Diversity and ice nucleation activity of Pseudomonas syringae in drone-based water samples from eight lakes in Austria.

Bacteria from the Pseudomonas syringae complex (comprised of at least 15 recognized species and more than 60 different pathovars of P. syringae sensu stricto) have been cultured from clouds, rain, snow, streams, rivers, and lakes. Some strains of P. syringae express an ice nucleation protein (hereafter referred to as ice+) that catalyzes the heterogeneous freezing of water. Though P. syringae has been sampled intensively from freshwater sources in the U.S. and France, little is known about the genetic diversity and ice nucleation activity of P. syringae in other parts of the world. We investigated the haplotype diversity and ice nucleation activity at -8 °C (ice+) of strains of P. syringae from water samples collected with drones in eight freshwater lakes in Austria. A phylogenetic analysis of citrate synthase (cts) sequences from 271 strains of bacteria isolated from a semi-selective medium for Pseudomonas revealed that 69% (188/271) belonged to the P. syringae complex and represented 32 haplotypes in phylogroups 1, 2, 7, 9, 10, 13, 14 and 15. Strains within the P. syringae complex were identified in all eight lakes, and seven lakes contained ice+ strains. Partial 16S rDNA sequences were analyzed from a total of 492 pure cultures of bacteria isolated from non-selective medium. Nearly half (43.5%; 214/492) were associated with the genus Pseudomonas. Five of the lakes (ALT, GRU, GOS, GOL, and WOR) were all distinguished by high levels of Pseudomanas (p ≤ 0.001). HIN, the highest elevation lake, had the highest percentage of ice+ strains. Our work highlights the potential for uncovering new haplotypes of P. syringae in aquatic habitats, and the use of robotic technologies to sample and characterize microbial life in remote settings.

A mycotoxin transporter (4D) from a library of deoxynivalenol-tolerant microorganisms.

New strategies are needed to mitigate the mycotoxin deoxynivalenol (DON) in feed and food products. Microbial DNA fragments were generated from a library of DON-tolerant microorganisms. These fragments were screened in DON-sensitive yeast strains for their ability to modify or transport DON. Fragments were cloned into a PCR8/TOPO vector, and recombined into the yeast vector, pYES-DEST52. Resulting yeast transformants were screened in the presence of 100 ppm DON. Transformants that were able to grow in the presence of DON were plated on a selective medium, and the cloned microbial DNA fragments were sequenced. BLAST queries of one microbial DNA fragment (4D) showed a high degree of similarity to an ABC transporter. A series of screening and inhibition assays were conducted with a transport inhibitor (propanol), to test the hypothesis that 4D is a mycotoxin transporter. DON concentrations did not change for yeast transformants expressing 4D. The ability of yeast transformants expressing 4D to transport DON was inhibited by the addition of propanol. Moreover, yeast transformants expressing a known efflux pump (PDR5) showed similar trends in propanol transport inhibition compared to 4D. Future work should consider mycotoxin transporters such as 4D to the development of transgenic plants to limit DON accumulation in seeds.

Diversity and Ice Nucleation Activity of Microorganisms Collected With a Small Unmanned Aircraft System (sUAS) in France and the United States.

Many microbes relevant to crops, domestic animals, and humans are transported over long distances through the atmosphere. Some of these atmospheric microbes catalyze the freezing of water at higher temperatures and facilitate the onset of precipitation. We collected microbes from the lower atmosphere in France and the United States with a small unmanned aircraft system (sUAS). 55 sampling missions were conducted at two locations in France in 2014 (an airfield in Pujaut, and the top of Puy de Dôme), and three locations in the U.S. in 2015 (a farm in Blacksburg, Virginia, and a farm and a lake in Baton Rouge, Louisiana). The sUAS was a fixed-wing electric drone equipped with a remote-operated sampling device that was opened once the aircraft reached the desired sampling altitude (40-50 meters above ground level). Samples were collected on agar media (TSA, R4A, R2A, and CA) with and without the fungicide cycloheximide. Over 4,000 bacterial-like colonies were recovered across the 55 sUAS sampling missions. A positive relationship between sampling time and temperature and concentrations of culturable bacteria was observed for sUAS flights conducted in France, but not for sUAS flights conducted in Louisiana. A droplet freezing assay was used to screen nearly 2,000 colonies for ice nucleation activity, and 15 colonies were ice nucleation active at temperatures warmer than -8°C. Sequences from portions of 16S rDNA were used to identify 503 colonies from 54 flights to the level of genus. Assemblages of bacteria from sUAS flights in France (TSA) and sUAS flights in Louisiana (R4A) showed more similarity within locations than between locations. Bacteria collected with sUAS on TSA in France and Virginia were significantly different across all levels of classification tested (P < 0.001 for class, order, family, and genus). Principal Coordinates Analysis showed a strong association between the genera Curtobacterium, Pantoea, and Pseudomonas from sUAS flights in Virginia, and Agrococcus, Lysinibacillus, and Paenibacillus from sUAS flights in France. Future work aims to understand the potential origin of the atmospheric microbial assemblages collected with sUAS, and their association with mesoscale atmospheric processes.

