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

Charakterisierung der Typ-IV-Sekretionssysteme und der kryptischen Plasmide von Helicobacter pylori hinsichtlich ihrer Beteiligung am DNA-Transfer

H. pylori kolonisiert die Magenmukosa von etwa 50% der Bevölkerung und ist die Ursache von vielen schweren Erkrankungen, wie z.B. chronischer Gastritis, Magengeschwüren und Magenkrebs. Lange Zeit wurde angenommen, dass das Milieu des Magens aufgrund der dort herrschenden Bedingungen steril sei. H. pylori hat Strategien entwickelt, um dieses Habitat zu besiedeln und stellt den dominierenden Bestandteil der Mikroflora im Magen dar. Eine entscheidende Voraussetzung für die erfolgreiche Kolonisierung ist die genetische Diversität und die damit verbundene Anpassung an die verschiedenen Mikronischen des Magens. Die Pathogenität von H. pylori wird nicht nur durch Toxine vermittelt, sondern resultiert aus der komplexen Interaktion zwischen dem Bakterium, dem Wirt und der Umwelt. Eine wichtige Bedeutung hierbei hat der Austausch von genetischem Material. Während die natürliche Kompetenz eine entscheidende Rolle für die Aufnahme von genetischem Material spielt, wird auch ein konjugativer Mechanismus zum Transfer von DNA diskutiert. Im Rahmen dieser Arbeit wurde erstmals der DNaseI-resistente Transfer der intrinsischen, kryptischen Plasmide pHel4 und pHel12 zwischen H. pylori-Stämmen nachgewiesen. Es konnte gezeigt werden, dass für diesen Mechanismus sowohl die Plasmid-, als auch die chromosomal-kodierten Relaxasen nicht essentiell sind. Möglicherweise wird die Rezirkularisierung der Plasmid-DNA im Rezipienten durch RecA durchgeführt. Um Informationen über die Maschinerie, welche den DNaseI-geschützten Transfer der intrinsischen Plasmide vermittelt zu erhalten, wurden alle in H. pylori P12 identifizierten T4SS mit Hilfe einer Kontraselektionsstrategie sequentiell deletiert und Kokultivierungsexperimente mit den entsprechenden Mutanten durchgeführt. Es konnte gezeigt werden, dass außer dem ComB-System keines der T4SS für den DNA-Transfer zwischen H. pylori-Stämmen essentiell ist. Dieses ist für die Aufnahme von DNA im Rezipienten verantwortlich und spielt auch eine Rolle für den DNA-Export durch den Donor. Das ComB-System stellt somit das entscheidende T4SS für den Transfer von DNA dar und hat eine duale Funktion hinsichtlich Transformation und einem Konjugations-ähnlichen Mechanismus im Donor und Rezipienten. Bemerkenswert ist, dass auch nach Deletion aller T4SS in H. pylori P12 DNA-Transfer stattfindet. Mögliche Kandidaten für einen alternativen DNA-Übertragungsweg, stellen Membranvesikel dar. Darüber hinaus konnte nachgewiesen werden, dass Tfs4 Plasmid-DNA in das umgebende Milieu sekretiert. Durch die Sekretion des Modul-artig aufgebauten kryptischen Plasmids pHel12 kann die Verbreitung von genetischem Material zwischen Stämmen unterstützt werden. Die Unabhängigkeit des DNA-Transfermechanismus von den Relaxasen, sowie die Resistenz gegenüber dem Angriff durch DNaseI lassen einen neuartigen, Konjugations-ähnlichen DNA-Transfermechanismus vermuten, der von der konventionellen Konjugation abgrenzt werden kann. Neben der Charakterisierung der DNA-Transfer-Mechanismen in H. pylori P12 wurden im Rahmen dieser Arbeit auch die kryptischen Plasmide pHel4 und pHel12 und die mögliche Funktion ihrer Genprodukte untersucht. Etwa 50% aller klinischen Isolate enthalten kryptische Plasmide. Ihre Funktion ist bisher allerdings nicht klar. Die Anwesenheit einer mob-Region deutete auf eine konjugative Übertragung der Plasmide hin. Darüber hinaus lässt die Struktur der Plasmide eine Rolle bei der Verbreitung von genetischem Material als Orte des „gene shufflings“ vermuten. Zudem konnten erste Hinweise bezüglich der mit den Plasmiden verbundenen Zytotoxizität bestätigt werden. So beeinflusst die Expression von orf4M aus pHel4 und orf12M aus pHel12 in eukaryotischen Zellen die zelluläre Integrität und führt schließlich zum Zelltod. Die kryptischen Plasmide stellen eine interessante Möglichkeit für H. pylori dar, genetische Information auszutauschen und möglicherweise die Wirtszelle zu beeinflussen

