Bacillus anthracis pXO1 minireplicon (MR) plasmid consisting of open reading frames (ORFs) GBAA_pXO1_0020 toGBAA_pXO1_0023 is not stably maintained in B. anthracis, whereas the full-size parent pXO1 plasmid (having 181,677 bp and 217 ORFs) is extremely stable under the same growth conditions. Two genetic tools developed for DNA manipulation in B. anthracis (Cre-loxP and Flp-FRT systems) were used to identify pXO1 regions important for plasmid stability. We localized a large segment of pXO1 that enables stable plasmid maintenance during vegetative growth. Further genetic analysis identified three genes that are necessary for pXO1 maintenance: amsP (GBAA_pXO1_0069), minP (GBAA_pXO1_0082), and sojP (GBAA_pXO1_0084). Analysis of conserved domains in the corresponding proteins indicated that only AmsP (activator of maintenance system of pXO1) is predicted to bind DNA, due to its strong helix-turn-helix domain. Two conserved domains were found in the
We previously identified three noncontiguous regions on Bacillus anthracis plasmid pXO1 that comprise a system for accurate plasmid partitioning and maintenance. However, deletion of these regions did not decrease retention of certain shortened pXO1 plasmids during vegetative growth. Using two genetic tools developed for DNA manipulation in B. anthracis (the Cre-loxP and Flp-FRT systems), we found two other noncontiguous pXO1 regions that together are sufficient for plasmid stability. This second pXO1 maintenance system includes the tubZ and tubR genes, characteristic of a type III partitioning system, and the IntXO recombinase gene (GBAA_RS29165), encoding a tyrosine recombinase, along with its adjacent 37-bp perfect stem-loop (PSL) target. Insertion of either the tubZ and tubR genes or the IntXO-PSL system into an unstable mini-pXO1 plasmid did not restore plasmid stability. The need for the two components of the second pXO1 maintenance system follows from the sequential roles of IntXO-PSL in generating monomeric circular daughter pXO1 molecules (thereby presumably preventing dimer catastrophe) and of TubZ/TubR in partitioning the monomers during cell division. We show that the IntXO recombinase deletes DNA regions located between two PSL sites in a manner similar to the actions of the Cre-loxP and Flp-FRT systems. IMPORTANCETyrosine recombinases catalyze cutting and joining reactions between short specific DNA sequences. Three types of reactions occur: integration and excision of DNA segments, inversion of DNA segments, and separation of monomeric forms from replicating circular DNA molecules. Here we show that the newly discovered site-specific IntXO-PSL recombinase system that contributes to the maintenance of the B. anthracis plasmid pXO1 can be used for genome engineering in a manner similar to that of the Cre-loxP or Flp-FRT system. T he large low-copy-number pXO1 plasmid (181,677 bp) of Bacillus anthracis encodes the anthrax toxin proteins and other virulence-related factors. A pXO1 minireplicon plasmid (pMR) comprised of two essential open reading frames (ORFs) (GBAA_RS28535 and GBAA_RS28545) (Fig. 1A) replicates but is not stably maintained in B. anthracis, whereas the full-size parent pXO1 plasmid (carrying 217 ORFs) is extremely stable under the same growth conditions (1). (Table S1 in the supplemental material lists all genes and proteins discussed in this work.) Recently we found that retention of pMR can be stabilized by insertion of several noncontiguous pXO1 regions containing three genes which work cooperatively to achieve plasmid maintenance: amsP (GBAA_RS28725), minP (GBAA_RS28775), and sojP (GBAA_ RS28785) (2). The minP and sojP genes encode proteins belonging to the ParA/MinD family described by Lutkenhaus (3). MinD is involved in spatial regulation of the cytokinetic Z ring, and ParA proteins are involved in chromosome and plasmid segregation (3).The amsP-, minP-, and sojP-encoded proteins, comprising the plasmid maintenance system (maintenance system I [MSI]) identified in our pr...
The immediate products of x-ray absorption in aqueous biological samples are free radicals including *OH, H 2 O 2 , *H and solvated electrons. Because their lifetimes and diffusion ranges are dependent on the local bio-molecular environment, imaging these free radicals in real-time while they are produced by a scanning x-ray nanobeam may provide a biological microscopy method of high resolution. As a first step towards this goal, we investigated the feasibility of imaging the initial free radical products of x-ray absorption in live cells using fluorescent free radical sensors. We selected six commercially available fluorescent sensors for screening tests of their sensitivities towards x-ray radiation in solution form. Two of the six dyes were found to have high sensitivities. One of the two was successfully used for dynamic confocal fluorescence imaging of x-ray generated free radicals in the intracellular space of mouse embryonic fibroblasts. Time series of fluorescence images before and during x-ray radiation were acquired. The rate of increase of cellular fluorescence showed both the initial production of free radicals by the physical ionization events as well as stimulated biological production of reactive oxygen species later on. The implications of the results for future development of microscopy techniques are discussed.
Introduction The incidence of and risk factors for exertional heat illness (EHI) and cold weather injury (CWI) in the U.S. Army have been well documented. The “heat season”, when the risk of EHI is highest and application of risk mitigation procedures is mandatory, has been arbitrarily defined as May 1 through September 30, while the “cold season” is understood to occur from October 1 to April 30 each year. The proportions of EHI and CWI that occur outside of the traditional heat and cold seasons are unknown. Additionally, it is unknown if either of the seasonal definitions are appropriate. The primary purpose of this study was to determine the proportion of EHI and of CWI that occur within the commonly accepted seasonal definitions. We also report the location-specific variability, seasonal definitions, and the demographic characteristics of the populations. Methods The U.S. Army installations with the highest frequency of EHI and of CWI from 2008 to 2013 were identified and used for analysis. In total there were 15 installations included in the study, with five installations used for analysis in both the EHI and CWI projects. In- and out-patient EHI and CWI data (ICD-9-CM codes 992.0 to 992.9 and ICD codes 991.0 to 991.9, respectively) were obtained from the Defense Medical Surveillance System. Installation-specific denominator data were obtained from the Defense Manpower Data Center, and incidence rates were calculated by week, for each installation. Segmental (piecewise) regression analysis was used to determine the start and end of the heat and cold seasons. Results Our analysis indicates that the heat season starts around April 22 and ends around September 9. The cold season starts on October 3 and ends on March 24. The majority (n = 6,445, 82.3%) of EHIs were diagnosed during the “heat season” of May 1 to September 30, while 10.3% occurred before the heat season started (January1 to April 30) and 7.3% occurred after the heat season ended (October 1 to December 31). Similar to EHI, 90.5% of all CWIs occurred within the traditionally defined cold season, while 5.7% occurred before and 3.8% occurred after the cold season. The locations with the greatest EHI frequency were Ft Bragg (n = 2,129), Ft Benning (n = 1,560), and Ft Jackson (n = 1,538). The bases with the largest proportion of CWI in this sample were Ft Bragg (17.8%), Ft Wainwright (17.2%), and Ft Jackson (12.7%). There were considerable inter-installation differences for the start and end dates of the respective seasons. Conclusions The present study indicates that the traditional heat season definition should be revised to begin ∼3 weeks earlier than the current date of May 1; our data indicate that the current cold season definition is appropriate. Inter-installation variability in the start of the cold season was much larger than that for the heat season. Exertional heat illnesses are a year-round problem, with ∼17% of all cases occurring during non-summer months, when environmental heat strain and vigilance are lower. This suggests that EHI mitigation policies and procedures require greater year-round emphasis, particularly at certain locations.
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