OprF Impacts Pseudomonas aeruginosa Biofilm Matrix eDNA Levels in a Nutrient-Dependent Manner.

The biofilm matrix is composed of exopolysaccharides, eDNA, membrane vesicles, and proteins. While proteomic analyses have identified numerous matrix proteins, their functions in the biofilm remain understudied compared to the other biofilm components. In the Pseudomonas aeruginosa biofilm, several studies have identified OprF as an abundant matrix protein and, more specifically, as a component of biofilm membrane vesicles. OprF is a major outer membrane porin of P. aeruginosa cells. However, current data describing the effects of OprF in the P. aeruginosa biofilm are limited. Here, we identify a nutrient-dependent effect of OprF in static biofilms, whereby ΔoprF cells form significantly less biofilm than wild type when grown in media containing glucose or low sodium chloride concentrations. Interestingly, this biofilm defect occurs during late static biofilm formation and is not dependent on the production of PQS, which is responsible for outer membrane vesicle production. Furthermore, while biofilms lacking OprF contain approximately 60% less total biomass than those of wild type, the number of cells in these two biofilms is equivalent. We demonstrate that P. aeruginosa ΔoprF biofilms with reduced biofilm biomass contain less eDNA than wild-type biofilms. These results suggest that the nutrient-dependent effect of OprF is involved in the maintenance of P. aeruginosa biofilms by retaining eDNA in the matrix. IMPORTANCE Many pathogens form biofilms, which are bacterial communities encased in an extracellular matrix that protects them against antibacterial treatments. The roles of several matrix components of the opportunistic pathogen Pseudomonas aeruginosa have been characterized. However, the effects of P. aeruginosa matrix proteins remain understudied and are untapped potential targets for antibiofilm treatments. Here, we describe a conditional effect of the abundant matrix protein OprF on late-stage P. aeruginosa biofilms. A ΔoprF strain formed significantly less biofilm in low sodium chloride or with glucose. Interestingly, the defective ΔoprF biofilms did not exhibit fewer resident cells but contained significantly less extracellular DNA (eDNA) than wild type. These results suggest that OprF is involved in matrix eDNA retention in biofilms.

Coexistence of vitreous and crystalline phases of H2O at ambient temperature.

Formation of vitreous ice during rapid compression of water at room temperature is important for biology and the study of biological systems. Here, we show that Raman spectra of rapidly compressed water at greater than 1 GPa at room temperature exhibits the signature of high-density amorphous ice, whereas the X-ray diffraction (XRD) pattern is dominated by crystalline ice VI. To resolve this apparent contradiction, we used molecular dynamics simulations to calculate full vibrational spectra and diffraction patterns of mixtures of vitreous ice and ice VI, including embedded interfaces between the two phases. We show quantitatively that Raman spectra, which probe the local polarizability with respect to atomic displacements, are dominated by the vitreous phase, whereas a small amount of the crystalline component is readily apparent by XRD. The results of our combined experimental and theoretical studies have implications for detecting vitreous phases of water, survival of biological systems under extreme conditions, and biological imaging. The results provide additional insight into the stable and metastable phases of H2O as a function of pressure and temperature, as well as of other materials undergoing pressure-induced amorphization and other metastable transitions.

Quantification of Organic Carbon Sequestered by Biogenic Iron Sulfide Minerals in Long-Term Anoxic Laboratory Incubations.

Organic carbon sequestration in sedimentary environments controls oxygen and carbon dioxide concentrations in the atmosphere. While minerals play an important role in the preservation of organic carbon, there is a lack of understanding about the formation and stability of organo-mineral interactions in anoxic environments, especially those involving authigenic iron sulfide minerals. In this study, we quantified organic carbon and nitrogen sequestered in biogenic iron sulfide minerals co-precipitated with sulfate-reducing bacteria (SRB) in freshwater and marine conditions in long-term laboratory experiments. The amounts of C and N associated with biogenic iron sulfide minerals increased with increasing cell biomass concentrations available in the media. C and N levels stabilized over the first 2 months of incubation and remained stable for up to 1 year. Crystalline mackinawite (FeS) formed in all experimental conditions and transformed to greigite only in some experimental conditions. We did not find evidence that this mineral transformation affected C and N levels, neither could we identify the factors that controlled greigite formation. Pyrite did not form in our experimental conditions. While C concentrations in minerals correlated with concentrations of reduced sulfate in both the freshwater and marine media, removal of OC by iron sulfide minerals was more efficient in freshwater than marine conditions. Removal of OC by iron sulfide minerals was also more efficient when cells were present (SRB biomass) in comparison with abiotic incubations with organic mixtures (e.g., tryptone, yeast extract, and casamino acids). Our study highlights the potential for biogenic iron sulfide minerals to quantitatively contribute to organic carbon preservation in anoxic environments.

