A) Mu::Cm(<i>Bam</i>1066) or (B) Mu<i>Bam</i>1066ΔSE::Cm. Mutant categories as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002642#pgen-1002642-g002" target="_blank">Figure 2B</a>. Error bars indicate standard deviation from the mean of triplicate data sets obtained from three independent colonies of the same strain. In (B), data for RNA/Other mutants are from a single colony/experiment. See Methods for details.</p

Strains used in this study.

<p>All strains listed as being from the Sandler lab are isogenic and are derivatives of JC13509. The genotype of JC13509 is <i>sulB103 lacMS286 φ80dIIlacBK1 argE3 hi-4 thi-1 xyl-5 mtl-1 rpsL31 tsx</i>. The <i>lacMS286 φ80dIIlacBK1</i> denote two partial non-overlapping deletions of the <i>lac</i> operon <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002642#pgen.1002642-Konrad1" target="_blank">[68]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002642#pgen.1002642-Zieg1" target="_blank">[69]</a>.</p

PCR assay for Mu integration in mutants defective in lysogen recovery.

<p>(A) Control PCR reactions monitoring integration at different time points after infection of wild type BW25113 with Mu::Cm, Mu::Cm(<i>Bam</i>1066) and Mu::Cm(<i>Aam</i>1093) phage. These phages can integrate-replicate, integrate but not replicate, or not integrate, respectively. (B) PCR results for wild type Mu::Cm integration 30 min after infection of mutants in the first three categories shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002642#pgen-1002642-g002" target="_blank">Figure 2B</a>. (C) As in (B) but with mutants in the Mu Receptor category. Control reactions with either no template (N), Mu, or genomic DNA templates from uninfected BW25113 host (G) are indicated, along with size markers (M). Reaction products were run on agarose gels and stained with ethidium bromide as described under Methods.</p

This figure shows the distributions of cells with different levels of constitutive SOS expression (detected as GFP fluorescence) expressed as the percentage of cells in the population.

<p>The graphs truncate the percentage of cells at 25%. The strains are in order from top of the graph to the bottom with the relevant part of the genotype in parentheses. Unless otherwise indicated, all strains were grown in minimal medium at 37°C with aeration. The strains are: SS1408 (<i>lexA51::Tn5</i>), SS4629 (<i>recA730</i>), SS4976 (<i>recAo1403 recA4142</i>), SS6013 (<i>recA4142</i>), SS6088 (<i>recAo1403 recA⁺</i>) and SS996 (<i>recA</i>⁺).</p

Same as for Figure 2.

<p>All grown in rich medium: SS996 (<i>recA</i>⁺), SS6080 (<i>del(recX)</i>), SS6013 (<i>recA4142</i>), SS6019 (<i>recA4142 del(recX)</i>), SS4976 (<i>recAo1403 recA4142</i>), SS5312 (<i>recAo1403 recA4142 del(recX)</i>).</p