Convergent evolution of heat-inducibility during subfunctionalization of the Hsp70 gene family

Background: Heat-shock proteins of the 70 kDa family (Hsp70s) are essential chaperones required for key cellular functions. In eukaryotes, four subfamilies can be distinguished according to their function and localisation in different cellular compartments: cytosol, endoplasmic reticulum, mitochondria and chloroplasts. Generally, multiple cytosol-type Hsp70s can be found in metazoans that show either constitutive expression and/or stress-inducibility, arguing for the evolution of different tasks and functions. Information about the hsp70 copy number and diversity in microbial eukaryotes is, however, scarce, and detailed knowledge about the differential gene expression in most protists is lacking. Therefore, we have characterised the Hsp70 gene family of Paramecium caudatum to gain insight into the evolution and differential heat stress response of the distinct family members in protists and to investigate the diversification of eukaryotic hsp70s focusing on the evolution of heat-inducibility. Results: Eleven putative hsp70 genes could be detected in P. caudatum comprising homologs of three major Hsp70-subfamilies. Phylogenetic analyses revealed five evolutionarily distinct Hsp70-groups, each with a closer relationship to orthologous sequences of Paramecium tetraurelia than to another P. caudatum Hsp70-group. These highly diverse, paralogous groups resulted from duplications preceding Paramecium speciation, underwent divergent evolution and were subject to purifying selection. Heat-shock treatments were performed to test for differential expression patterns among the five Hsp70-groups as well as for a functional conservation within Paramecium. These treatments induced exceptionally high mRNA up-regulations in one cytosolic group with a low basal expression, indicative for the major heat inducible hsp70s. All other groups showed comparatively high basal expression levels and moderate heat-inducibility, signifying constitutively expressed genes. Comparative EST analyses for P. tetraurelia hsp70s unveiled a corresponding expression pattern, which supports a functionally conserved evolution of the Hsp70 gene family in Paramecium. Conclusions: Our analyses suggest an independent evolution of the heat-inducible cytosol-type hsp70s in Paramecium and in its close relative Tetrahymena, as well as within higher eukaryotes. This result indicates convergent evolution during hsp70 subfunctionalization and implies that heat-inducibility evolved several times during the course of eukaryotic evolution

High variability of plasmid uptake rates in Escherichia coli isolated from sewage and river sediments

The horizontal transfer of plasmids is a key mechanism behind the spread of antibiotic resistance in bacteria. So far, transfer rate constants were measured for a variety of plasmids, donors and recipients. The employed strains typically had a long history in laboratories. Existing data are, therefore, not necessarily representative for real-world environments. Moreover, information on the inter-strain variability of plasmid transfer rates is scarce. Using a high-throughput approach, we studied the uptake of RP4 by various Escherichia coli recipients using Serratia marcescens as the donor. The recipient strains were isolated from human-borne sewage and river sediments. The rate constants of plasmid transfer generally followed a log-normal distribution with considerable variance. The rate constants for good and poor recipients (95 and 5% quantile) differed by more than three orders of magnitude. Specifically, the inter-strain variability of the rate constant was large in comparison to alterations induced by low-level antibiotic exposure. We did not find evidence for diverging efficiencies of plasmid uptake between E. coli recipients of different origin. On average, strains isolated from river bottom sediments were equally efficient in the acquisition of RP4 as isolates extracted from sewage. We conclude that E. coli strains persisting in the aquatic environment and those of direct human origin share a similar intrinsic potential for the conjugative uptake of certain plasmids. In view of the large inter-strain variability, we propose to work towards probabilistic modeling of the environmental spread of antibiotic resistance.Peer reviewe

Sewage from Airplanes Exhibits High Abundance and Diversity of Antibiotic Resistance Genes

Airplane sanitary facilities are shared by an international audience. We hypothesized the corresponding sewage to be an extraordinary source of antibiotic-resistant bacteria (ARB) and resistance genes (ARG) in terms of diversity and quantity. Accordingly, we analyzed ARG and ARB in airplane-borne sewage using complementary approaches: metagenomics, quantitative polymerase chain reaction (qPCR), and cultivation. For the purpose of comparison, we also quantified ARG and ARB in the inlets of municipal treatment plants with and without connection to airports. As expected, airplane sewage contained an extraordinarily rich set of mobile ARG, and the relative abundances of genes were mostly increased compared to typical raw sewage of municipal origin. Moreover, combined resistance against third-generation cephalosporins, fluorochinolones, and aminoglycosides was unusually common (28.9%) among Escherichia coli isolated from airplane sewage. This percentage exceeds the one reported for German clinical isolates by a factor of 8. Our findings suggest that airplane-borne sewage can effectively contribute to the fast and global spread of antibiotic resistance.Peer reviewe