Interactions Between Iron Sulfide Minerals and Organic Carbon: Implications for Biosignature Preservation and Detection.

Microbe-mineral interactions can produce unique composite materials, which can preserve biosignatures. Geological evidence suggests that iron sulfide (Fe-S) minerals are abundant in the subsurface of Mars. On Earth, the formation of Fe-S minerals is driven by sulfate-reducing microorganisms (SRM) that produce reactive sulfide. Moreover, SRM metabolites, as well as intact cells, can influence the morphology, particle size, aggregation, and composition of biogenic Fe-S minerals. In this work, we evaluated how simple and complex organic molecules-hexoses and amino acid/peptide mixtures, respectively-influence the formation of Fe-S minerals (simulated prebiotic conditions), and whether the observed patterns mimic the biological influence of SRM. To this end, organo-mineral aggregates were characterized with X-ray diffraction, scanning electron microscopy, and scanning transmission X-ray microscopy coupled to near-edge X-ray absorption fine structure spectroscopy. Overall, Fe-S minerals were found to have a strong affinity for proteinaceous organic matter. Fe-S minerals precipitated at simulated prebiotic conditions yielded organic carbon distributions that were more homogeneous than treatments with whole SRM cells. In prebiotic experiments, spectroscopy detected potential organic transformations during Fe-S mineral formation, including conversion of hexoses to sugar acids and polymerization of amino acids/peptides into larger peptides/proteins. In addition, prebiotic mineral-carbon assemblages produced nanometer-scaled filamentous aggregated morphologies. On the contrary, in biotic treatments with cells, organic carbon in minerals displayed a more heterogeneous distribution. Notably, "hot spots" of organic carbon and oxygen-containing functional groups, with the size, shape, and composition of microbial cells, were preserved in mineral aggregates. We propose a list of characteristics that could be used to help distinguish biogenic from prebiotic/abiotic Fe-S minerals and help refine the search of extant or extinct microbial life in the martian subsurface.

Pressure effects on sulfur-oxidizing activity of Thiobacillus thioparus.

Carbon capture and storage technologies are crucial for reducing carbon emission from power plants as a response to global climate change. The CarbFix project (Iceland) aims at examining the geochemical response of injected CO2 into subsurface reservoirs. The potential role of the subsurface biosphere has been little investigated up to now. Here, we used Thiobacillus thioparus that became abundant at the CarbFix1 pilot site after injection of CO2 and purified geothermal gases in basaltic aquifer at 400-800 m depth (4-8 MPa). The capacity of T. thioparus to produce sulfate, through oxidation of thiosulfate, was measured by Raman spectroscopy as a function of pressure up to 10 MPa. The results show that the growth and metabolic activity of T. thioparus are influenced by the initial concentration of the electron donor thiosulfate. It grows best at low initial concentration of thiosulfate (here 5 g.l-1 or 31.6 mM) and best oxidizes thiosulfate into sulfate at 0.1 MPa with a yield of 14.7 ± 0.5%. Sulfur oxidation stops at 4.3 ± 0.1 MPa (43 bar). This autotrophic specie can thereby react to CO2 and H2 S injection down to 430 m depth and may contribute to induced biogeochemical cycles during subsurface energy operations.

Biogeochemical and physical controls on methane fluxes from two ferruginous meromictic lakes.

Meromictic lakes with anoxic bottom waters often have active methane cycles whereby methane is generally produced biogenically under anoxic conditions and oxidized in oxic surface waters prior to reaching the atmosphere. Lakes that contain dissolved ferrous iron in their deep waters (i.e., ferruginous) are rare, but valuable, as geochemical analogues of the conditions that dominated the Earth's oceans during the Precambrian when interactions between the iron and methane cycles could have shaped the greenhouse regulation of the planet's climate. Here, we explored controls on the methane fluxes from Brownie Lake and Canyon Lake, two ferruginous meromictic lakes that contain similar concentrations (max. >1 mM) of dissolved methane in their bottom waters. The order Methanobacteriales was the dominant methanogen detected in both lakes. At Brownie Lake, methanogen abundance, an increase in methane concentration with respect to depths closer to the sediment, and isotopic data suggest methanogenesis is an active process in the anoxic water column. At Canyon Lake, methanogenesis occurred primarily in the sediment. The most abundant aerobic methane-oxidizing bacteria present in both water columns were associated with the Gammaproteobacteria, with little evidence of anaerobic methane oxidizing organisms being present or active. Direct measurements at the surface revealed a methane flux from Brownie Lake that was two orders of magnitude greater than the flux from Canyon Lake. Comparison of measured versus calculated turbulent diffusive fluxes indicates that most of the methane flux at Brownie Lake was non-diffusive. Although the turbulent diffusive methane flux at Canyon Lake was attenuated by methane oxidizing bacteria, dissolved methane was detected in the epilimnion, suggestive of lateral transport of methane from littoral sediments. These results highlight the importance of direct measurements in estimating the total methane flux from water columns, and that non-diffusive transport of methane may be important to consider from other ferruginous systems.

