The impact of space flight on survival and interaction of Cupriavidus metallidurans CH34 with basalt, a volcanic moon analog rock

Microbe-mineral interactions have become of interest for space exploration as microorganisms could be used to biomine from extra-terrestrial material and extract elements useful as micronutrients in life support systems. This research aimed to identify the impact of space flight on the long-term survival of Cupriavidus metallidurans CH34 in mineral water and the interaction with basalt, a lunar-type rock in preparation for the ESA spaceflight experiment, BIOROCK. Therefore, C. metallidurans CH34 cells were suspended in mineral water supplemented with or without crushed basalt and send for 3 months on board the Russian FOTON-M4 capsule. Long-term storage had a significant impact on cell physiology and energy status (by flow cytometry analysis, plate count and intracellular ATP measurements) as 60% of cells stored on ground lost their cell membrane potential, only 17% were still active, average ATP levels per cell were significantly lower and cultivability dropped to 1%. The cells stored in the presence of basalt and exposed to space flight conditions during storage however showed less dramatic changes in physiology, with only 16% of the cells lost their cell membrane potential and 24% were still active, leading to a higher cultivability (50%) and indicating a general positive effect of basalt and space flight on survival. Microbe-mineral interactions and biofilm formation was altered by spaceflight as less biofilm was formed on the basalt during flight conditions. Leaching from basalt also changed (measured with ICP-OES), showing that cells release more copper from basalt and the presence of cells also impacted iron and magnesium concentration irrespective of the presence of basalt. The flight conditions thus could counteract some of the detrimental effects observed after the 3 month storage conditions

Effect of shear stress on Pseudomonas aeruginosa isolated from the cystic fibrosis lung

Chronic colonization of the lungs by Pseudomonas aeruginosa is one of the major causes of morbidity and mortality in cystic fibrosis (CF) patients. To gain insights into the characteristic biofilm phenotype of P. aeruginosa in the CF lungs, mimicking the CF lung environment is critical. We previously showed that growth of the non-CF-adapted P. aeruginosa PAO1 strain in a rotating wall vessel, a device that simulates the low fluid shear (LS) conditions present in the CF lung, leads to the formation of in-suspension, self-aggregating biofilms. In the present study, we determined the phenotypic and transcriptomic changes associated with the growth of a highly adapted, transmissible P. aeruginosa CF strain in artificial sputum medium under LS conditions. Robust self-aggregating biofilms were observed only under LS conditions. Growth under LS conditions resulted in the upregulation of genes involved in stress response, alginate biosynthesis, denitrification, glycine betaine biosynthesis, glycerol metabolism, and cell shape maintenance, while genes involved in phenazine biosynthesis, type VI secretion, and multidrug efflux were downregulated. In addition, a number of small RNAs appeared to be involved in the response to shear stress. Finally, quorum sensing was found to be slightly but significantly affected by shear stress, resulting in higher production of autoinducer molecules during growth under high fluid shear (HS) conditions. In summary, our study revealed a way to modulate the behavior of a highly adapted P. aeruginosa CF strain by means of introducing shear stress, driving it from a biofilm lifestyle to a more planktonic lifestyle. IMPORTANCE: Biofilm formation by Pseudomonas aeruginosa is one of the hallmarks of chronic cystic fibrosis (CF) lung infections. The biofilm matrix protects this bacterium from antibiotics as well as from the immune system. Hence, the prevention or reversion of biofilm formation is believed to have a great impact on treatment of chronic P. aeruginosa CF lung infections. In the present study, we showed that it is possible to modulate the behavior of a highly adapted transmissible P. aeruginosa CF isolate at both the transcriptomic and phenotypic levels by introducing shear stress in a CF-like environment, driving it from a biofilm to a planktonic lifestyle. Consequently, the results obtained in this study are of great importance with regard to therapeutic applications that introduce shear stress in the lungs of CF patients

Variation in genomic islands contribute to genome plasticity in cupriavidus metallidurans

