Gp55.2 binds to DNA, inhibits Topo I relaxation activity but does not affect DNA gyrase supercoiling activity.

<p>(A) Electrophoretic mobility shift assays (EMSA). Mixtures containing 300 ng (= 79 fmol) of linear, form I’, or form I pDB29 DNA and the indicated amount of gp55.2-His₆ (95 ng = 6.9 pmol, namely one gp55.2 molecule per 65.6 bp for the amount DNA used in this assay) were incubated and analyzed as described in Materials and Methods. An arrow indicates the migration position of the form II DNA contaminating the form I DNA. (B) Relaxation assays mixtures containing 600 ng (= 158 fmol) form I pDB29 DNA and the indicated units of Topo I (0.47 U = 415 fmol) were incubated in the presence (+) or absence (–) of 855 ng of gp55.2-His₆ (= 63 pmol, one gp55.2 molecule per 14.5 bp of DNA), and the DNA products were analyzed as described in Materials and Methods. The migration positions of form I, form I’ and form II DNA are indicated on the left; linear DNA migration position is indicated by an arrowhead. Results representative of two independent experiments are shown. (C) Supercoiling assays mixtures containing 158 fmol form I’ pDB29 DNA and the indicated units of DNA gyrase (0.42 U = 196 fmol) were incubated in the presence (+) or absence (–) of 855 ng of gp55.2-His₆ and the DNA products were analyzed as described in the Materials and Methods section. Results representative of two independent experiments are shown. (D) Representation of form I, form I’ and form II plasmid DNA. Note that the treatment of form I plasmid DNA by a eukaryotic Topo I to obtain form I’ DNA results, at equilibrium, in a distribution of relaxed DNA topoisomers (A, middle panel) whose maximum corresponds to the fully relaxed form I’ plasmid illustrated in D.</p

Loss of gene <i>55</i>.<i>2</i> function reduces T4 phage fitness.

<p>(A) CR63 cells grown in M9 medium were infected with T4 K10 (wt) or T4 K10-<i>55</i>.<i>2</i> (<i>55</i>.<i>2</i>) at a moi of 6 at 30°C. Intracellular phage accumulation was followed at the indicated time points; free phages at 25 min represented < 12% of the total infective centers. Data represents mean and ranges of two (wt) and four (<i>55</i>.<i>2</i>) independent experiments. (B) Competition experiment. A mix of T4 K10 and T4 K10-<i>55</i>.<i>2</i> with an initial ratio of 1:9 was grown on <i>E</i>. <i>coli</i> CR63 in M9S medium at 37°C at low moi (< 0.1) over successive growth cycles. The percentage of <i>55</i>.<i>2+</i> phages was determined by PCR and digestion as described in Materials and Methods. Data represent mean and standard deviation of four independent experiments; the dotted line represents the result of a simulation were the <i>55</i>.<i>2</i> mutant has a 16% disadvantage per growth cycle. The intracellular phage accumulation over a single growth cycle in these conditions is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124309#pone.0124309.s005" target="_blank">S5A Fig</a>.</p

<i>55</i>.<i>2</i> expression affects the control of DNA topology and plasmid copy number in <i>E</i>. <i>coli</i>.

<p>(A) Plasmid topoisomers analysis. Left panel: plasmid DNA was extracted from exponentially growing DH5α harboring pYM58 (<i>55</i>.<i>2</i>_IS) or pDB2114 (<i>55</i>.<i>2</i>) plasmids. Plasmid topoisomers were resolved on TBE agarose gels containing the indicated amount of CLQ. The position of migration of relaxed and/or nicked circular DNA is indicated (R). Right panel: Densitometry analysis of the topoisomer distribution on 1.5 μg ml^-1 CLQ gel of four independent samples of pYM58 or pDB2114 plasmid DNA (gel images are depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124309#pone.0124309.s004" target="_blank">S4A Fig</a>). Plotted is the average (lines) and standard deviation (shaded area) of relative plasmid density as a function of negative supercoiling. (B) 2D electrophoretic separation of plasmid topoisomers. Left panel: schematic representation of a 2D gel. The migration positions of negatively supercoiled (–), positively supercoiled (+), and form II topoisomers (N) are indicated. Right panel: gel images show the 2D topoisomer distribution of plasmid DNA samples prepared as in A. Chloroquine concentration was 1.5 μg ml^-1 and 25 μg ml^-1 in the first and second dimension, respectively. (C) Plasmid copy number analysis. Linearized plasmid DNA samples from the experiment shown in A were quantified and normalized to the amount (A_600nm) of bacteria used to extract the plasmids. The data represents means and standard error of four independent cultures. (D) Plasmid DNA was extracted from overnight cultures of DHB3, DB503, and DH5α transformed with pBAD101 (<i>55</i>.<i>2</i> –) or pDB2114-101 (<i>55</i>.<i>2</i> +), and a reporter plasmid (pDB868-2). Linearized plasmids were analyzed by agarose gel electrophoresis.</p

