Unexpected aspects in the dynamics of horizontal gene transfer of prokaryotes: the impact of outer membrane vesicles.

Horizontal gene transfer (HGT) was observed by incubation of an amino acid-deficient strain of Escherichia coli (AB1157) with particles gained from an oligotrophic environment, when all deficiencies were restored with frequencies up to 1.94â€¯× 10-5 and no preference for a single marker. Hence, the DNA transfer to the revertant cells was carried out by generalized transduction. Those particles display structural features of outer membrane vesicles (OMVs) but contain high amounts of DNA. Due to a process called serial transduction, the revertant's particles were likewise transferring genetic information to deficient E. coli AB1157 cells. These results indicate a new way of HGT, in which mobilized DNA is transferred in particles from the donor to the recipient. Extracted OMV-associated DNA of known alpha-, and gamma-proteobacterials, Ahrensia kielensis and Pseudoalteromonas marina, respectively, was larger than 30 kbp with all sequences in single copy and identified as prokaryotic sequences. Inserted viral sequences were not found.

Towards a framework for assessment and management of cumulative human impacts on marine food webs.

Effective ecosystem-based management requires understanding ecosystem responses to multiple human threats, rather than focusing on single threats. To understand ecosystem responses to anthropogenic threats holistically, it is necessary to know how threats affect different components within ecosystems and ultimately alter ecosystem functioning. We used a case study of a Mediterranean seagrass (Posidonia oceanica) food web and expert knowledge elicitation in an application of the initial steps of a framework for assessment of cumulative human impacts on food webs. We produced a conceptual seagrass food web model, determined the main trophic relationships, identified the main threats to the food web components, and assessed the components' vulnerability to those threats. Some threats had high (e.g., coastal infrastructure) or low impacts (e.g., agricultural runoff) on all food web components, whereas others (e.g., introduced carnivores) had very different impacts on each component. Partitioning the ecosystem into its components enabled us to identify threats previously overlooked and to reevaluate the importance of threats commonly perceived as major. By incorporating this understanding of system vulnerability with data on changes in the state of each threat (e.g., decreasing domestic pollution and increasing fishing) into a food web model, managers may be better able to estimate and predict cumulative human impacts on ecosystems and to prioritize conservation actions.

Hacia un Marco de Trabajo para la Evaluación y el Manejo de los Impactos Humanos Acumulativos sobre las Redes Alimenticias Marinas Resumen El manejo efectivo con base en los ecosistemas requiere entender la respuesta de los ecosistemas a múltiples amenazas humanas en lugar de enfocarse en amenazas individuales. Para entender holísticamente la respuesta de los ecosistemas a las múltiples amenazas antropogénicas es necesario saber cómo estas amenazas afectan a los diferentes componentes dentro de los ecosistemas y cómo alteran finalmente el funcionamiento de los ecosistemas. Usamos el estudio de caso de la red alimenticia del pasto marino del Mediterráneo (Posidonia oceanica) y la obtención de conocimiento de expertos en una aplicación de los pasos iniciales de un método para la evaluación de los impactos humanos acumulativos sobre las redes alimenticias. Produjimos un modelo de red alimenticia de pastos marinos, determinamos las principales relaciones tróficas, identificamos a las principales amenazas para los componentes de la red y evaluamos la vulnerabilidad de los componentes a esas amenazas. Algunas amenazas tuvieron impactos altos (p. ej.: infraestructura costera) o bajos (p. ej.: escorrentía agrícola) sobre todos los componentes de la red, mientras que otros (p. ej.: carnívoros introducidos) tuvieron impactos muy diferentes sobre cada componente. Partir al ecosistema en sus componentes nos permitió identificar amenazas no vistas previamente y reevaluar la importancia de las amenazas percibidas comúnmente como mayores. Al incorporar este entendimiento de la vulnerabilidad del sistema con datos sobre los cambios en el estado de cada amenaza (p. ej.: disminución de la contaminación doméstica e incremento de la pesca) al modelo de red alimenticia, los manejadores pueden ser capaces de estimar y predecir de mejor manera los impactos humanos acumulativos sobre los ecosistemas y priorizar las acciones de conservación.

DNA-bearing membrane vesicles produced by Ahrensia kielensis and Pseudoalteromonas marina.