<p>Abstract</p> <p>Background</p> <p>Different <it>Cupriavidus metallidurans </it>strains isolated from metal-contaminated and other anthropogenic environments were genotypically and phenotypically compared with <it>C. metallidurans </it>type strain CH34. The latter is well-studied for its resistance to a wide range of metals, which is carried for a substantial part by its two megaplasmids pMOL28 and pMOL30.</p> <p>Results</p> <p>Comparative genomic hybridization (CGH) indicated that the extensive arsenal of determinants involved in metal resistance was well conserved among the different <it>C. metallidurans </it>strains. Contrary, the mobile genetic elements identified in type strain CH34 were not present in all strains but clearly showed a pattern, although, not directly related to a particular biotope nor location (geographical). One group of strains carried almost all mobile genetic elements, while these were much less abundant in the second group. This occurrence was also reflected in their ability to degrade toluene and grow autotrophically on hydrogen gas and carbon dioxide, which are two traits linked to separate genomic islands of the Tn<it>4371</it>-family. In addition, the clear pattern of genomic islands distribution allowed to identify new putative genomic islands on chromosome 1 and 2 of <it>C. metallidurans </it>CH34.</p> <p>Conclusions</p> <p>Metal resistance determinants are shared by all <it>C. metallidurans </it>strains and their occurrence is apparently irrespective of the strain's isolation type and place. <it>Cupriavidus metallidurans </it>strains do display substantial differences in the diversity and size of their mobile gene pool, which may be extensive in some (including the type strain) while marginal in others.</p

Antimicrobial efficacy against Pseudomonas aeruginosa biofilm formation in a three-dimensional lung epithelial model and the influence of fetal bovine serum

In vitro models that mimic in vivo host-pathogen interactions are needed to evaluate candidate drugs that inhibit bacterial virulence traits. We established a new approach to study Pseudomonas aeruginosa biofilm susceptibility on biotic surfaces, using a three-dimensional (3-D) lung epithelial cell model. P. aeruginosa formed antibiotic resistant biofilms on 3-D cells without affecting cell viability. The biofilm-inhibitory activity of antibiotics and/or the anti-biofilm peptide DJK-5 were evaluated on 3-D cells compared to a plastic surface, in medium with and without fetal bovine serum (FBS). In both media, aminoglycosides were more efficacious in the 3-D cell model. In serum-free medium, most antibiotics (except polymyxins) showed enhanced efficacy when 3-D cells were present. In medium with FBS, colistin was less efficacious in the 3-D cell model. DJK-5 exerted potent inhibition of P. aeruginosa association with both substrates, only in serum-free medium. DJK-5 showed stronger inhibitory activity against P. aeruginosa associated with plastic compared to 3-D cells. The combined addition of tobramycin and DJK-5 exhibited more potent ability to inhibit P. aeruginosa association with both substrates. In conclusion, lung epithelial cells influence the efficacy of most antimicrobials against P. aeruginosa biofilm formation, which in turn depends on the presence or absence of FBS

Soil microbial community structure and functionality changes in response to long-term metal and radionuclide pollution

Microbial communities are essential for a healthy soil ecosystem. Metals and radionuclides can exert a persistent pressure on the soil microbial community. However, little is known on the effect of long-term co-contamination of metals and radionuclides on the microbial community structure and functionality. We investigated the impact of historical discharges of the phosphate and nuclear industry on the microbial community in the Grote Nete river basin in Belgium. Eight locations were sampled along a transect to the river edge and one location further in the field. Chemical analysis demonstrated a metal and radionuclide contamination gradient and revealed a distinct clustering of the locations based on all metadata. Moreover, a relation between the chemical parameters and the bacterial community structure was demonstrated. Although no difference in biomass was observed between locations, cultivation-dependent experiments showed that communities from contaminated locations survived better on singular metals than communities from control locations. Furthermore, nitrification, a key soil ecosystem process seemed affected in contaminated locations when combining metadata with microbial profiling. These results indicate that long-term metal and radionuclide pollution impacts the microbial community structure and functionality and provides important fundamental insights into microbial community dynamics in co-metal-radionuclide contaminated sites

Characterization of the bacterial communities on recent Icelandic volcanic deposits of different ages

