Bridged filaments of histone-like nucleoid structuring protein pause RNA polymerase and aid termination in bacteria.

Bacterial H-NS forms nucleoprotein filaments that spread on DNA and bridge distant DNA sites. H-NS filaments co-localize with sites of Rho-dependent termination in Escherichia coli, but their direct effects on transcriptional pausing and termination are untested. In this study, we report that bridged H-NS filaments strongly increase pausing by E. coli RNA polymerase at a subset of pause sites with high potential for backtracking. Bridged but not linear H-NS filaments promoted Rho-dependent termination by increasing pause dwell times and the kinetic window for Rho action. By observing single H-NS filaments and elongating RNA polymerase molecules using atomic force microscopy, we established that bridged filaments surround paused complexes. Our results favor a model in which H-NS-constrained changes in DNA supercoiling driven by transcription promote pausing at backtracking-susceptible sites. Our findings provide a mechanistic rationale for H-NS stimulation of Rho-dependent termination in horizontally transferred genes and during pervasive antisense and noncoding transcription in bacteria

P1 Ref Endonuclease: A Molecular Mechanism for Phage-Enhanced Antibiotic Lethality

<div><p>Ref is an HNH superfamily endonuclease that only cleaves DNA to which RecA protein is bound. The enigmatic physiological function of this unusual enzyme is defined here. Lysogenization by bacteriophage P1 renders <i>E</i>. <i>coli</i> more sensitive to the DNA-damaging antibiotic ciprofloxacin, an example of a phenomenon termed phage-antibiotic synergy (PAS). The complementary effect of phage P1 is uniquely traced to the P1-encoded gene <i>ref</i>. Ref is a P1 function that amplifies the lytic cycle under conditions when the bacterial SOS response is induced due to DNA damage. The effect of Ref is multifaceted. DNA binding by Ref interferes with normal DNA metabolism, and the nuclease activity of Ref enhances genome degradation. Ref also inhibits cell division independently of the SOS response. Ref gene expression is toxic to <i>E</i>. <i>coli</i> in the absence of other P1 functions, both alone and in combination with antibiotics. The RecA proteins of human pathogens <i>Neisseria gonorrhoeae</i> and <i>Staphylococcus aureus</i> serve as cofactors for Ref-mediated DNA cleavage. Ref is especially toxic during the bacterial SOS response and the limited growth of stationary phase cultures, targeting aspects of bacterial physiology that are closely associated with the development of bacterial pathogen persistence.</p></div

Validation of algorithms to ascertain clinical conditions and medical procedures used during pregnancy

PURPOSE: To evaluate the validity of health plan administrative and claims data to identify pre-gestational and gestational diabetes, obesity, and ultrasounds among pregnant women. METHODS: A retrospective study was conducted using the administrative and claims data of three health plans participating in the HMO Research Network. Diagnoses, drug dispensings, and procedure codes were used to identify diabetes, obesity, and ultrasounds among women who were pregnant between January 2006 and December 2008. A random sample of medical charts (n = 222) were abstracted. Positive predictive values (PPVs) were calculated. Sensitivity also was calculated for obesity among women for whom body mass index data were available in electronic medical records at two sites. RESULTS: Overall, 190 of 222 cases of diabetes (86%) were confirmed (82% for gestational diabetes and 74% for pre-gestational diabetes). The PPV for codes to identify ultrasounds was 80%. Whereas the PPV for obesity-related diagnosis codes was high (93%), and the sensitivity was low (33%). CONCLUSIONS: Health plan administrative and claims data can be used to accurately identify pre-gestational and gestational diabetes and ultrasounds. Obesity is not consistently coded

Ref protein can cleave DNA bound by RecA from pathogenic bacterial species.

<p>Agarose gel of circular single-stranded (css) DNA (4 μMnt) incubated with RecA protein from different bacterial species (2.4 μM) and Ref (100 nM) for 20 minutes at 37°C. All cssDNA bound by <i>E</i>. <i>coli</i> and <i>N</i>. <i>gonorrhoeae</i> RecA protein was degraded within 1 hour. P1 Ref protein showed slightly reduced nuclease activity on DNA bound by RecA from <i>Staphylococcus aureus</i> and minimal low activity on DNA bound by RecA from <i>Pseudomonas aeruginosa</i>.</p

Expression of the N-terminal DNA binding domain of Ref induces the SOS response.

