Differential effect of silver nanoparticles on the microbiome of adult and developing planaria.

Silver nanoparticles (AgNPs) are widely incorporated in household, consumer and medical products. Their unintentional release via wastewaters raises concerns on their environmental impact, particularly for aquatic organisms and their associated bacterial communities. It is known that the microbiome plays an important role in its host's health and physiology, e.g. by producing essential nutrients and providing protection against pathogens. A thorough understanding of the effects of AgNPs on bacterial communities and on their interactions with the host is crucial to fully assess AgNP toxicity on aquatic organisms. Our results indicate that the microbiome of the invertebrate Schmidtea mediterranea, a freshwater planarian, is affected by AgNP exposure at the tested 10 µg/ml concentration. Using targeted amplification of the bacterial 16S rRNA gene V3-V4 region, two independent experiments on the microbiomes of adult worms revealed a consistent decrease in Betaproteobacteriales after AgNP exposure, mainly attributed to a decrease in Curvibacter and Undibacterium. Although developing tissues and organisms are known to be more sensitive to toxic compounds, three independent experiments in regenerating worms showed a less pronounced effect of AgNP exposure on the microbiome, possibly because underlying bacterial community changes during development mask the AgNP induced effect. The presence of a polyvinyl-pyrrolidone (PVP) coating did not significantly alter the outcome of the experiments compared to those with uncoated particles. The observed variation between the different experiments underlines the highly variable nature of microbiomes and emphasises the need to repeat microbiome experiments, within and between physiological states of the animal.

Insights into selenate removal mechanism of hydrogen-based membrane biofilm reactor for nitrate-polluted groundwater treatment based on anaerobic biofilm analysis.

The selenate removal mechanism of hydrogen-based membrane biofilm reactor (MBfR) for nitrate-polluted groundwater treatment was studied based on anaerobic biofilm analysis. A laboratory-scale MBfR was operated for over 60 days with electron balance, structural analysis, and bacterial community identification. Results showed that anaerobic biofilm had an excellent removal of both selenate (95%) and nitrate (100%). Reduction of Selenate → Selenite → Se0 with hydrogen was the main pathway of anaerobic biofilm for selenate removal with amorphous Se0 precipitate accumulating in the biofilm. The element selenium was observed to be evenly distributed along the cross-sectional thin biofilm. A part of selenate (3%) was also reduced into methyl-selenide by heterotrophic bacteria. Additionally, Hydrogenophaga bacteria of ß-Proteobacteria, capable of both nitrate and selenate removal, worked as the dominant species (over 85%) in the biofilm and contributed to the stable removal of both nitrate and selenate. With the selenate input, bacteria with a capacity for both selenate and nitrate removal were also developed in the anaerobic biofilm community.

Pyomelanin production: Insights into the incomplete aerobic l-phenylalanine catabolism of a photosynthetic bacterium, Rubrivivax benzoatilyticus JA2.

Rubrivivax benzoatilyticus JA2 is a metabolically versatile bacterium, thrives on a wide array of organic compounds under different growth modes. Though genomic insights revealed the aromatic compound catabolic potential of strain JA2 under anaerobic/aerobic conditions, the studies are largely restricted to anaerobic metabolism. The previous study on phenylalanine metabolism in strain JA2 indicated melanin-like pigment production under aerobic conditions; however, characterization of pigment and its biosynthetic pathway is not explored. The current study aims at the characterization of pigment and elucidation of its biosynthetic pathway. Strain JA2 utilized l-phenylalanine as source of nitrogen under anaerobic/aerobic conditions but not as a carbon source. Strain JA2 produced a brown-pigment under phenylalanine-amended aerobic conditions. Spectroscopic and physicochemical analysis identified the purified brown-pigment as a melanin. Further, the genomic insights revealed the presence of a complete set of genes related to pyomelanin synthesis. Identification of key metabolites l-tyrosine, 4-hydroxyphenylpyruvic acid and homogentisic acid and their respective enzyme activities further supports the pyomelanin synthesis. Moreover, the precursors feeding, pathway specific inhibitor studies confirmed the pyomelanin synthesis in strain JA2. Our study revealed an incomplete catabolism of phenylalanine; absence of ring cleavage gene, homogentisate dioxygenase leading to homogentisate accumulation thereby pyomelanin synthesis in strain JA2.

Experimental evidence of the quantitative relationship between the prokaryote ingestion rate and the food vacuole content in mixotrophic phytoflagellates.