Background: Basalt is the most common igneous rock on the Earth's surface covering. Basalt-associated microorganisms drive the cycling and sequestration of different elements such as nitrogen, carbon and other nutrients, which facilitate subsequent pioneer and plant development, impacting long-term regulation of the Earth's temperature and biosphere. The initial processes of colonization and subsequent rock weathering by microbial communities are still poorly understood and relatively few data are available on the diversity and richness of the communities inhabiting successive and chronological lava flows. In this study, the bacterial communities present on lava deposits from different eruptions of the 1975-84 Krafla Fires (32-, 35-and 39-year old, respectively) at the Krafla, Iceland, were determined. Results: Three sites were sampled for each deposit (32-, 35-and 39-year old), two proximal sites (at 10 m distance) and one more distant site (at 100 m from the two other sites). The determined chemical composition and metal concentrations were similar for the three basalt deposits. No significant differences were observed in the total number of cells in each flow. 16S rRNA gene amplicon sequencing showed that the most abundant classified phylum across the 3 flows was Proteobacteria, although predominance of Acidobacteria, Actinobacteria and Firmicutes was observed for some sampling sites. In addition, a considerable fraction of the operational taxonomic units remained unclassified. Alpha diversity (Shannon, inverse Simpson and Chao), HOMOVA and AMOVA only showed a significant difference for Shannon between the 32-and 39-year old flow (p < 0.05). Nonmetric multidimensional scaling (NMDS) analysis showed that age significantly (p = 0.026) influenced the leftward movement along NMDS axis 1. Conclusions: Although NMDS indicated that the (relatively small) age difference of the deposits appeared to impact the bacterial community, this analysis was not consistent with AMOVA and HOMOVA, indicating no significant difference in community structure. The combined results drive us to conclude that the (relatively small) age differences of the deposits do not appear to be the main factor shaping the microbial communities. Probably other factors such as spatial heterogeneity, associated carbon content, exogenous rain precipitations and wind also affect the diversity and dynamics

Synthetic Biology Toolbox, Including a Single-Plasmid CRISPR-Cas9 System to Biologically Engineer the Electrogenic, Metal-Resistant Bacterium Cupriavidus metallidurans CH34

Cupriavidus metallidurans CH34 exhibits extraordinary metabolic versatility, including chemolithoautotrophic growth; degradation of BTEX (benzene, toluene, ethylbenzene, xylene); high resistance to numerous metals; biomineralization of gold, platinum, silver, and uranium; and accumulation of polyhydroxybutyrate (PHB). These qualities make it a valuable host for biotechnological applications such as bioremediation, bioprocessing, and the generation of bioelectricity in microbial fuel cells (MFCs). However, the lack of genetic tools for strain development and studying its fundamental physiology represents a bottleneck to boosting its commercial applications. In this study, inducible and constitutive promoter libraries were built and characterized, providing the first comprehensive list of biological parts that can be used to regulate protein expression and optimize the CRISPR-Cas9 genome editing tools for this host. A single-plasmid CRISPR-Cas9 system that can be delivered by both conjugation and electroporation was developed, and its efficiency was demonstrated by successfully targeting the pyrE locus. The CRISPR-Cas9 system was next used to target candidate genes encoding type IV pili, hypothesized by us to be involved in extracellular electron transfer (EET) in this organism. Single and double deletion strains (ΔpilA, ΔpilE, and ΔpilAE) were successfully generated. Additionally, the CRISPR-Cas9 tool was validated for constructing genomic insertions (ΔpilAE::gfp and ΔpilAE::λPrgfp). Finally, as type IV pili are believed to play an important role in extracellular electron transfer to solid surfaces, C. metallidurans CH34 ΔpilAE was further studied by means of cyclic voltammetry using disposable screen-printed carbon electrodes. Under these conditions, we demonstrated that C. metallidurans CH34 could generate extracellular currents; however, no difference in the intensity of the current peaks was found in the ΔpilAE double deletion strain when compared to the wild type. This finding suggests that the deleted type IV pili candidate genes are not involved in extracellular electron transfer under these conditions. Nevertheless, these experiments revealed the presence of different redox centers likely to be involved in both mediated electron transfer (MET) and direct electron transfer (DET), the first interpretation of extracellular electron transfer mechanisms in C. metallidurans CH34

Differential proteomics and physiology of Pseudomonas putida KT2440 under filament-inducing conditions

Abstract Background Pseudomonas putida exerts a filamentous phenotype in response to environmental stress conditions that are encountered during its natural life cycle. This study assessed whether P. putida filamentation could confer survival advantages. Filamentation of P. putida was induced through culturing at low shaking speed and was compared to culturing in high shaking speed conditions, after which whole proteomic analysis and stress exposure assays were performed. Results P. putida grown in filament-inducing conditions showed increased resistance to heat and saline stressors compared to non-filamented cultures. Proteomic analysis showed a significant metabolic change and a pronounced induction of the heat shock protein IbpA and recombinase RecA in filament-inducing conditions. Our data further indicated that the associated heat shock resistance, but not filamentation, was dependent of RecA. Conclusions This study provides insights into the altered metabolism of P. putida in filament-inducing conditions, and indicates that the formation of filaments could potentially be utilized by P. putida as a survival strategy in its hostile, recurrently changing habitat.</p