<p>A. Induction of the SOS response in P1 lysogens by 8 ng/mL ciprofloxacin. <i>E</i>. <i>coli</i> strains EAR15, EAR16, and EAR17 (non-lysogen, P1<i>ref</i>^<i>+</i>lysogen, and P1<i>Δref</i> lysogen, respectively) were grown at 30°C. Each strain contained a plasmid (pEAW903) expressing SuperGlo GFP from the <i>recN</i> promoter, allowing the SOS response to be reported as fluorescence. After reaching log phase, ciprofloxacin (8 ng/mL) or water was added (time = 0) and growth continued under the same conditions. Δfluorescence (as calculated in Methods) average and standard deviation of three biological and four technical replicates is reported. B. SOS induction by strains expressing Ref in the absence of phage P1. <i>E</i>. <i>coli</i> strains EAR86 (EV), EAR87 (pRef) EAR88 (pRefΔC110), EAR 120 (pRefΔN76), EAR121 (pRef^nuc-) and EAR123 (pRbsR) harboring protein expression plasmids and an SOS reporter plasmid were grown to log-phase at 30°C. Cultures were treated with 1% arabinose to induce protein expression (time = 0). Fluorescence and optical density were measured as in panel A. Normalized fluorescence average and standard deviation of at least two biological and two technical replicates is reported for each condition. C. Microscopy of <i>E</i>. <i>coli</i> cells expressing Ref variants. Log phase cultures of EAR61 (EV), EAR62 (pRef), EAR73 (pRefΔC110), EAR98 (pRefΔN76), and EAR105 (pRef^nuc-) at 1x10⁸ CFU/mL were treated with 1% arabinose. Cells were outgrown for 4 hours and incubated with DAPI before imaging at 600x magnification using brightfield and fluorescence channels. Scale bar = 10 μm, representative images shown. D. Quantification of cell length data from (C) was obtained using the MicrobeTracker plugin for MatLab [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005797#pgen.1005797.ref066" target="_blank">66</a>]. Each counted cell is represented by a single data point with the average and standard deviation for the data shown. At least 100 cells were measured for each condition, except WT Ref in which only 39 cells could be found. **** = p-value <0.0001.</p

Ref expression causes cell filamentation independent of the <i>E</i>. <i>coli</i> SOS response.

<p><i>A</i>. Microscopy of cells expressing Ref or RefΔC110. WT <i>E</i>. <i>coli</i> strains EAR61 (EV), EAR62 (pRef), EAR73 (pRefΔC110), <i>ΔrecA</i> strains EAR64 (EV), EAR65 (pRef), EAR74 (pRefΔC110), <i>sulA</i>^<i>-</i> strains EAR77 (EV), EAR78 (pRef), EAR79 (pRefΔC110), and <i>sulA</i>^<i>-</i> <i>lexA3</i> strains EAR69 (EV), EAR70 (pRef), and EAR75 (pRefΔC110) were grown to 1x10⁸ CFU/mL, treated with 1% arabinose, outgrown for 4 hours, and images were obtained as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005797#pgen.1005797.g004" target="_blank">Fig 4C</a>. Scale bar = 10 μm, representative images shown. B. Quantification of cell length data from (A) was obtained using the MicrobeTracker plugin for MatLab. Each counted cell is represented by a single data point with the average and standard deviation for the data shown. An average of 90 cells (range: 39–188) were counted for each condition. ** = p-value <0.0001 when compared to EV in same background, * = p-value <0.05 when compared to same vector in WT background. C. Cell survival after expression of Ref. WT <i>E</i>. <i>coli</i> strains EAR61 (EV), EAR62 (pRef), EAR73 (pRefΔC110), <i>ΔrecA</i> strains EAR64 (EV), EAR65 (pRef), EAR74 (pRefΔC110) were treated as in (A). Cells were plated for viability and the average and standard deviation of at least three biological replicates for each condition are reported (error bars small and not visible in some cases). Significant p-values are noted in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005797#pgen.1005797.s002" target="_blank">S1 Table</a>.</p

Ref is toxic to <i>E</i>. <i>coli</i> in stationary phase and enhances lethal effects of DNA-damaging antibiotics.