Outer membrane vesicles (OMVs) derived from the alphaproteobacterium Ahrensia kielensis and from Pseudoalteromonas marina, a gammaproteobacterium, were sampled from liquid cultures in order to extract the MV-associated DNA, establish a shotgun library, and sequence randomly chosen clones to determine the origins of their DNA. We show that OMVs from A. kielensis and from P. marina both harbour DNA larger than 20 or 30 kbp. Transmission electron microscopical inspection of OMVs of A. kielensis and P. marina showed two types of vesicles: bilayered OMVs with a diameter between 30 and 250 nm and double bilayered OMVs ranging between 80 and 200 nm. Bilayered OMVs are either characterized by the presence of a large electron-dense substance or are elctron translucent. Double bilayered OMVs contained an electron dense substance in the core region surrounded by the second bilayer. 30,094 bp of the genome from OMV of A. kielensis and 45,981 bp of that from P. marina were sequenced. The results indicated that all sequences were single copy and that all sequences, with one exception, were similar to prokaryotic sequences, inserted viral sequences were not detected.

Mobilizable bacterial DNA packaged into membrane vesicles induces serial transduction.

Experiments where the amino acid deficient strain E. coli AB1157 was exposed to a particle fraction harvested from a marine oligotrophic environment, ranging in diameter size between 100-130 nm indicated evidence for horizontal gene transfer resulting in revertant cells with restoration of all genetic deficiencies with frequencies up to 1.94 × 10(-5). None of the markers was preferentially transferred indicating that the DNA-transfer is performed by generalized transduction. The highest transfer frequency obtained for single markers was 1.04 × 10(-2). All revertant strains were able to produce particles of comparable size that were again infectious, appearing at the beginning of the stationary phase. Ultra structural investigation showed a structural resemblance with membrane vesicles, however, Field Gel Electrophoresis indicated that the DNA content of some of the particles was 370 kbp; much higher than that of the so far known previously described membrane vesicles providing evidence of a new mechanism for horizontal gene transfer.

Evidence for particle-induced horizontal gene transfer and serial transduction between bacteria.

Incubation of the amino acid-deficient strain Escherichia coli AB1157 with particles harvested from an oligotrophic environment revealed evidence of horizontal gene transfer (HGT) with restoration of all deficiencies in revertant cells with frequencies up to 1.94 × 10(-5). None of the markers were preferentially transferred, indicating that the DNA transfer is performed by generalized transduction. The highest gene transfer frequencies were obtained for single markers, with values up to 1.04 × 10(-2). All revertants were able to produce particles of comparable size, appearing at the beginning of the stationary phase. Examination of the revertants using electron microscopy showed bud-like structures with electron-dense bodies. The particles that display the structural features of membrane vesicles were again infectious to E. coli AB1157, producing new infectious particles able to transduce genetic information, a phenomenon termed serial transduction. Thus, the <0.2-µm particle fraction from seawater contains a particle size fraction with high potential for gene transfer. Biased sinusoidal field gel electrophoresis indicated a DNA content for the particles of 370 kbp, which was higher than that of known membrane vesicles. These findings provide evidence of a new method of HGT, in which mobilizable DNA is trafficked from donor to recipient cells via particles.

Development of the bacterial compartment along the Danube River: a continuum despite local influences.

Microbial food webs dominate heterotrophic food webs in large rivers with bacterial metabolism being a key component of carbon processing. Thus, analysis of bacterial population dynamics is critical to understanding patterns and mechanisms of material cycling and energy fluxes in large rivers. Within the frame of the Joint Danube Survey (JDS) 2007, the longitudinal development of the natural bacterial community in the Danube in terms of bacterial numbers, morphotype composition, and heterotrophic production of the suspended and particle-attached fractions was followed at a fine spatial resolution of approximately 30 km for the first time in such a large river along a 2,600-km stretch. Twenty-one major tributaries and branches were also included. This allowed us to investigate whether bacterial standing stock and production undergo continuous, linear changes or whether discontinuities and local processes like the merging of tributaries or the potential impact of sewage input drive the bacterial population in the Danube. The presented investigation revealed surprising continuous patterns of changes of bacterial parameters along the Danube River. Despite the presence of impoundments or hydropower plants, large municipalities, and the discharge of large tributaries, most bacterial parameters (standing stock, morphotype succession, and attached bacterial production) developed gradually, indicating that mainly broad-scale drivers and not local conditions shape and control the bacterial community in the midstream of this large river. As most important broad-scale drivers, nutrients (inorganic and organic) and changes in particle concentrations were identified. These data are also in remarkable accordance with the patterns of changes of the genetic bacterial community composition, observed during the first JDS (2001) 6 years before. In contrast, bacterial activity did not follow a continuous trend and was mainly controlled by the input of sewage from large cities in the middle section, leading to a bloom of phytoplankton. The observed patterns and the comparison between the Danube, its tributaries and other large rivers worldwide indicate that the bacterial community in rivers has a powerful indicator function for estimating the ecological status of large river ecosystems once enough information has been collected at various temporal and spatial scales.