Transport and kinase activities of CbrA of Pseudomonas putida KT2440

The CbrA/CbrB system is a two-component signal transduction system known to participate in the regulation of the cellular carbon/nitrogen balance and to play a central role in carbon catabolite repression in Pseudomonas species. CbrA is composed of a domain with similarity to proteins of the solute/sodium symporter family (SLC5) and domains typically found in bacterial sensor kinases. Here, the functional properties of the sensor kinase CbrA and its domains are analyzed at the molecular level using the system of the soil bacterium P. putida KT2440 as a model. It is demonstrated that CbrA can bind and transport L-histidine. Transport is specific for L-histidine and probably driven by an electrochemical proton gradient. The kinase domain is not required for L-histidine uptake by the SLC5 domain of CbrA, and has no significant impact on transport kinetics. Furthermore, it is shown that the histidine kinase can autophosphorylate and transfer the phosphoryl group to the response regulator CbrB. The SLC5 domain is not essential for these activities but appears to modulate the autokinase activity. A phosphatase activity of CbrA is not detected. None of the activities is significantly affected by L-histidine. The results demonstrate that CbrA functions as a L-histidine transporter and sensor kinase

Effekte einer Düngung mit elementarem Schwefel auf einer 4-schnittigen Dauerwiese im Alpenraum

Schwefel ist ein wichtiger Pflanzennährstoff und die Immissionen aus der Atmosphäre gingen seit 1980 stark zurück. Unter diesen Rahmenbedingungen wurde der Einsatz eines elementaren Schwefeldüngers ("Sulfogüll plus" von BvG) auf einer 4-schnittigen Dauerwiese getestet. Vier Schwefeldüngungsstufen von 0 (S0), 30 (S30), 60 (S60) und 90 (S90) kg S/ha stellten die Varianten dar. Variante S60 und S90 erreichten die signifikant höchste TM- (S60 10.779 und S90 11.073 kg/ha) XP- (S60 1.749 und S90 1798 kg/ha) und Energie-Erträge (S60 64.008 und S90 65.370 MJ/ha) im Vergleich zur Variante S0 (TM: 9.947 kg/ha, XP: 1.569 kg/ha, Energie: 58.434 MJ NEL/ha). Ein Ziel der Bio-Landwirtschaft ist, möglichst viel Eiweiß aus dem Grünland zu gewinnen. In diesem Fall dürfte die Schwefeldüngung in Zukunft eine wichtige Rolle spielen

Correlation of Pretreatment Polarographically Measured Oxygen Pressures with Quantified Contrast-Enhanced Power Doppler Ultrasonography in Spontaneous Canine Tumors and their Impact on Outcome After Radiation Therapy

Purpose: : To evaluate the use of noninvasive quantified contrast-enhanced power Doppler ultrasonography as a surrogate in the estimation of tumor hypoxia measured by invasive pO2 histography in canine tumors. Material and Methods: : Data of pretreatment tumor oxygenation status, tumor vascularity and blood volume, and tumor response after radiation therapy was collected in 48 spontaneous malignant oral tumors (Table 1). Tumor oxygenation status was correlated to vascularity and blood volume, and influences on outcome after treatment were analyzed. Results: : Although vascularity and blood volume correlated moderately with median pO2 (r = 0.51 and 0.61; p = 0.001 and < 0.0001) and percentage of pO2 readings ≤ 2.5, 5, and 10 mmHg (r = -0.37 to -0.42; p < 0.01-0.03) for all tumors, they did not correlate within the different histology groups (p = 0.06-0.9). For all tumors, pretreatment oxygenation status, vascularity and blood volume were not found to be of prognostic value (Tables 2 and 3). Conclusion: : These analyses show that quantified contrast-enhanced power Doppler ultrasonography does not represent a noninvasive indirect method to assess tumor hypoxia measured by invasive pO2 histography. Both technologies were nonprognostic indicators in spontaneous malignant canine oral tumor

Influence of Pretreatment Polarographically Measured Oxygenation Levels in Spontaneous Canine Tumors Treated with Radiation Therapy

Background and Purpose:: The level of hypoxia in primary tumors has been described to influence response to treatment. The aim of the present study was to investigate the impact of pretreatment oxygen level measurements in spontaneous canine tumors on treatment outcome. Materials and Methods:: Data of pretreatment tumor oxygenation status and local tumor response after primary radiation therapy in a group of spontaneously occurring tumors in dogs (n = 52) was collected. Radiation therapy was given with curative (14-17 × 3-3.5 Gy) or palliative intent (3 × 8 Gy or 4-5 × 6 Gy). Progression-free interval and overall survival were correlated to polarographically measured tumor oxygenation status. Results:: In the curatively irradiated group, tumors with median pO2 values ≤ 10 mmHg tended to have shorter median progression- free interval compared to better oxygenated tumors (246 vs. 739 days). The same trend could be shown for overall survival (330 vs. 745 days), indicating a cutoff value in this region. In the group treated with lower doses of radiation, the level of oxygen was no longer found to be of prognostic value; however, in this group hemoglobin had a significant impact on outcome. Conclusion:: In curatively irradiated spontaneous canine tumors, tumor hypoxia was found to be a prognostic indicator, independent of tumor histologies and volum