Impact of Chemoeffectors on Bacterial Motility, Transport, and Contaminant Degradation in Sand-Filled Percolation Columns.

Chemoeffector-mediated bacterial motility and tactic swimming are major drivers for contaminant accessibility and biodegradation at submillimeter scales. In sand-filled percolated columns we tested how and to what degree chemoeffectors influenced bacterial transport and thereby promoted accessibility and degradation of distantly located 14C-naphthalene (NAH) at the centimeter scale. Sunflower root exudates and silver nanoparticles (AgNPs) were used as chemoeffectors to stimulate opposing effects of motility and tactic swimming of NAH-degrading Pseudomonas putida G7. Sunflower exudates prompted smooth bacterial movement and positive taxis, while AgNPs induced tortuous movement and repellent responses. Compared to chemoeffector-free controls exudates reduced deposition and stimulated bacterial transport during percolation experiments. AgNPs, however, provoked bacterial deposition and concomitant saturation of the collector surfaces (filter blocking) that led to progressively increased percolation of cells. Despite mechanistic differences, both motility patterns supported bacterial transport and promoted mineralization rates of NAH desorbing from a source placed at the column outlet. Observed mineralization rates in the presence of the chemoeffectors were 5-fold higher than those in their absence and similar to NAH-mineralization in well-stirred batch assays. Our results indicate that chemically mediated, small-scale bacterial motility patterns may become relevant for long-distance bacterial transport and the biodegradation of patchy contaminants at higher scales, respectively.

Swimming performance of Bradyrhizobium diazoefficiens is an emergent property of its two flagellar systems.

Many bacterial species use flagella for self-propulsion in aqueous media. In the soil, which is a complex and structured environment, water is found in microscopic channels where viscosity and water potential depend on the composition of the soil solution and the degree of soil water saturation. Therefore, the motility of soil bacteria might have special requirements. An important soil bacterial genus is Bradyrhizobium, with species that possess one flagellar system and others with two different flagellar systems. Among the latter is B. diazoefficiens, which may express its subpolar and lateral flagella simultaneously in liquid medium, although its swimming behaviour was not described yet. These two flagellar systems were observed here as functionally integrated in a swimming performance that emerged as an epistatic interaction between those appendages. In addition, each flagellum seemed engaged in a particular task that might be required for swimming oriented toward chemoattractants near the soil inner surfaces at viscosities that may occur after the loss of soil gravitational water. Because the possession of two flagellar systems is not general in Bradyrhizobium or in related genera that coexist in the same environment, there may be an adaptive tradeoff between energetic costs and ecological benefits among these different species.

Tactic response of bacteria to zero-valent iron nanoparticles.

The microbial assessment of pollutant toxicity rarely includes behavioral responses. In this study, we investigated the tactic response of Pseudomonas putida G7, a representative of soil bacterium, towards engineered zero-valent iron nanoparticles (nZVIs), as a new end-point assessment of toxicity. The study integrated the characterization of size distribution and charge of nZVIs and tactic reaction response by means of inverted capillary assay and computer-assisted motion analysis of motility behavior. Iron nanoparticles (diameter ≤ 100 nm) were prepared in the absence of oxygen to prevent aggregation, and then exposed in aerobic conditions. We first demonstrate that iron nanoparticles can elicit a negative tactic response in bacteria at low but environmentally-relevant, sub-lethal concentrations (1-10 µg/L). Cells were repelled by nZVIs in the concentration gradients created inside the capillaries, and a significant increase in turning events, characteristic of negative taxis, was detected under exposure to nZVIs. These tactic responses were not detectable after sustained exposure of the nanoparticles to oxygen. This new behavioral assessment may be prospected for the design of sensitive bioassays for nanomaterial toxicity.

Impact of dissolved organic matter on bacterial tactic motility, attachment, and transport.

Bacterial dispersal is a key driver of the ecology of microbial contaminant degradation in soils. This work investigated the role of dissolved organic matter (DOM) in the motility, attachment, and transport of the soil bacterium Pseudomonas putida G7 in saturated porous media. The study is based on the hypothesis that DOM quality is critical to triggering tactic motility and, consequently, affects bacterial transport and dispersal. Sunflower root exudates, humic acids (HA), and the synthetic oleophilic fertilizer S-200 were used as representatives of fresh, weathered, and artificially processed DOM with high nitrogen and phosphorus contents, respectively. We studied DOM levels of 16-130 mg L(-1), which are representative of DOM concentrations typically found in agricultural soil pore water. In contrast to its responses to HA and S-200, strain G7 exhibited a tactic behavior toward root exudates, as quantified by chemotaxis assays and single-cell motility observations. All DOM types promoted bacterial transport through sand at high concentrations (∼ 130 mg L(-1)). At low DOM concentrations (∼ 16 mg L(-1)), the enhancement occurred only in the presence of sunflower root exudates, and this enhancement did not occur with G7 bacteria devoid of flagella. Our results suggest that tactic DOM effectors strongly influence bacterial transport and the interception probability of motile bacteria by collector surfaces.