Microbiological water quality along the Danube River: integrating data from two whole-river surveys and a transnational monitoring network.

The River Danube is, with 2780 km, the second longest river in Europe. Its catchment area covers 801 500 km(2), with approximately 81 million inhabitants in 19 countries. River water for anthropogenic use, transportation and recreation is of major importance in all of these countries. Microbiological contamination from faecal pollution by anthropogenic sources is considered to be a crucial problem throughout the Danube River basin. Thus, detailed knowledge on the extent and the origin of microbial pollution is essential for watershed management. The determination of faecal indicator concentrations along the Danube and its major tributaries during two whole-river surveys and 16 permanent stations allowed for the first time to draw a clear picture of the faecal pollution patterns along the whole longitudinal profile of this important international river. By including a variety of environmental variables in statistical analysis, an integrative picture of faecal pollution in the Danube River basin could be evolved. Four hot spots and six stretches of differing faecal pollution were identified, mainly linked with input from large municipalities. Significant decline of microbiological pollution was observed in the upper and lower Danube stretches over the investigation period. In contrast, a significant increase in the middle part was evident. The planned implementation of new wastewater treatment plants and advanced wastewater treatment measures according to the European Union urban wastewater directive will have a great potential to reduce microbial faecal pollution in the Danube and thus improving water quality.

Drosophila P transposons of the urochordata Ciona intestinalis.

P transposons belong to the eukaryotic DNA transposons, which are transposed by a cut and paste mechanism using a P-element-coded transposase. They have been detected in Drosophila, and reside as single copies and stable homologous sequences in many vertebrate species. We present the P elements Pcin1, Pcin2 and Pcin3 from Ciona intestinalis, a species of the most primitive chordates, and compare them with those from Ciona savignyi. They showed typical DNA transposon structures, namely terminal inverted repeats and target site duplications. The coding region of Pcin1 consisted of 13 small exons that could be translated into a P-transposon-homologous protein. C. intestinalis and C. savignyi displayed nearly the same phenotype. However, their P elements were highly divergent and the assumed P transposase from C. intestinalis was more closely related to the transposase from Drosophila melanogaster than to the transposase of C. savignyi. The present study showed that P elements with typical features of transposable DNA elements may be found already at the base of the chordate lineage.

Effects of deposit-feeding macrofauna on benthic bacteria, viruses, and protozoa in a silty freshwater sediment.

In microcosm experiments, we simultaneously tested the effects of increased numbers of deposit-feeding macrofauna (chironomids, oligochaetes and cladocerans) on the standing stock, activities and interactions of heterotrophic bacteria, viruses, and bacterivorous protozoa (heterotrophic nanoflagellates and ciliates) in the aerobic layer of a silty littoral freshwater sediment. On average, bacterial secondary production was stimulated between 11 and 29% by all macrofaunal groups compared to control experiments without macrofauna addition. Bacterial standing stock increased significantly by 8 and 13% in case of chironomids and cladocerans, respectively. Oligochaetes and chironomids produced significant negative effects on viral abundance while the results with cladocerans were inconsistent. The addition of oligochaetes and chironomids resulted in a significant decrease by on average 68 and 32% of viral decay rates, respectively, used as a measure of viral production. The calculated contribution of virus-induced lysis to benthic bacterial mortality was low, with 2.8 to 11.8% of bacterial secondary production, and decreased by 39 to 81% after the addition of macrofauna compared to the control. The abundances of heterotrophic nanoflagellates were significantly reduced by 20% by all tested macrofauna groups, while ciliates showed inconsistent results. The importance of heterotrophic nanoflagellate grazing on benthic bacteria was very low (<1% of bacterial secondary production) and was further reduced by elevated numbers of macrofauna. Thus, the selected deposit feeding macrofauna groups seem to have several direct and indirect and partly antagonistic effects on the benthic bacterial compartment through the enhancement of bacterial production and the reduction of virus-induced cell lysis and protozoan grazing.

Contribution of virus-induced lysis and protozoan grazing to benthic bacterial mortality estimated simultaneously in microcosms.