The toxicity of <i>55</i>.<i>2</i> in <i>E</i>. <i>coli</i> is suppressed by an increase in the copy number of <i>topA</i>.

<p>(A) Liquid growth assay. Overnight cultures of DB503 cells harboring pBAD101 (ctr) or pDB2114-101 (<i>55</i>.<i>2</i>) plasmids were diluted in fresh LB medium and optical density (A_600nm) was measured at the indicated times. At A_600nm = 0.3, cultures were split in two and one half was induced with 0.2% arabinose (vertical arrow). When A_600nm reached ≈ 1, cultures were diluted 10-fold in prewarmed medium plus or minus arabinose. The graph depicts the data of a representative experiment. (B) Reversibility assay. During a growth curve assay, aliquots of arabinose-induced DB503 cultures, harboring pBAD101 (<i>55</i>.<i>2</i> –) or pDB2114-101 (<i>55</i>.<i>2</i> +) plasmids, were withdrawn at the indicated times, washed in cold media without arabinose, and adjusted to the same A_600nm. Serial 10-fold dilutions were spotted on LB plates without arabinose. (C) DB503 cells transformed with pBAD101 (vector) or pDB2114-101 (<i>55</i>.<i>2</i>) and one of the compatible plasmids, pDB868-2 (vector, 1–2), pDB34-8-4 (<i>topA</i>, 3–4), or pDB34-8 (<i>topA</i>, 5–6) were streaked on LB plates with or without 0.2% arabinose. (D) Plasmid based lethality assay. Overnight cultures of AS1047 (<i>topA</i> +) or AS1050 (<i>topA</i>-) transformed with pBAD33-K (<i>55</i>.<i>2</i> –) or pDB2114-33-K (<i>55</i>.<i>2</i> +) were diluted and outgrown as indicated in the Materials and Methods section. Aliquots were diluted, and plated on M63 plates supplemented with glucose and X-gal. The number of blue and white colonies was scored after 36h at 37°C. Representative photographs are shown in the upper panel (the position of a rare white colony in inset IV is indicated by an arrow). The lower panel depicts percentage of white colonies; average and standard deviation are from three independent experiments.</p

Gp55.2 binds to DNA, inhibits Topo I relaxation activity but does not affect DNA gyrase supercoiling activity.

<p>(A) Electrophoretic mobility shift assays (EMSA). Mixtures containing 300 ng (= 79 fmol) of linear, form I’, or form I pDB29 DNA and the indicated amount of gp55.2-His₆ (95 ng = 6.9 pmol, namely one gp55.2 molecule per 65.6 bp for the amount DNA used in this assay) were incubated and analyzed as described in Materials and Methods. An arrow indicates the migration position of the form II DNA contaminating the form I DNA. (B) Relaxation assays mixtures containing 600 ng (= 158 fmol) form I pDB29 DNA and the indicated units of Topo I (0.47 U = 415 fmol) were incubated in the presence (+) or absence (–) of 855 ng of gp55.2-His₆ (= 63 pmol, one gp55.2 molecule per 14.5 bp of DNA), and the DNA products were analyzed as described in Materials and Methods. The migration positions of form I, form I’ and form II DNA are indicated on the left; linear DNA migration position is indicated by an arrowhead. Results representative of two independent experiments are shown. (C) Supercoiling assays mixtures containing 158 fmol form I’ pDB29 DNA and the indicated units of DNA gyrase (0.42 U = 196 fmol) were incubated in the presence (+) or absence (–) of 855 ng of gp55.2-His₆ and the DNA products were analyzed as described in the Materials and Methods section. Results representative of two independent experiments are shown. (D) Representation of form I, form I’ and form II plasmid DNA. Note that the treatment of form I plasmid DNA by a eukaryotic Topo I to obtain form I’ DNA results, at equilibrium, in a distribution of relaxed DNA topoisomers (A, middle panel) whose maximum corresponds to the fully relaxed form I’ plasmid illustrated in D.</p