Sulphur and carbon isotopes as tracers of past sub-seafloor microbial activity.

Microbial life below the seafloor has changed over geological time, but these changes are often not obvious, as they are not recorded in the sediment. Sulphur (S) isotope values in pyrite extracted from a Plio- to Holocene sequence of the Peru Margin (Ocean Drilling Program, ODP, Site 1229) show a down-core pattern that correlates with the pattern of carbon (C) isotopes in diagenetic dolomite. Early formation of the pyrite is indicated by the mineralogical composition of iron, showing a high degree of pyritization throughout the sedimentary sequence. Hence, the S-record could not have been substantially overprinted by later pyrite formation. The S- and C-isotope profiles show, thus, evidence for two episodes of enhanced microbial methane production with a very shallow sulphate-methane transition zone. The events of high activity are correlated with zones of elevated organic C content in the stratigraphic sequence. Our results demonstrate how isotopic signatures preserved in diagenetic mineral phases provide information on changes of past biogeochemical activity in a dynamic sub-seafloor biosphere.

Experimental diagenesis of organo-mineral structures formed by microaerophilic Fe(II)-oxidizing bacteria.

Twisted stalks are organo-mineral structures produced by some microaerophilic Fe(II)-oxidizing bacteria at O2 concentrations as low as 3 µM. The presence of these structures in rocks having experienced a diagenetic history could indicate microbial Fe(II)-oxidizing activity as well as localized abundance of oxygen at the time of sediment deposition. Here we use spectroscopy and analytical microscopy to evaluate if--and what kind of--transformations occur in twisted stalks through experimental diagenesis. Unique mineral textures appear on stalks as temperature and pressure conditions increase. Haematite and magnetite form from ferrihydrite at 170 °C-120 MPa. Yet the twisted morphology of the stalks, and the organic matrix, mainly composed of long-chain saturated aliphatic compounds, are preserved at 250 °C-140 MPa. Our results suggest that iron minerals might play a role in maintaining the structural and chemical integrity of stalks under diagenetic conditions and provide spectroscopic signatures for the search of ancient life in the rock record.

Iron reduction by the deep-sea bacterium Shewanella profunda LT13a under subsurface pressure and temperature conditions.

Microorganisms influence biogeochemical cycles from the surface down to the depths of the continental rocks and oceanic basaltic crust. Due to the poor recovery of microbial isolates from the deep subsurface, the influence of physical environmental parameters, such as pressure and temperature, on the physiology and metabolic potential of subsurface inhabitants is not well constrained. We evaluated Fe(III) reduction rates (FeRRs) and viability, measured as colony-forming ability, of the deep-sea piezophilic bacterium Shewanella profunda LT13a over a range of pressures (0-125 MPa) and temperatures (4-37∘C) that included the in situ habitat of the bacterium isolated from deep-sea sediments at 4500 m depth below sea level. S. profunda LT13a was active at all temperatures investigated and at pressures up to 120 MPa at 30∘C, suggesting that it is well adapted to deep-sea and deep sedimentary environments. Average initial cellular FeRRs only slightly decreased with increasing pressure until activity stopped, suggesting that the respiratory chain was not immediately affected upon the application of pressure. We hypothesize that, as pressure increases, the increased energy demand for cell maintenance is not fulfilled, thus leading to a decrease in viability. This study opens up perspectives about energy requirements of cells in the deep subsurface.

Pressure as an environmental parameter for microbial life--a review.

Microbial life has been prevailing in the biosphere for the last 3.8 Ga at least. Throughout most of the Earth's history it has experienced a range of pressures; both dynamic pressure when the young Earth was heavily bombarded, and static pressure in subsurface environments that could have served as a refuge and where microbial life nowadays flourishes. In this review, we discuss the extent of high-pressure habitats in early and modern times and provide a short overview of microbial survival under dynamic pressures. We summarize the current knowledge about the impact of microbial activity on biogeochemical cycles under pressures characteristic of the deep subsurface. We evaluate the possibility that pressure can be a limiting parameter for life at depth. Finally, we discuss the open questions and knowledge gaps that exist in the field of high-pressure geomicrobiology.