In contrast to the water column, the fate of bacterial production in freshwater sediments is still a matter of debate. Thus, the importance of virus-induced lysis and protozoan grazing of bacteria was investigated for the first time simultaneously in a silty sediment layer of a mesotrophic oxbow lake. Microcosms were installed in the laboratory in order to study the dynamics of these processes over 15 days. All microbial and physicochemical parameters showed acceptable resemblance to field data observed during a concomitant in situ study, and similar conclusions can be drawn with respect to the quantitative impact of viruses and protozoa on the bacterial compartment. Viral decay rates ranged from undetectable to 0.078 h(-1) (average, 0.033 h(-1)), and the control of bacterial production from below the detection limit to 36% (average, 12%). The contribution of virus-induced lysis of bacteria to the dissolved organic matter pool as well as to benthic bacterial nutrition was low. Ingestion rates of protozoan grazers ranged from undetectable to 24.7 bacteria per heterotrophic nanoflagellate (HNF) per hour (average, 4.8 bacteria HNF(-1) h(-1)) and from undetectable to 73.3 bacteria per ciliate per hour (average, 11.2 bacteria ciliate(-1) h(-1)). Heterotrophic nanoflagellate and ciliates together cropped up to 5% (average, 1%) of bacterial production. The viral impact on bacteria prevailed over protozoan grazing by a factor of 2.5-19.9 (average, 9.5). In sum, these factors together removed up to 36% (average, 12%) of bacterial production. The high number of correlations between viral and protozoan parameters is discussed in view of a possible relationship between virus removal and the presence of protozoan grazers.

Benthic and pelagic viral decay experiments: a model-based analysis and its applicability.

The viral decay in sediments, that is, the decrease in benthic viral concentration over time, was recorded after inhibiting the production of new viruses. Assuming that the viral abundance in an aquatic system remains constant and that viruses from lysed bacterial cells replace viruses lost by decay, the decay of viral particles can be used as a measure of viral production. Decay experiments showed that this approach is a useful tool to assess benthic viral production. However, the time course pattern of the decay experiments makes their interpretation difficult, regardless of whether viral decay is determined in the water column or in sediments. Different curve-fitting approaches (logarithmic function, power function, and linear regression) to describe the time course of decay experiments found in the literature are used in the present study and compared to a proposed "exponential decay" model based on the assumption that at any moment the decay is proportional to the amount of viruses present. Thus, an equation of the form dVA/dt = -k x VA leading to the time-integrated form VAt = VA0 x e(-k x t) was used, where k represents the viral decay rate (h(-1)), VAt is the viral abundance (viral particles ml(-1)) at time t (h), and VA0 is the initial viral abundance (viral particles ml(-1)). This approach represents the best solution for an accurate curve fitting based on a mathematical model for a realistic description of viral decay occurring in aquatic systems. Decay rates ranged from 0.0282 to 0.0696 h(-1) (mean, 0.0464 h(-1)). Additionally, a mathematical model is presented that enables the quantification of the viral control of bacterial production. The viral impact on bacteria based on decay rates calculated from the different mathematical approaches varied widely within one and the same decay experiment. A comparison of the viral control of bacterial production in different aquatic environments is, therefore, improper when different mathematical formulas are used to interpret viral decay experiments.

Does virus-induced lysis contribute significantly to bacterial mortality in the oxygenated sediment layer of shallow oxbow lakes?

Despite the recognition that viruses are ubiquitous components of aquatic ecosystems, the number of studies on viral abundance and the ecological role of viruses in sediments is scarce. In this investigation, the interactions between viruses and bacteria were studied in the oxygenated silty sediment layer of a mesotrophic oxbow lake. A long-term study (13 months) and a diel study revealed that viruses are a numerically important and dynamic component of the microbial community. The abundance and decay rates ranged from 4.3 x 10(9) to 7.2 x 10(9) particles ml of wet sediment(-1) and from undetectable to 22.2 x 10(7) particles ml(-1) h(-1), respectively, and on average the values were 2 orders of magnitude higher than the values for the overlying water. In contrast to our expectations, viruses did not contribute significantly to the bacterial mortality in the sediment, since on average only 6% (range, 0 to 25%) of the bacterial secondary production was controlled by viruses. The low impact of viruses on the bacterial community may be associated with the quantitatively low viral burden that benthic bacteria have to cope with compared to the viral burden with which bacterial assemblages in the water column are confronted. The virus-to-bacterium ratio of the sediment varied between 0.9 and 3.2, compared to a range of 5.0 to 12.4 obtained for the water column. We speculate that despite high numbers of potential hosts, the possibility of encountering a host cell is limited by the physical conditions in the sediment, which is therefore not a favorable environment for viral proliferation. Our data suggest that viruses do not play an important role in the processing and transfer of bacterial carbon in the oxygenated sediment layer of the environment investigated.

Erythrobacter citreus sp. nov., a yellow-pigmented bacterium that lacks bacteriochlorophyll a, isolated from the western Mediterranean Sea.