The verification that many phytoflagellates ingest prokaryotes has changed the view of the microbial loop in aquatic ecosystems. Still, progress is limited because the phagotrophic activity is difficult to quantify in natural assemblages. Linking the food vacuole content in protist with the ingestion rate of prokaryotes would provide a crucial step forward. In this study, using the catalysed reporter deposition - fluorescence in situ hybridization protocol (CARD-FISH), which allows the visualization of labelled prokaryotes inside protists without relying on incubation procedures, we experimentally relate the food vacuole content of prokaryotes (Vc ) to the population-averaged ingestion rates (Ir ) estimated using bacteria-size fluorescent microspheres. The two variables relate according to the equation Ir = 7.52 Vc 0.9 , which indicates a prokaryote half-life of about 6 min in the protist vacuole. Five mixotrophic flagellate species from natural and culture populations were evaluated seven times during 24 h; they provided a broad range of average vacuole content (0.01 to 2.02 prokaryote protist-1 ) and ingestion rates (0.18 to 23 prokaryote protist-1 h-1 ). Consequently, the relationship found can be applied to quantify the mixotrophy activity in a large variety of field and experimental studies.

Dicyandiamide has more inhibitory activities on nitrification than thiosulfate.

Dicyandiamide (DCD) and thiosulfates are two type of nitrification inhibitors (NIs) that have been widely used in agriculture to improve nitrogen (N) fertilizer use efficiency and mitigate negative effect of N on environment. Little information is available concerning the comparison of the efficacy of DCD and thiosulfate on N transformations in soil. The aim of this study was to compare the effects of DCD and thiosulfate (K2S2O3) on changes of NH4+-N, nitrification inhibition and N recovery in a latosolic red soil. An incubation experiment was conducted with four treatments of control (CK), N, N+DCD, and N+K2S2O3. Soil samples were collected periodically over 50 d to determine concentrations of mineral N, and the amoA gene abundance of ammonia monooxygenase (AMO) for ammonia-oxidizing bacteria (AOB) was estimated by qPCR after 10 d incubation. In the N treatment, 67.8% of the applied N as NH4+-N disappeared from the mineral N pool and only 2.7% and 30.8% of the applied N was accumulated as NO2--N and NO3--N, respectively. Addition of DCD and thiosulfate to the soil prevented NH4+-N disappearance by 63.0% and 13.6%, respectively. DCD suppressed the production of NO2--N by 97.41%, whereas thiosulfate increased accumulation of NO2--N by 14.6%. Application of N along with DCD and thiosulfate inhibited nitrification, respectively, by 72.6% and 33.1%, resulting in the delay of the nitrification process for 30 days and 10 days, respectively. Apparent N recovery in N treatment was 66.2%, which increased by 55.2% and 4.8% by DCD and thiosulfate, respectively. Numbers of AOB amoA gene copy was significantly inhibited by both DCD and thiosulfate, and the stronger inhibition induced by DCD than thiosulfate was recorded. Results indicated that both DCD and thiosulfate were effective inhibitors for NH4+-N oxidation, NO3--N production, mineral N losses and AOB growth. DCD showed a more pronounced effect on nitrification inhibition than thiosulfate.

Effects of nitrification inhibitors on gross N nitrification rate, ammonia oxidizers, and N2O production under different temperatures in two pasture soils.

Australian pasture soil for cattle and sheep industries constitutes the principal land use with considerable N fertilizer consumption, which is one of the causes of local environmental problems. Nitrification plays a key role in regulating soil inorganic N concentration and its environmental diffusion. The effects of different nitrification inhibitors (NIs) on gross N nitrification (ngross) rate and N2O production under different temperatures in pasture soils remain unclear. A laboratory incubation experiment was conducted to determine the effect of NIs (dicyandiamide [DCD], 3,4-dimethylpyrazole phosphate [DMPP], and 3-methylpyrazol and 1H-1,2,4-triazol [3MP + TZ]) on N2O emissions, ngross and net N nitrification (nnet) rates, and the abundance of ammonia oxidizers, namely, ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB), in two Australian pasture soils incubated at temperatures of 15, 25, and 35 °C. All NIs reduced both ngross and nnet rates and N2O production rate from the two pasture soils but to different extents. The inhibitory rates of NIs on ngross and nnet reached 6.80-63.8 and 5.91-62.3%, respectively, whereas that on N2O production rate totaled 4.5-41.4% in the tested soils. NIs reduced nitrification and N2O production by inhibiting the growth of AOB rather than AOA. The inhibitory effects of NIs were temperature-dependent, that is, decreasing with increasing temperature from 15 to 35 °C. In general, DMPP performed better than DCD and 3MP + TZ at 15 and 35 °C, whereas DCD performed more effectively than the other two NIs at 25 °C. Our results suggest that the utilization of NIs will depend on the conditions present, especially soil temperature. Additionally, AOB is the target of inhibition when mitigating nitrification and N2O emission by applying NIs in pasture soils.

Cultivable bacteria from Pectinatella magnifica and the surrounding water in South Bohemia indicate potential new Gammaproteobacterial, Betaproteobacterial and Firmicutes taxa.

