Characteristics of a wicked problem.

<p>Characteristics of a wicked problem.</p

Ciprofloxacin MICs of <i>E</i>. <i>coli</i> clinical isolates.

<p>Box and whisker plots show the range of ciprofloxacin MICs for FQS and FQR clinical isolates. Data are from Becnel Boyd et al. 2009 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006805#ppat.1006805.ref012" target="_blank">12</a>]. Plots are divided into four quartiles, each representing 25% of the MICs (Q1: end of lower whisker to edge of box; Q2: edge of box to median line; Q3: median line to edge of box; Q4: edge of box to end of upper whisker). The length of the box is referred to as the IQR. Circles indicate outliers (here MICs higher than Q4 plus 1.5x IQR). Ciprofloxacin-susceptible isolates (MIC ≤1 μg/ml), as determined in the microbiology laboratory of the hospital; ciprofloxacin-nonsusceptible isolates were categorized as FQR. CIP, ciprofloxacin; FQR, fluoroquinolone-resistant; FQS, fluoroquinolone-susceptible; IQR, interquartile range; MIC, minimum inhibitory concentration.</p

Schematic showing known ciprofloxacin resistance mechanisms in <i>E</i>. <i>coli</i>.

<p>(A) Ciprofloxacin-susceptible <i>E</i>. <i>coli</i>. The inner and outer membranes intrinsically protect the bacterium. Also depicted are the AcrAB-TolC efflux pump, porin, and DNA gyrase (or topoisomerase IV) interacting with the DNA nucleoid (in blue). Ciprofloxacin (green diamond) can diffuse through the membranes but also accesses the cell via porins. Ciprofloxacin forms a ternary complex with the topoisomerase bound to DNA, resulting in cell death. (B) Chromosomally encoded ciprofloxacin resistance mechanisms. Altered porin(s), mutant gyrase (and perhaps also topoisomerase IV), and increased numbers of AcrAB-TolC efflux pumps are shown. Ciprofloxacin access is reduced via alterations (deletion, down-regulation, or mutation) in porins. Ciprofloxacin that enters the cell can be removed through increased numbers of efflux pumps. Ciprofloxacin that reaches the mutant topoisomerase(s) is less effective against the mutant version of the enzyme than the drug-susceptible version shown in A. (C) Plasmid-borne ciprofloxacin resistance mechanisms. Plasmids can harbor genes encoding the ciprofloxacin efflux pumps QepA or OqxAB, the Qnr protein—which binds gyrase by mimicking B-form DNA—or Aac(6’)-Ib-cr, an aminoglycoside-modifying acetyltransferase that acetylates and inactivates ciprofloxacin. Aac(6’)-Ib-cr, aminoglycoside 6’-N-acetyltransferase type lb-cr; AcrAB-TolC, Acriflavin-resistant Proteins AB Tolerant to Colicin E mutant; OqxAB, olaquindox-resistant efflux pump proteins A and B; QepA, quinolone efflux pump A; Qnr, quinolone resistance protein.</p

Escherichia coli DNA ligase B may mitigate damage from oxidative stress.

Escherichia coli encodes two DNA ligases, ligase A, which is essential under normal laboratory growth conditions, and ligase B, which is not. Here we report potential functions of ligase B. We found that across the entire Enterobacteriaceae family, ligase B is highly conserved in both amino acid identity and synteny with genes associated with oxidative stress. Deletion of ligB sensitized E. coli to specific DNA damaging agents and antibiotics resulted in a weak mutator phenotype, and decreased biofilm formation. Overexpression of ligB caused a dramatic extension of lag phase that eventually resumed normal growth. The ligase function of ligase B was not required to mediate the extended lag phase, as overexpression of a ligase-deficient ligB mutant also blocked growth. Overexpression of ligB during logarithmic growth caused an immediate block of cell growth and DNA replication, and death of about half of cells. These data support a potential role for ligase B in the base excision repair pathway or the mismatch repair pathway

Effect of <i>lig</i>B overexpression on <i>E</i>. <i>coli</i> growth.

<p>(A) <i>E</i>. <i>coli</i> MG1655 either without (■) or with (●) plasmid pCA24N JW3622 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180800#pone.0180800.ref048" target="_blank">48</a>] (pLigB), which encodes <i>ligB</i> under control of the T5 <i>lac</i> promoter, were grown in the absence or presence of IPTG. As a negative control, <i>E</i>. <i>coli</i> MG1655 with pCA24N [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180800#pone.0180800.ref048" target="_blank">48</a>] (pEmpty) (○), which is the parental “empty” vector, was included. Average lag time was quantified using a custom R program (21) and plotted as a function of IPTG concentration. Error bars denote the standard deviation calculated from experiments done in triplicate repeated at least two times. Where error bars are not shown, they were smaller than the symbol. (B) Representative growth curves of MG1655 with overexpression of pLigB and of ligase-deficient pLigB K124A with increasing levels of IPTG induction (lighter color (lower) to darker color (higher)). The experiment was repeated three times each in triplicate with the same results.</p

Effect of <i>ligB</i> deletion on bacterial response to DNA damaging agents.

<p>Survival of the parent strain MG1655 (●) compared to the isogenic Δ<i>ligB</i> strain (○). Percent survival was determined by dividing the number of CFU/ml with the indicated treatment by the number of CFU/ml without treatment and these data are shown on a semi-log plot as a function of increasing DNA damaging agent. Error bars denote the standard deviation calculated from experiments done in triplicate (repeated three times with similar results). Comparing the parent and the mutant strain for each specific condition by Student’s T-test, <i>p</i> < 0.05(_*).</p

Effect of Δ<i>ligB</i> on growth in the presence of cadmium.

<p>(A) Growth of <i>E</i>. <i>coli</i> MG1655 or the isogenic Δ<i>ligB</i> strain was measured at OD₄₅₀ [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180800#pone.0180800.ref019" target="_blank">19</a>] in the presence of the various indicated concentrations of cadmium. Growth without cadmium is in black and increasing cadmium is depicted from light blue (lowest) to dark blue (highest). The experiment shown was repeated three additional times (each in triplicate) with similar results. (B) Averaged growth lag times of <i>E</i>. <i>coli</i> MG1655 (●) or the isogenic Δ<i>ligB</i> mutant (○) as a function of cadmium concentration. Lag times were calculated with a custom R program [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180800#pone.0180800.ref018" target="_blank">18</a>]. Error bars denote standard deviation.</p

Effect of <i>ligB</i> deletion on antibiotic MICs.

<p>Effect of <i>ligB</i> deletion on antibiotic MICs.</p

<i>E</i>. <i>coli</i> ligase B.

<p>(A) Ligase B amino acid sequence similarity tree. Ligase B peptide sequences from 46 species of Enterobacteriaceae are shown compared to the outgroup, ligase A. The distance of 0.2 substitutions per site is indicated. (B) The conserved <i>ligB</i> gene neighborhood (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180800#pone.0180800.s002" target="_blank">S2 Fig</a>), represented by the region in <i>E</i>. <i>coli</i> MG1655.</p