Fitness Impact of Obligate Intranuclear Bacterial Symbionts Depends on Host Growth Phase

According to text book definition, parasites reduce the fitness of their hosts whereas mutualists provide benefits. But biotic and abiotic factors influence symbiotic interactions, thus under certain circumstances parasites can provide benefits and mutualists can harm their host. Here we addressed the question which intrinsic biotic factors shape a symbiosis and are crucial for the outcome of the interaction between the obligate intranuclear bacterium Holospora caryophila (Alphaproteobacteria; Rickettsiales) and its unicellular eukaryotic host Paramecium biaurelia (Alveolata; Ciliophora). The virulence of H. caryophila, i.e., the negative fitness effect on host division and cell number, was determined by growth assays of several P. biaurelia strains. The performances of genetically identical lines either infected with H. caryophila or symbiont-free were compared. Following factors were considered as potentially influencing the outcome of the interaction: (1) host strain, (2) parasite strain, and (3) growth phases of the host. All three factors revealed a strong effect on the symbiosis. In presence of H. caryophila, the Paramecium density in the stationary growth phase decreased. Conversely, a positive effect of the bacteria during the exponential phase was observed for several host × parasite combinations resulting in an increased growth rate of infected P. biaurelia. Furthermore, the fitness impact of the tested endosymbionts on different P. biaurelia lines was not only dependent on one of the two involved strains but distinct for the specific combination. Depending on the current host growth phase, the presence of H. caryophila can be harmful or advantageous for P. biaurelia. Thus, under the tested experimental conditions, the symbionts can switch from the provision of benefits to the exploitation of host resources within the same host population and a time-span of less than 6 days

High variability of plasmid uptake rates in Escherichia coli isolated from sewage and river sediments

The horizontal transfer of plasmids is a key mechanism behind the spread of antibiotic resistance in bacteria. So far, transfer rate constants were measured for a variety of plasmids, donors and recipients. The employed strains typically had a long history in laboratories. Existing data are, therefore, not necessarily representative for real-world environments. Moreover, information on the inter-strain variability of plasmid transfer rates is scarce. Using a high-throughput approach, we studied the uptake of RP4 by various Escherichia coli recipients using Serratia marcescens as the donor. The recipient strains were isolated from human-borne sewage and river sediments. The rate constants of plasmid transfer generally followed a log-normal distribution with considerable variance. The rate constants for good and poor recipients (95 and 5% quantile) differed by more than three orders of magnitude. Specifically, the inter-strain variability of the rate constant was large in comparison to alterations induced by low-level antibiotic exposure. We did not find evidence for diverging efficiencies of plasmid uptake between E. coli recipients of different origin. On average, strains isolated from river bottom sediments were equally efficient in the acquisition of RP4 as isolates extracted from sewage. We conclude that E. coli strains persisting in the aquatic environment and those of direct human origin share a similar intrinsic potential for the conjugative uptake of certain plasmids. In view of the large inter-strain variability, we propose to work towards probabilistic modeling of the environmental spread of antibiotic resistance

The Genome Sequence of "Candidatus Fokinia solitaria": Insights on Reductive Evolution in Rickettsiales

"Candidatus Fokinia solitaria" is an obligate intracellular endosymbiont of a unicellular eukaryote, a ciliate of the genus Paramecium. Here, we present the genome sequence of this bacterium and subsequent analysis. Phylogenomic analysis confirmed the previously reported positioning of the symbiont within the "Candidatus Midichloriaceae" family (order Rickettsiales), as well as its high sequence divergence from other members of the family, indicative of fast sequence evolution. Consistently with this high evolutionary rate, a comparative genomic analysis revealed that the genome of this symbiont is the smallest of the Rickettsiales to date. The reduced genome does not present flagellar genes, nor the pathway for the biosynthesis of lipopolysaccharides (present in all the other so far sequenced members of the family "Candidatus Midichloriaceae") or genes for the Krebs cycle (present, although not always complete, in Rickettsiales). These results indicate an evolutionary trend toward a stronger dependence on the host, in comparison with other members of the family. Two alternative scenarios are compatible with our results; "Candidatus Fokinia solitaria" could be either a recently evolved, vertically transmitted mutualist, or a parasite with a high host-specificity

