mwr Xer site-specific recombination is hypersensitive to DNA supercoiling

The multiresistance plasmid pJHCMW1, first identified in a Klebsiella pneumoniae strain isolated from a neonate with meningitis, includes a Xer recombination site, mwr, with unique characteristics. Efficiency of resolution of mwr-containing plasmid dimers is strongly dependent on the osmotic pressure of the growth medium. An increase in supercoiling density of plasmid DNA was observed as the osmotic pressure of the growth culture decreased. Reporter plasmids containing directly repeated mwr, or the related cer sites were used to test if DNA topological changes were correlated with significant changes in efficiency of Xer recombination. Quantification of Holliday junctions showed that while recombination at cer was efficient at all levels of negative supercoiling, recombination at mwr became markedly less efficient as the level of supercoiling was reduced. These results support a model in which modifications at the level of supercoiling density caused by changes in the osmotic pressure of the culture medium affects resolution of mwr-containing plasmid dimers, a property that separates mwr from other Xer recombination target sites

The NASA Solar Cruiser Mission - Solar Sail Propulsion Enabling Heliophysics Missions

Solar Cruiser is a Small Satellite Technology Demonstration Mission (TDM) to mature solar sail propulsion technology to enable near-term, high-priority breakthrough science missions as defined in the Solar and Space Physics Decadal Survey. Solar sails have the potential to provide high ΔV for many types of missions. Solar sails are large, mirror-like structures made of a lightweight material that reflects sunlight to propel the spacecraft. The continuous solar photon pressure provides thrust with no need for the heavy, expendable propellants used by conventional chemical and electric propulsion systems. Solar Cruiser will demonstrate a “sailcraft” platform with pointing control and attitude stability comparable to traditional platforms, upon which a new class of Heliophysics missions may fly. It will show sailcraft operation (acceleration, navigation, station keeping, heliocentric plane change) scalability of sail technologies such as the boom, membrane, and deployer to enable more demanding missions, such as high inclination solar imaging. Solar Cruiser will launch as a secondary payload with NASA’s Interstellar Mapping and Acceleration Probe (IMAP) in early 2025. The sailcraft will separate from the launch vehicle on a near-L1 trajectory (Sun-Earth Lagrangian Point 1; sunward of L1 along the Sun-Earth Line) and complete its primary mission in 11 months or less

Klebsiella pneumoniae Multiresistance Plasmid pMET1: Similarity with the Yersinia pestis Plasmid pCRY and Integrative Conjugative Elements

Dissemination of antimicrobial resistance genes has become an important public health and biodefense threat. Plasmids are important contributors to the rapid acquisition of antibiotic resistance by pathogenic bacteria.The nucleotide sequence of the Klebsiella pneumoniae multiresistance plasmid pMET1 comprises 41,723 bp and includes Tn1331.2, a transposon that carries the bla(TEM-1) gene and a perfect duplication of a 3-kbp region including the aac(6')-Ib, aadA1, and bla(OXA-9) genes. The replication region of pMET1 has been identified. Replication is independent of DNA polymerase I, and the replication region is highly related to that of the cryptic Yersinia pestis 91001 plasmid pCRY. The potential partition region has the general organization known as the parFG locus. The self-transmissible pMET1 plasmid includes a type IV secretion system consisting of proteins that make up the mating pair formation complex (Mpf) and the DNA transfer (Dtr) system. The Mpf is highly related to those in the plasmid pCRY, the mobilizable high-pathogenicity island from E. coli ECOR31 (HPI(ECOR31)), which has been proposed to be an integrative conjugative element (ICE) progenitor of high-pathogenicity islands in other Enterobacteriaceae including Yersinia species, and ICE(Kp1), an ICE found in a K. pneumoniae strain causing primary liver abscess. The Dtr MobB and MobC proteins are highly related to those of pCRY, but the endonuclease is related to that of plasmid pK245 and has no significant homology with the protein of similar function in pCRY. The region upstream of mobB includes the putative oriT and shares 90% identity with the same region in the HPI(ECOR31).The comparative analyses of pMET1 with pCRY, HPI(ECOR31), and ICE(Kp1 )show a very active rate of genetic exchanges between Enterobacteriaceae including Yersinia species, which represents a high public health and biodefense threat due to transfer of multiple resistance genes to pathogenic Yersinia strains

The power of engagement: implementation of a career ladder program

At Baystate Health in Massachusetts, the development and implementation of a career ladder program was implemented to reduce turnover and to improve employee satisfaction, morale, and recruitment efforts. There was significant initial expenditure in the program, as a result of promoting the large number of employees with significant experience and seniority. A smaller number of staff are expected to apply for advancement during successive cycles, allowing for decreased incremental expense going forward. Critical to the success of the program was understanding the time commitment, getting senior organizational support and staff buy-in, and justifying the associated expenses. The development and initiation of the program has done much to support a positive work environment with increased morale and higher performance among significant numbers of staff at all levels

Sublethal Concentrations of the Aminoglycoside Amikacin Interfere with Cell Division without Affecting Chromosome Dynamics

