Broad host range plasmids can invade an unexpectedly diverse fraction of a soil bacterial community

This is the author accepted manuscript. The final version is available from Springer Nature via the DOI in this recordConjugal plasmids can provide microbes with full complements of new genes and constitute potent vehicles for horizontal gene transfer. Conjugal plasmid transfer is deemed responsible for the rapid spread of antibiotic resistance among microbes. While broad host range plasmids are known to transfer to diverse hosts in pure culture, the extent of their ability to transfer in the complex bacterial communities present in most habitats has not been comprehensively studied. Here, we isolated and characterized transconjugants with a degree of sensitivity not previously realized to investigate the transfer range of IncP- and IncPromA-type broad host range plasmids from three proteobacterial donors to a soil bacterial community. We identified transfer to many different recipients belonging to 11 different bacterial phyla. The prevalence of transconjugants belonging to diverse Gram-positive Firmicutes and Actinobacteria suggests that inter-Gram plasmid transfer of IncP-1 and IncPromA-type plasmids is a frequent phenomenon. While the plasmid receiving fractions of the community were both plasmid- and donor- dependent, we identified a core super-permissive fraction that could take up different plasmids from diverse donor strains. This fraction, comprising 80% of the identified transconjugants, thus has the potential to dominate IncP- and IncPromA-type plasmid transfer in soil. Our results demonstrate that these broad host range plasmids have a hitherto unrecognized potential to transfer readily to very diverse bacteria and can, therefore, directly connect large proportions of the soil bacterial gene pool. This finding reinforces the evolutionary and medical significances of these plasmids.This work was funded by the Villum Kann Rasmussen Foundation Center of Excellence CREAM (Center for Environmental and Agricultural Microbiology)

Host defence responses in pigs experimentally infected with S. typhimurium

Salmonella Typhimurium may induce persistent infections (organ carriers) in pigs, and healthy carriers seems to be a problem in controling the infection (8). Evasion or disturbance of the host defence responses may be nessesary for establishment of a persistent infection. Functional changes in circulating neutrophils from pigs infected with Salmonella Typhimurium have been demonstrated (1). The aim of the study was to investigate host defence mechanisms in pigs that have cleared the infection, persistently infected pigs and uninfected pigs, studies that may contribute to the explanation of how the carrier-state is established. Phagocytosis and oxidative burst in peripheral blood or cells isolated from peripheral blood were investigated in three inoculation experiments

Effect of continuous nutrient enrichment on microalgae colonizing hard substrates

In order to understand the effect of changing nutrient conditions on benthic microalgae on hard substrates, in-situ experiments with artificial substrates were conducted in Kiel Fjord, Western Baltic Sea. As an extension of previous investigations, we used artificial substrates without silicate and thus were able to supply nutrient media with different Si:N ratios to porous substrates, from where they trickled out continuously. The biofilm developing on these substrates showed a significant increase in biovolume due to N + P enrichment, while Si alone had only minor effects. The stoichiometric composition of the biomass indicated nitrogen limitation during most of the year. The C:N ratios were lowered by the N + P addition. The algae were dominated by diatoms in most cases, but rhodophytes and chlorophytes also became important. The nutrient treatment affected the taxonomic composition mostly at the species level. The significance of the results with regard to coastal eutrophication is discussed

Metal stressors consistently modulate bacterial conjugal plasmid uptake potential in a phylogenetically conserved manner.

Published onlineJOURNAL ARTICLEThe environmental stimulants and inhibitors of conjugal plasmid transfer in microbial communities are poorly understood. Specifically, it is not known whether exposure to stressors may cause a community to alter its plasmid uptake ability. We assessed whether metals (Cu, Cd, Ni, Zn) and one metalloid (As), at concentrations causing partial growth inhibition, modulate community permissiveness (that is, uptake ability) against a broad-host-range IncP-type plasmid (pKJK5). Cells were extracted from an agricultural soil as recipient community and a cultivation-minimal filter mating assay was conducted with an exogenous E. coli donor strain. The donor hosted a gfp-tagged pKJK5 derivative from which conjugation events could be microscopically quantified and transconjugants isolated and phylogenetically described at high resolution via FACS and 16S rRNA amplicon sequencing. Metal stress consistently decreased plasmid transfer frequencies to the community, while the transconjugal pool richness remained unaffected with OTUs belonging to 12 bacterial phyla. The taxonomic composition of the transconjugal pools was distinct from their respective recipient communities and clustered dependent on the stress type and dose. However, for certain OTUs, stress increased or decreased permissiveness by more than 1000-fold and this response was typically correlated across different metals and doses. The response to some stresses was, in addition, phylogenetically conserved. This is the first demonstration that community permissiveness is sensitive to metal(loid) stress in a manner that is both partially consistent across stressors and phylogenetically conserved.The ISME Journal advance online publication, 2 August 2016; doi:10.1038/ismej.2016.98.We thank J Magid for access to the CRUCIAL field plot, LK Jensen for technical assistance in the laboratory and SM Milani for assistance in FACS sorting. This work was funded by the Villum Kann Rasmussen Foundation Center of Excellence CREAM (Center for Environmental and Agricultural Microbiology). UK is currently supported through an MRC/BBSRC grant (MR/N007174/1)