Der Einfluss von Klimavariabilität auf aquatische Nahrungsnetze: Der Einfluss von Klimavariabilität auf aquatische Nahrungsnetze

In den gemäßigten Breiten zeigte sich die allgemeine Erwärmung der letzten Jahrzehnte insbesondere im Winter und im zeitigen Frühjahr. Dementsprechend traten Veränderungen in der Phänologie, dem zeitlichen Verlauf von Populations- und Entwicklungsprozessen von Organismen (z. B. Zeitpunkt der Knospung bei Pflanzen oder der Laichperiode bei Fischen), vor allem im Frühjahr auf. Obwohl generell eine frühere und beschleunigte Entwicklung als Reaktion auf die Erwärmung beobachtet wurde, zeigten sich doch Unterschiede in der Sensitivität von Organismen. Dadurch kann es in Nahrungsnetzen zu Match- oder Mismatch- Situationen in Räuber-Beute Beziehungen kommen. Am Beispiel der komplexen Interaktionen im Nahrungsnetz der Talsperre Saidenbach wird der Einfluss verschiedener Erwärmungsszenarien auf Schlüsselorganismen und deren Interaktionen im Nahrungsnetz und letztlich auf die Wassergüte in dieser Trinkwassertalsperre im Rahmen des DFG-Schwerpunktprogramms AQUASHIFT analysiert.In temperate regions, the warming trends of the last decades have been observed primarily in winter and early spring. Accordingly, changes in the phenology of individual species, e.g. sprouting in plants or spawning of fish, occurred mainly in spring. Although the general pattern is earlier and faster development in response to warming, differences in sensitivity have been apparent between species, potentially giving rise to match or mismatch scenarios in predator-prey relations. The impact of warming scenarios on key species, their interactions and ultimately on the water quality is studied at Saidenbach Reservoir within the framework of the DFG priority program AQUASHIFT

Application of a Multiplex PCR with Specific PCR Primers for the Detection of the Genus Paramecium and the Paramecium aurelia Complex

The representatives of the genus Paramecium are well-studied ciliates and can be used in water quality assessment and the determinations of saprobic levels. For these applications, a clear and unambiguous identification of ciliate assemblages is essential, which is typically based on morphological characters requiring a sound taxonomic knowledge and experience in species determination including microscopic identification of both living and stained specimens. Therefore, we developed and applied specific PCR primers for the detection of species belonging to the genus Paramecium and the Paramecium aurelia complex. These primers were successfully tested with different Paramecium species including representatives of the P. aurelia complex as well as closely related species like Frontonia sp. and Tetrahymena sp. in both experimental and environmental samples. These primers can be used in a simultaneous approach achieving fast and reliable results with regard to determination of ciliate community and water assessment

Multidrug-Resistant and Clinically Relevant Gram-Negative Bacteria Are Present in German Surface Waters

Water is considered to play a role in the dissemination of antibiotic-resistant Gram-negative bacteria including those encoding Extended-spectrum beta-lactamases (ESBL) and carbapenemases. To investigate the role of water for their spread in more detail, we characterized ESBL/Carbapenemase-producing bacteria from surface water and sediment samples using phenotypic and genotypic approaches. ESBL/Carbapenemase-producing isolates were obtained from water/sediment samples. Species and antibiotic resistance were determined. A subset of these isolates (n = 33) was whole-genome-sequenced and analyzed for the presence of antibiotic resistance genes and virulence determinants. Their relatedness to isolates associated with human infections was investigated using multilocus sequence type and cgMLST-based analysis. Eighty-nine percent of the isolates comprised of clinically relevant species. Fifty-eight percent exhibited a multidrug-resistance phenotype. Two isolates harbored the mobile colistin resistance gene mcr-1. One carbapenemase-producing isolate identified as Enterobacter kobei harbored bla(VIM-)(1). Two Escherichia coli isolates had sequence types (ST) associated with human infections (ST131 and ST1485) and a Klebsiella pneumoniae isolate was classified as hypervirulent. A multidrug-resistant (MDR) Pseudomonas aeruginosa isolate encoding known virulence genes associated with severe lung infections in cystic fibrosis patients was also detected. The presence of MDR and clinically relevant isolates in recreational and surface water underlines the role of aquatic environments as both reservoirs and hot spots for MDR bacteria. Future assessment of water quality should include the examination of the multidrug resistance of clinically relevant bacterial species and thus provide an important link regarding the spread of MDR bacteria in a One Health context.Peer reviewe