Multiple pathways of plasmid DNA transfer in Helicobacter pylori

Many Helicobacter pylori (Hp) strains carry cryptic plasmids of different size and gene content, the function of which is not well understood. A subgroup of these plasmids (e.g. pHel4, pHel12), contain a mobilisation region, but no cognate type IV secretion system (T4SS) for conjugative transfer. Instead, certain H. pylori strains (e.g. strain P12 carrying plasmid pHel12) can harbour up to four T4SSs in their genome (cag-T4SS, comB, tfs3, tfs4). Here, we show that such indigenous plasmids can be efficiently transferred between H. pylori strains, even in the presence of extracellular DNaseI eliminating natural transformation. Knockout of a plasmid-encoded mobA relaxase gene significantly reduced plasmid DNA transfer in the presence of DNaseI, suggesting a DNA conjugation or mobilisation process. To identify the T4SS involved in this conjugative DNA transfer, each individual T4SS was consecutively deleted from the bacterial chromosome. Using a marker-free counterselectable gene deletion procedure (rpsL counterselection method), a P12 mutant strain was finally obtained with no single T4SS (P12ΔT4SS). Mating experiments using these mutants identified the comB T4SS in the recipient strain as the major mediator of plasmid DNA transfer between H. pylori strains, both in a DNaseI-sensitive (natural transformation) as well as a DNaseI-resistant manner (conjugative transfer). However, transfer of a pHel12::cat plasmid from a P12ΔT4SS donor strain into a P12ΔT4SS recipient strain provided evidence for the existence of a third, T4SS-independent mechanism of DNA transfer. This novel type of plasmid DNA transfer, designated as alternate DNaseI-Resistant (ADR) mechanism, is observed at a rather low frequency under in vitro conditions. Taken together, our study describes for the first time the existence of three distinct pathways of plasmid DNA transfer between H. pylori underscoring the importance of horizontal gene transfer for this species

Empirical Analysis Revealing Privileged Chemical Space of Cosmetic Preservatives

Most cosmetic products require preservation to prevent microbial contamination and to ensure consumer safety. Due to regulatory restrictions and rejection by consumers, preservative options have become limited and the development of novel solutions is needed. This search can be guided by knowledge about favorable chemical space for cosmetic preservatives. Therefore, we used preservatives allowed in the EU as training set and calculated various molecular properties. Empirical analysis revealed two separated areas of privileged chemical space with the net charge as distinctive property. The first area comprises the group of neutral and anionic preservatives and is characterized by low molecular size as well as limited hydrogen-bonding capacity, polarity, and flexibility. The second area includes cationic preservatives, which are rather diffusely distributed regarding molecular weight and hydrogen-bonding, however, all members share high flexibility. Both groups significantly differ from antibiotics, reflecting the specific requirement of cosmetic preservation. The molecular properties defining the privileged chemical space are easy to calculate, and thus, can provide guidance for the development of novel preservatives

Bacterial strains used in this study.

<p>Bacterial strains used in this study.</p

Power Wheelchair Use in Persons with ALS: Changes over Time

<div><p></p><p>Objectives: To survey persons with ALS at 1 month and 6 months after receiving power wheelchairs to determine long term use, comfort and function as well as the power wheelchair impact on daily tasks and quality of life.</p><p>Methods: 33-question survey and Psychosocial Impact of Assistive Devices Scale (PIADS) was sent 1 month after getting a new power wheelchair, follow up survey sent at 6 months.</p><p>Results: Based on satisfaction and feature use survey results, at one month, 81% of users found the power wheelchair overall comfort to he high, 88% found their overall mobility to be improved and 95% found it easy to use. Their quality of life increased and pain decreased at one month and six months. According to the PIADS, the power wheelchair gave users increased ability to participate and sense of competence.</p><p>Conclusions: This study has important results for the ALS community, as it is the first to assess power wheelchair users at one month and six months after power wheelchair procurement. Our results demonstrate the impact the power wheelchair has on mobility, psychosocial issues, functional abilities and quality of life for a person with ALS.</p></div

Role of chromosomally encoded relaxases for transfer of plasmid pHel12.

<p>(A) DNA transfer rates of plasmid pHel12::<i>cat</i> from a P12Δ<i>recA</i> donor carrying precise marker-free deletions of <i>tfs3</i> or <i>tfs4</i> including the adjacent relaxase genes <i>virD2</i> (see Materials and Methods for construction of the deletions) into a P12 recipient strain carrying a chromosomal kanamycin resistance gene (<i>aphA</i>-3). (B) DNA transfer of plasmid pHel12::<i>cat</i> from a P12Δ<i>recA</i> donor carrying marker-free deletions of both, <i>tfs3</i> and <i>tfs4</i> including the adjacent relaxase genes <i>virD2</i> into a P12 recipient strain. Transfer rates were determined as the number of transconjugants/cfu/donor. Data shown are mean values of at least three independent experiments including standard deviations. *, p<0.05; ns, not significant (p>0.05) according to students t-test.</p

Marker-free deletions of all T4SS in the <i>H. pylori</i> P12 (P12ΔT4SS) donor and recipient strain prove the existence of an alternate DNaseI-resistant (ADR) pathway of DNA transfer.

<p>(A) Schematic depiction of DNA exchange between <i>H. pylori</i> P12 donor and recipient strains both of which are devoid of any T4SS to support natural transformation or conjugative plasmid transfer. The recipient strain carries a kanamycin resistance gene (<i>aphA-3</i>) in the chromosomal <i>moeB</i> locus, the donor strain either carries a <i>cat</i> gene in pHel12 (pHel12::<i>cat</i>) (B), or <i>cat</i> replaces the <i>mobA</i> gene (pHel12Δ<i>mobA</i>::<i>cat)</i> (C). DNA transfer rates of plasmid pHel12::<i>cat</i> (B) or pHel12Δ<i>mobA</i>::<i>cat</i> (C) from a P12ΔT4SS donor strain into a P12ΔT4SS recipient strain carrying an <i>aphA-3</i> cassette in the <i>moeB</i> locus are shown. Transfer rates were determined as the number of transconjugants/cfu/ml. Data shown are mean values of at least three independent experiments including standard deviations.</p

Confirmation of pHel plasmid transfer into transconjugants.

<p>(A) Isolation of plasmid DNA from transconjugants after co-cultivation of <i>H. pylori</i> P12 donor and recipient strains. Isolation of plasmid DNA after co-cultivation of <i>H. pylori</i> P12 pHel12::<i>cat</i> (donor) with <i>H. pylori</i> P12[<i>moeB::aphA-3</i>]. Presence of the donor plasmid pHel12::<i>cat</i> in the recipient strain (red arrow, lane 3,4) and of the recipient plasmid pHel12 (black arrow, lanes 3 and 4) is shown. Lane 1: 1 kb ladder, lane 2: pHel12, lane 3: transconjugant 1, lane 4: transconjugant 2. (B) Scheme explaining the PCR for the specific detection of donor- and recipient plasmid in transconjugants using oligos SR57 and SR58. (C) PCR fragments obtained with primers SR57/SR58 for the donor plasmid (red arrow, 1.3 kb) and for the original pHel12 plasmid of the recipient strain (black arrow, 0.4 kb). Lane 1, 1 kb ladder, lane 2, pHel12 used as template, lane 3 and 4, plasmid DNA from transconjugants 1 and 2 used as template.</p