Bioavailability of pollutants and chemotaxis.

The exposure of bacteria to pollutants induces frequently chemoattraction or chemorepellent reactions. Recent research suggests that the capacity to degrade a toxic compound has co-evolved in some bacteria with the capacity to chemotactically react to it. There is an increasing amount of data which show that chemoattraction to biodegradable pollutants increases their bioavailability which translates into an enhancement of the biodegradation rate. Pollutant chemoreceptors so far identified are encoded on degradation or resistance plasmids. Genetic engineering of bacteria, such as the transfer of chemoreceptor genes, offers thus the possibility to optimize biodegradation processes.

Chemical effectors cause different motile behavior and deposition of bacteria in porous media.

We tested the hypothesis whether chemically induced motility patterns of bacteria may affect their transport in porous media. Naphthalene-degrading Pseudomonas putida G7 cells were exposed to glucose, salicylate, and silver nanoparticles (AgNPs) and their motility was assessed by computer-assisted, quantitative swimming and capillary-based taxis determinations. Exposure to salicylate induced smooth movement with few acceleration events and positive taxis, whereas cells exposed to AgNPs exhibited tortuous movement and a repellent response. Although metabolized by strain G7, glucose did not cause attraction and induced a hyper-motile mode of swimming, characterized by a high frequency of acceleration events, high swimming speed (>60 µm s(-1)), and a high tortuosity in the trajectories. Chemically induced motility behavior correlated with distinct modes of attachment to sand in batch assays and breakthrough curves in percolation column experiments. Salicylate significantly reduced deposition of G7 cells in column experiments whereas glucose and AgNPs enhanced attachment and caused filter blocking that resulted in a progressive decrease in deposition. These findings are relevant for bioremediation scenarios that require an optimized outreach of introduced inoculants and in other environmental technologies, such as water disinfection and microbially enhanced oil recovery.

Bacterial chemotaxis towards aromatic hydrocarbons in Pseudomonas.

Bacterial chemotaxis is an adaptive behaviour, which requires sophisticated information-processing capabilities that cause motile bacteria to either move towards or flee from chemicals. Pseudomonas putida DOT-T1E exhibits the capability to move towards different aromatic hydrocarbons present at a wide range of concentrations. The chemotactic response is mediated by the McpT chemoreceptor encoded by the pGRT1 megaplasmid. Two alleles of mcpT are borne on this plasmid and inactivation of either one led to loss of this chemotactic phenotype. Cloning of mcpT into a plasmid complemented not only the mcpT mutants but also its transfer to other Pseudomonas conferred chemotactic response to high concentrations of toluene and other chemicals. Therefore, the phenomenon of chemotaxis towards toxic compounds at high concentrations is gene-dose dependent. In vitro experiments show that McpT is methylated by CheR and McpT net methylation was diminished in the presence of hydrocarbons, what influences chemotactic movement towards these chemicals.

Bacterial tactic response to silver nanoparticles.

In this study, we investigated the tactic response of Pseudomonas putida G7, a representative soil bacterium, towards silver nanoparticles (AgNPs). The study integrated the characterization of surface area and size distribution of AgNPs, toxicity determinations, based on ATP production, and assessment of the repellent reaction by means of an inverted capillary assay ('chemical-in-pond' method), and changes in the motility behaviour determined by computer-assisted motion analysis. Our data demonstrate, for the first time, that nanoparticles can elicit a negative tactic response in bacteria at low but environmentally relevant, sublethal concentrations. Data obtained by the chemical-in-pond method indicated that cells exposed to 0.1 mg l(-1) of two AgNPs preparations, differing in particle size (maximum diameter ≤ 100 nm and ≤ 150 nm respectively), were repelled in the gradients created inside the capillaries. However, cells exposed to similar low concentration of AgNO3 did not demonstrate any detectable repellent response, although it reduced cell viability by 20%, a decrease comparable to that caused by AgNPs. Computer analysis of swimming behaviour of cells exposed to AgNPs (0.2 mg l(-1) ) revealed a significant increase in turning events, as compared with unexposed controls, which is characteristic of bacterial repellent response. Greater AgNPs concentrations (up to 100 mg l(-1) ) also induced changes in the swimming behaviour, although they did not induce any detectable repellent response as determined by the chemical-in-pond assays. In contrast, AgNO3 failed to induce the repellent swimming behaviour within the wide range of concentrations tested (0.001-100 mg l(-1) ), and caused a significant inhibition of cell motility at a concentration above 0.1 mg l(-1) . The evidence presented here suggests there are likely to be alternative mechanisms by which nano-scale silver induces a repellent response, which is more direct than the toxic response of macro-forms of silver, attributed to ion formation and exposure.