Aminoglycosides bind to the 16S rRNA at the tRNA acceptor site (A site) and disturb protein synthesis by inducing codon misreading. We investigated Escherichia coli cell elongation and division, as well as the dynamics of chromosome replication and segregation, in the presence of sublethal concentrations of amikacin (AMK). The fates of the chromosome ori and ter loci were monitored by visualization by using derivatives of LacI and TetR fused to fluorescent proteins in E. coli strains that carry operator arrays at the appropriate locations. The results showed that cultures containing sublethal concentrations of AMK contained abnormally elongated cells. The chromosomes in these cells were properly located, suggesting that the dynamics of replication and segregation were normal. FtsZ, an essential protein in the process of cell division, was studied by using an ectopic FtsZ-cyan fluorescent protein fusion. Consistent with a defect in cell division, we revealed that the Z ring failed to properly assemble in these elongated cells

Differences in Resolution of mwr-Containing Plasmid Dimers Mediated by the Klebsiella pneumoniae and Escherichia coli XerC Recombinases: Potential Implications in Dissemination of Antibiotic Resistance Genes

Xer-mediated dimer resolution at the mwr site of the multiresistance plasmid pJHCMW1 is osmoregulated in Escherichia coli containing either the Escherichia coli Xer recombination machinery or Xer recombination elements from K. pneumoniae. In the presence of K. pneumoniae XerC (XerC(Kp)), the efficiency of recombination is lower than that in the presence of the E. coli XerC (XerC(Ec)) and the level of dimer resolution is insufficient to stabilize the plasmid, even at low osmolarity. This lower efficiency of recombination at mwr is observed in the presence of E. coli or K. pneumoniae XerD proteins. Mutagenesis experiments identified a region near the N terminus of XerC(Kp) responsible for the lower level of recombination catalyzed by XerC(Kp) at mwr. This region encompasses the second half of the predicted α-helix B and the beginning of the predicted α-helix C. The efficiencies of recombination at other sites such as dif or cer in the presence of XerC(Kp) or XerC(Ec) are comparable. Therefore, XerC(Kp) is an active recombinase whose action is impaired on the mwr recombination site. This characteristic may result in restriction of the host range of plasmids carrying this site, a phenomenon that may have important implications in the dissemination of antibiotic resistance genes

Genetic structures located upstream of <i>parF</i> and <i>parG.</i>

<p>A. The direct repeats within the pMET1 putative <i>parH</i>-like locus are shown in red. The diagram also shows the −35 and −10 sequences, as well as the inverted repeats (arrows). The inverted repeat within the putative <i>parH</i> locus is shown in blue. The beginning of the ParF amino acid sequence including the deviant Walker motif A and motif A' are shown. B. Logo plot <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001800#pone.0001800-Crooks1" target="_blank">[60]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001800#pone.0001800-Schneider1" target="_blank">[61]</a> of a multiple alignment of the direct repeats shown in red.</p

Genetic map of pMET1 and comparison to plasmid pCRY and chromosomal elements.

<p>A. The genetic maps of pMET1 and pCRY are compared showing the homologous regions. The arrows indicate genes locations and orientation. Genes with different functions are shown with different colors and if the genes in the different structures shown are homologus they are represented with the same colors. Yellow: mobilization; green: replication and partition; red: antibiotic resistance; purple: virB/pilX-like; blue: transposition; grey: unknown. Since pCRY is smaller than pMET1, to represent it in circular form a dotted line was added to fill the gap. Solid line represents non-homologous DNA. B. Comparison of a pMET1 region with chromosomal HPIs or ICEs is shown using a linearized version of the plasmid. The HPIs shown are those from <i>E. coli</i> ECOR31 (HPI_ECOR31) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001800#pone.0001800-Schubert1" target="_blank">[43]</a>, <i>K. pneumoniae</i> NTUH-K2044 (ICE_Kp1) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001800#pone.0001800-Lin1" target="_blank">[44]</a>, and <i>Y. pestis</i> KIM (HPI_Yp)<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001800#pone.0001800-Schubert1" target="_blank">[43]</a>. The diagram shows the HP core regions, which are not at scale and are represented as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001800#pone.0001800-Schubert1" target="_blank">[43]</a>, and the RB-HPIs. The sequence described in this manuscript has been deposited in GenBank, accession number is EU383016.</p

Replication region of pMET1.

<p>A. The bar shows a genetic map of the pMET1 replication region and the GC content plot generated using a window size of 100 bp on top. Recombinant clones were obtained by inserting the indicated fragments into pCR2.1 or ligated to the pUC4K <i>aph</i> cassette. The ability to be maintained in <i>E. coli</i> C2110 (a <i>polA</i> mutant) of the recombinant plasmids made using pCR2.1 as vector is indicated to the right by a + or − sign. The ability to generate kanamycin resistant colonies in <i>E. coli</i> TOP10 of the indicated fragments when ligated to the <i>aph</i> cassette from pUC4K is also represented by a + or − sign. B. BLASTP comparison of the amino acid sequences of the putative RepA proteins from pMET1 and pCRY.</p