Pig α₁-Acid Glycoprotein: Characterization and First Description in Any Species as a Negative Acute Phase Protein.

The serum protein α1-acid glycoprotein (AGP), also known as orosomucoid, is generally described as an archetypical positive acute phase protein. Here, porcine AGP was identified, purified and characterized from pooled pig serum. It was found to circulate as a single chain glycoprotein having an apparent molecular weight of 43 kDa by SDS-PAGE under reducing conditions, of which approximately 17 kDa were accounted for by N-bound oligosaccharides. Those data correspond well with the properties of the protein predicted from the single porcine AGP gene (ORM1, Q29014 (UniProt)), containing 5 putative glycosylation sites. A monoclonal antibody (MAb) was produced and shown to quantitatively and specifically react with all microheterogenous forms of pig AGP as analyzed by 2-D electrophoresis. This MAb was used to develop an immunoassay (ELISA) for quantification of AGP in pig serum samples. The adult serum concentrations of pig AGP were in the range of 1-3 mg/ml in a number of conventional pig breeds while it was lower in Göttingen and Ossabaw minipigs (in the 0.3 to 0.6 mg/ml range) and higher in young (2-5 days old) conventional pigs (mean: 6.6 mg/ml). Surprisingly, pig AGP was found to behave as a negative acute phase protein during a range of experimental infections and aseptic inflammation with significant decreases in serum concentration and in hepatic ORM1 expression during the acute phase response. To our knowledge this is the first description in any species of AGP being a negative acute phase protein

An intriguing relationship between the cyclic diguanylate signaling system and horizontal gene transfer

The second messenger cyclic diguanylate (c-di-GMP) is ubiquitously used by bacteria to modulate and shift between different phenotypes including motility, biofilm formation and virulence. Here we show that c-di-GMP-associated genes are widespread on plasmids and that enzymes that synthesize or degrade c-di-GMP are preferentially encoded on transmissible plasmids. Additionally, expression of enzymes that synthesize c-di-GMP was found to increase both biofilm formation and, interestingly, conjugative plasmid transfer rates

Cell Size and the Initiation of DNA Replication in Bacteria

In eukaryotes, DNA replication is coupled to the cell cycle through the actions of cyclin-dependent kinases and associated factors. In bacteria, the prevailing view, based primarily from work in Escherichia coli, is that growth-dependent accumulation of the highly conserved initiator, DnaA, triggers initiation. However, the timing of initiation is unchanged in Bacillus subtilis mutants that are ∼30% smaller than wild-type cells, indicating that achievement of a particular cell size is not obligatory for initiation. Prompted by this finding, we re-examined the link between cell size and initiation in both E. coli and B. subtilis. Although changes in DNA replication have been shown to alter both E. coli and B. subtilis cell size, the converse (the effect of cell size on DNA replication) has not been explored. Here, we report that the mechanisms responsible for coordinating DNA replication with cell size vary between these two model organisms. In contrast to B. subtilis, small E. coli mutants delayed replication initiation until they achieved the size at which wild-type cells initiate. Modest increases in DnaA alleviated the delay, supporting the view that growth-dependent accumulation of DnaA is the trigger for replication initiation in E. coli. Significantly, although small E. coli and B. subtilis cells both maintained wild-type concentration of DnaA, only the E. coli mutants failed to initiate on time. Thus, rather than the concentration, the total amount of DnaA appears to be more important for initiation timing in E. coli. The difference in behavior of the two bacteria appears to lie in the mechanisms that control the activity of DnaA