<p>A. Cell survival during Ref and variant expression. WT <i>E</i>. <i>coli</i> strains EAR61 (EV), EAR62 (pRef), EAR73 (pRefΔC110), EAR98 (pRefΔN76), EAR105 (pRef^nuc-), EAR104 (pRbsR), and <i>ΔrecA</i> strains EAR64 (EV) and EAR65 (pRef) were grown to log-phase, adjusted to 5x10⁵ CFU/mL, treated with 1% arabinose, and outgrown for one day at 30°C. Cells were plated and the average and standard deviation of CFU/mL for at least three biological replicates for each condition are reported. Significant p-values can be found in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005797#pgen.1005797.s002" target="_blank">S1 Table</a>. B. WT <i>E</i>. <i>coli</i> strains EAR61 (EV) and EAR62 (pRef) were grown as described in (A), but with the addition of water or 8 ng/mL ciprofloxacin in addition to 1% arabinose. Cells were plated and data was reported as in (A). * = p-value<0.001, when compared to EV with same treatment. C. Same as (B) except 5 μg/mL mitomycin C was used instead of ciprofloxacin. * = p-value<0.001, when compared to EV with same treatment. D. Same as (B) except 0.5 μg/mL trimethoprim was used instead of ciprofloxacin. * = p-value<0.001, when compared to EV with same treatment. E. Images of cultures from (B) after overnight incubation. F. Quantification of viable cells in panel (E). Average and standard deviation of 4–8 biological replicate cultures are reported. * = p-value<0.001, when compared to EV with same treatment. G. Microscopy of cells in panel (E), obtained as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005797#pgen.1005797.g004" target="_blank">Fig 4C</a>. Scale bar = 10 μm.</p

Phage-antibiotic synergy with bacteriophage P1 and ciprofloxacin.

<p>A. Survival of P1 lysogens after treatment with 8 ng/mL ciprofloxacin. Log phase cultures of <i>E</i>. <i>coli</i> MG1655, EAR2 (P1<i>ref+)</i> or EAW195 (P1<i>Δref</i>), were adjusted to 5x10⁵ CFU/mL and were treated with water (open markers) or 8 ng/mL ciprofloxacin (filled markers) with six hours outgrowth. The average CFU/mL and standard deviation of three biological replicates is reported (error bars are present, but smaller than markers in most cases). * = p-value<0.0001, when compared to MG1655 treated with 8 ng/mL ciprofloxacin. B. Optical density of P1 lysogens treated with ciprofloxacin. Log phase cultures of above strains were treated with water (open markers) or 4 ng/mL ciprofloxacin (filled markers) with outgrowth at 30°C. The average OD₅₉₅ and standard deviation of five biological and two technical replicates for each condition is reported. C. Optical density of P1 lysogens induced with a temperature shift. Log phase cultures of strains in (B) were shifted from 30°C to 42°C to induce temperature-sensitive phage lysis and optical density was tracked as in (B). The average and standard deviation of three biological and three technical replicates is reported.</p

Multiple roles for Ref protein during the P1 lytic cycle.

<p>When a P1 lysogen is exposed to a DNA damaging agent (such as cipro), RecA filaments form near the damaged DNA sites. This causes (1) <i>E</i>. <i>coli</i> SOS response induction, (2) induction of phage lytic cycle, (3) inhibition of cell division. Ref protein is produced during the phage lytic cycle and performs three functions: (1) the Ref N-terminal DNA binding domain binds bacterial DNA, inhibiting other DNA metabolism enzymes, (2) the nuclease active site of Ref cleaves RecA-bound DNA to enhance the bacterial SOS response, (3) Ref inhibits cell division mainly <i>via</i> its C-terminal domain, and possibly through an interaction with the bacterial divisome.</p

<i>ref</i> is a P1 lytic cycle gene.

<p>A. Rt-qPCR of P1 <i>ref</i> gene expression during temperature-induced lytic development of phage P1Cm C1.100. Total RNA was isolated and cDNA was obtained at times indicated. Portions of the <i>ref</i> (P1 phage) and <i>cysG</i> (<i>E</i>. <i>coli</i>) genes were amplified in triplicate using qPCR. Fold induction (y-axis) was calculated using the method of Livak and Schmittgen [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005797#pgen.1005797.ref056" target="_blank">56</a>]. The average and standard deviation of three biological replicates is reported. B. Maximum fold-induction values for other P1 genes during the lytic switch, C1 repressor controlled genes = red. Fold-induction data was obtained and calculated as in A. Values within 20% of the maximum fold-induction for each gene in each biological replicate were averaged to obtain the maximum fold induction time and value for each gene. C. Lysogens formed upon infection of MG1655 with P1Cm C1.100. MG1655 culture was supplemented with CaCl₂ and incubated with P1Cm C1.100 phage, followed by the addition of sodium citrate to chelate calcium ions and prevent superinfection. After recovery in LB, cells were serially diluted and plated on LB and LB/chloramphenicol (shown here) to select for phage lysogens. D. Colonies from plates in C were counted and the lysogenization efficiency (lysogens/viable cells) average and standard deviation from three biological replicate infections are reported. * = p-value<0.0001.</p