Strain-specific genes of Helicobacter pylori: genome evolution driven by a novel type IV secretion system and genomic island transfer

The availability of multiple bacterial genome sequences has revealed a surprising extent of variability among strains of the same species. The human gastric pathogen Helicobacter pylori is known as one of the most genetically diverse species. We have compared the genome sequence of the duodenal ulcer strain P12 and six other H. pylori genomes to elucidate the genetic repertoire and genome evolution mechanisms of this species. In agreement with previous findings, we estimate that the core genome comprises about 1200 genes and that H. pylori possesses an open pan-genome. Strain-specific genes are preferentially located at potential genome rearrangement sites or in distinct plasticity zones, suggesting two different mechanisms of genome evolution. The P12 genome contains three plasticity zones, two of which encode type IV secretion systems and have typical features of genomic islands. We demonstrate for the first time that one of these islands is capable of self-excision and horizontal transfer by a conjugative process. We also show that excision is mediated by a protein of the XerD family of tyrosine recombinases. Thus, in addition to its natural transformation competence, conjugative transfer of genomic islands has to be considered as an important source of genetic diversity in H. pylori

The rainy season in the Southern Peruvian Andes: A climatological analysis based on the new Climandes index

The rainy season is of high importance for livelihoods in the Southern Peruvian Andes (SPA), especially for agriculture, which is mainly rain fed and one of the main income sources in the region. Therefore, knowledge and predictions of the rainy season such as its onset and ending are crucial for planning purposes. However, such information is currently not readily available for the local population. Moreover, an evaluation of existing rainy season indices shows that they are not optimally suited for the SPA and may not be directly applicable in a forecasting context. Therefore, we develop a new index, named Climandes index, which is tailored to the SPA and designed to be of use for operational monitoring and forecasting purposes. Using this index, we analyse the climatology and trends of the rainy season in the SPA. We find that the rainy season starts roughly between September and January with durations between 3 and 8 months. Both onset and duration show a pronounced northeast-southwest gradient, regions closer to the Amazon Basin have a considerably longer rainy season. The inter-annual variability of the onset is very high, that is, 2–5 months depending on the station, while the end of the rainy season shows a much lower variability (i.e., 1.5–3 months). The spatial patterns of total precipitation amount and dry spells within the rainy season are only weakly related to its timing. Trends in rainy season characteristics since 1965 are mostly weak and not significant, but generally indicate a tendency towards a shortening of the rainy season in the whole study area due to a later onset and an increase in precipitation sums during the rainy season in the northwestern study area

A combined view on precipitation and temperature climatology and trends in the southern Andes of Peru

In the southern Peruvian Andes, communities are highly dependent on climatic conditions due to the mainly rain-fed agriculture and the importance of glaciers and snow melt as a freshwater resource. Longer-term trends and year-to-year variability of precipitation or temperature severely affect living conditions. This study evaluates seasonal precipitation and temperature climatologies and trends in the period 1965/66–2017/18 for the southern Peruvian Andes using quality-controlled and homogenized station data and new observational gridded data. In this region, precipitation exhibits a strong annual cycle with very dry winter months and most of the precipitation falling from spring to autumn. Spatially, a northeast–southwest gradient in austral spring is observed, related to an earlier start of the rainy season in the northeastern partof the study area. Seasonal variations of maximum temperature are weak withan annual maximum in austral spring, which is related to reduced cloud coverin austral spring compared to summer. On the contrary, minimum tempera-tures show larger seasonal variations, possibly enhanced through changes inlongwave incoming radiation following the precipitation cycle. Precipitationtrends since 1965 exhibit low spatial consistency except for austral summer,when in most of the study area increasing precipitation is observed, and in aus-tral spring, when stations in the central-western region of the study area regis-ter decreasing precipitation. All seasonal and annual trends in maximum temperature are larger than trends in minimum temperature. Maximum temperature exhibits strong trends in austral winter and spring, whereas minimum temperature trends are strongest in austral winter. We hypothesize, that these trends are related to precipitation changes, as decreasing (increasing) precipita-tion in spring (summer) may enhance maximum (minimum) temperature trends through changes in cloud cover. El Niño Southern Oscillation (ENSO), however, has modifying effects onto precipitation and temperature, and thereby leads to larger trends in maximum temperatures