Linking microbial heterotrophic activity and sediment lithology in oxic, oligotrophic sub-seafloor sediments of the north atlantic ocean.

Microbial heterotrophic activity was investigated in oxic sub-seafloor sediments at North Pond, a sediment pond situated at 23°N on the western flank of the Mid-Atlantic Ridge. The North Pond sediments underlie the oligotrophic North Atlantic Gyre at 4580-m water depth and cover a 7-8 million-year-old basaltic crust aquifer through which seawater flows. Discrete samples for experimentation were obtained from up to ~9 m-long gravity cores taken at 14 stations in the North Pond area. Potential respiration rates were determined in sediment slurries incubated under aerobic conditions with (14)C-acetate. Microbial heterotrophic activity, as defined by oxidation of acetate to CO(2) (with O(2) as electron acceptor), was detected in all 14 stations and all depths sampled. Potential respiration rates were generally low (<0.2 nmol of respired acetate cm(-3) d(-1)) in the sediment, but indicate that microbial heterotrophic activity occurs in deep-sea, oxic, sub-seafloor sediments. Furthermore, discernable differences in activity existed between sites and within given depth profiles. At seven stations, activity was increased by several orders of magnitude at depth (up to ~12 nmol of acetate respired cm(-3) d(-1)). We attempted to correlate the measures of activity with high-resolution color and element stratigraphy. Increased activities at certain depths may be correlated to variations in the sediment geology, i.e., to the presence of dark clay-rich layers, of sandy layers, or within clay-rich horizons presumably overlying basalts. This would suggest that the distribution of microbial heterotrophic activity in deeply buried sediments may be linked to specific lithologies. Nevertheless, high-resolution microbial examination at the level currently enjoyed by sedimentologists will be required to fully explore this link.

In situ Raman and X-ray spectroscopies to monitor microbial activities under high hydrostatic pressure.

Until recently, monitoring of cells and cellular activities at high hydrostatic pressure (HHP) was mainly limited to ex situ observations. Samples were analyzed prior to and following the depressurization step to evaluate the effect of the pressure treatment. Such ex situ measurements have several drawbacks: (i) it does not allow for kinetic measurements and (ii) the depressurization step often leads to artifactual measurements. Here, we describe recent advances in diamond anvil cell (DAC) technology to adapt it to the monitoring of microbial processes in situ. The modified DAC is asymmetrical, with a single anvil and a diamond window to improve imaging quality and signal collection. Using this novel DAC combined to Raman and X-ray spectroscopy, we monitored the metabolism of glucose by baker's yeast and the reduction of selenite by Agrobacterium tumefaciens in situ under HHP. In situ spectroscopy is also a promising tool to study piezophilic microorganisms.

[Prevention of Bacillus Calmette-Guérin immunotherapy complications]. / Prévention des complications du Bacille de Calmette-Guérin.

Bacille Calmette-Guérin (BCG) in intravesical instillations is the reference treatment for urothelial carcinoma with a high risk of progression. Compliance with this treatment is altered by its potentially serious locoregional or general side effects. Prevention of these complications requires implementing rules of good practice for the instillations. The undesirable side effects should be recognized early. Their treatment should be rapid and adapted to the patient. The results of the French randomized, placebo-controlled ITB01 study showed that the class II side effects of BCG were significantly reduced by administration of ofloxacin after each instillation of BCG. The number of class III side effects requiring antitubercular treatment was also reduced in the patients in this study who had received ofloxacin.

Development of a low-pressure diamond anvil cell and analytical tools to monitor microbial activities in situ under controlled P and T.

We have designed a new low-pressure Diamond Anvil Cell (DAC), calibrated two novel pressure calibrants and validated the use of semi-quantitative Raman and X-ray spectroscopies to monitor the fate of microbes, their metabolism or their cellular components under controlled pressures and temperatures in the 0.1-1.4 GPa and 20-300 degrees C P,T range. The low-pressure DAC has a 250- to 600-microm-thick observation diamond window to allow for lower detection limits and improved microscopic imaging. This new design allows the determination of cellular growth parameters from automated image analysis, which can be correlated with the spectroscopic data obtained on metabolism, ensuring high quality data collection on microbial activity under pressure. The novel pressure sensors offer the ease of use of the well-known ruby scale, while being more sensitive and reacting to pressure variations instantaneously.