Two facultatively oligotrophic, intensely yellow-pigmented bacterial strains, RE35F/1T and RE10F/45, have been previously isolated from the western Mediterranean Sea (Bay of Calvi, Corsica, France) by 0.2 microm membrane filtration. The organisms were gram-negative, catalase- and oxidase-positive, strictly aerobic, rod-shaped and non-motile. Their respiratory lipoquinone profiles consisted exclusively of ubiquinone-10 (Q-10) and the G+C contents of their DNAs were 62.0 and 62.4 mol%, respectively. Among the cellular fatty acids, octadecenoic acid (18:1omega7c) was the major component. Both isolates also contained hydroxy fatty acids (14:0 2-OH, 18:1 2-OH and 16:0 iso 3-OH) and branched fatty acids (15:0 anteiso, 16:0 anteiso and 17:0 anteiso). Polar lipid fingerprints were characterized by the presence of a sphingoglycolipid. Comparative analyses of their 16S rRNA gene sequences indicated that both isolates were phylogenetically closely related (sequence similarity of 99.9%) and formed a coherent cluster with aerobic bacteriochlorophyll a-containing species of the Erythrobacter/Porphyrobacter/Erythromicrobium cluster within the family Sphingomonadaceae. The closest relative was Erythrobacter litoralis DSM 8509T (97.4 and 97.5% 16S rRNA gene sequence similarity between this strain and RE35F/1T and RE10F/45, respectively). DNA-DNA reassociation studies confirmed that strains RE35F/1T and RE10F/45 represent a single species (79.6% DNA homology), but also demonstrated that they do not belong to the species Erythrobacter litoralis (25.2 and 34.2% DNA homology, respectively). Notably, both RE35F/1T and RE10F/45 lacked bacteriochlorophyll a. Based upon phenotypic and molecular evidence, a novel species of the genus Erythrobacter, Erythrobacter citreus sp. nov., is proposed. Strain RE35F/1T (= CIP 107092T = DSM 14432T) is the type strain.

Vibrio calviensis sp. nov., a halophilic, facultatively oligotrophic 0.2 microm-fiIterabIe marine bacterium.

A gram-negative, facultatively anaerobic, straight to slightly curved rod-shaped bacterium (RE35F/12T) sensitive to vibriostatic agent O/129 was previously isolated from sea water (Western Mediterranean Sea, Bay of Calvi, Corsica, France) by 0.2 microm-membrane filtration. Strain RE35/F12T (= CIP 107077T = DSM 14347T) was facultatively oligotrophic, halophilic, required Na+ for growth and produced acid but no gas from D-glucose under anaerobic conditions. Comparative 165 rRNA gene-sequence analyses demonstrated that the bacterium is most closely related (94.3%) to Vibrio scophthalmi. Similarities to the sequences of all other established Vibrio species ranged from 93.6% (with Vibrio aestuarianus) to 90.7% (with Vibrio rumoiensis). Strain RE35/F12T occupies a distinct phylogenetic position; this is similar to the case of Vibrio hollisae, because RE35F/12T represents a relatively long subline of descent sharing a branching point with the outskirts species V. hollisae. The G+C content of the DNA was 49.5 mol%. Ubiquinone Q-8 was the main respiratory lipoquinone, and 16:1omega9cis, 16:0 and 18:1trans9, cis11 were the major cellular fatty acids, 16:1omega9cis being predominant. The polyamine pattern was characterized by the presence of the triamine sym-norspermidine. On the basis of the polyphasic information summarized above, a new Vibrio species is described for which the name Vibrio calviensis sp. nov. is proposed.

[Growth and Age Determination in the Sea Fan Eunicella cavolinii]. / Wachstum und Altersbestimmung der Gorgonie Eunicella cavolinii.

Growth of the sea fan Eunicella cavolinii was studied on the south coast of Filicudi, an island north-west of Sicily. Colonies, ranging from 1 to 100 cm in total length were observed for 2 years and 10 series of measurements taken during this period, including all seasons of the year. Each branch was measured separately.Total growth and the frequency of development of new branches indicated noncontinious growth over the year. Differences between the mean growth rates at different times of the year were treated by an analysis of variance for dependent samples and the significance tested by a Duncan test. Between February and April the gorgonians grow significantly faster than during the rest of the year. Measurements of temperature and of water movement show a correlation of the period of maximum growth with minimum temperature and maximum water movement. Reduced growth during the summer is probably due to high light intensities. The intrinsic rate of natural increase could be calculated and the age of the colonies estimated. An empirical growth curve is approximated by a theoretical model. Estimates of the age of colonies up to the maximum possible size are thus possible.