Pectinatella magnifica is a freshwater bryozoan, which has become a subject of scientific interest because of its invasive expansion worldwide. To obtain a comprehensive overview of its influence on environments, information on associated bacteria is needed. In this study, cultivable bacteria associated with P. magnifica were investigated. In total, 253 isolates were selected for preliminary identification by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry and clustered based on repetitive extragenic palindromic-PCR profiles. Among these, 169 strains were selected and identified using 16S rRNA gene comparative analyses. The sequences were grouped into 76 phylotypes and affiliated with 67 species. The majority of isolated bacteria belonged to Gammaproteobacteria, followed by Betaproteobacteria, Firmicutes, Bacteroidetes and Actinobacteria. Most strains within the Betaproteobacteria were isolated exclusively from bryozoan colonies. Aeromonas was the genus predominantly isolated from both P. magnifica and the water samples. Based on 16S rDNA similarity values, 15 putative new species belonging to the genera Aeromonas, Aquitalea, Clostridium, Herbaspirillum, Chromobacterium, Chryseobacterium, Morganella, Paludibacterium, Pectobacterium, Rahnella, Rhodoferax and Serratia, and putative new genera belonging to families Clostridiaceae and Sporomusaceae were revealed. The majority of the detected bacteria were species widely distributed in the environments; nevertheless, a possible symbiotic association of two new putative species with P. magnifica cannot be excluded.

The reduced genome of Candidatus Kinetoplastibacterium sorsogonicusi, the endosymbiont of Kentomonas sorsogonicus (Trypanosomatidae): loss of the haem-synthesis pathway.

Trypanosomatids of the genera Angomonas and Strigomonas (subfamily Strigomonadinae) have long been known to contain intracellular beta-proteobacteria, which provide them with many important nutrients such as haem, essential amino acids and vitamins. Recently, Kentomonas sorsogonicus, a divergent member of Strigomonadinae, has been described. Herein, we characterize the genome of its endosymbiont, Candidatus Kinetoplastibacterium sorsogonicusi. This genome is completely syntenic with those of other known Ca. Kinetoplastibacterium spp., but more reduced in size (~742 kb, compared with 810-833 kb, respectively). Gene losses are not concentrated in any hot-spots but are instead distributed throughout the genome. The most conspicuous loss is that of the haem-synthesis pathway. For long, removing haemin from the culture medium has been a standard procedure in cultivating trypanosomatids isolated from insects; continued growth was considered as an evidence of endosymbiont presence. However, we demonstrate that, despite bearing the endosymbiont, K. sorsogonicus cannot grow in culture without haem. Thus, the traditional test cannot be taken as a reliable criterion for the absence or presence of endosymbionts in trypanosomatid flagellates. It remains unclear why the ability to synthesize such an essential compound was lost in Ca. K. sorsogonicusi, whereas all other known bacterial endosymbionts of trypanosomatids retain them.

Epibiont growth on filamentous bacteria found in activated sludge: a morphological approach.

Occurrence of epibiont attachment on filamentous bacteria is a common phenomenon in activated sludge. In this study, an attempt has been made to elucidate the intrinsic nature of the attachment between the epibionts and filamentous bacteria based on microscopic observations. Characterization of the epiflora based on fluorescence in situ hybridization using group level probes revealed that the epibionts colonizing these filamentous bacteria largely belongs to the class Alphaproteobacteria, followed by Beta and Gammaproteobacteria. The ultrastructural examination using transmission electron microscopy pointed to the existence of a possible cell-to-cell interaction between epibionts and the selected filaments. Common bacterial appendages such as pili and fimbria were absent at the interface and further noted was the presence of cell membrane extensions on epibiont bacteria protruding towards the targeted filamentous cell. Fibrillar structures resembling amyloid-like proteins were observed within the filament cells targeted by the epibionts. An interaction was apparent between amyloid such as proteins and epibionts with regards to the direction of fibrillar structures and the distance of approaching epibiont bacteria. Due to the lack of visual evidence in support of penetration, the role of these amyloid-like fibrils as potential attachment sites for the epibionts was taken into consideration, and required further validation using conformational antibodies.

Dominant Candidatus Accumulibacter phosphatis Enriched in Response to Phosphate Concentrations in EBPR Process.

Candidatus Accumulibacter phosphatis (Accumulibacter), which plays an important role in enhanced biological phosphorus removal in wastewater treatment plants, is phylogenetically classified into two major types (Types I and II). Phosphate concentrations affect the Accumulibacter community of the biomass enriched in treatment plants. Therefore, in the present study, Accumulibacter enrichments were conducted using a down-flow hanging sponge reactor under five conditions and a wide range of controlled phosphate concentrations in order to investigate how phosphate governs the community. We found that excessive phosphate levels inhibited Accumulibacter activity, that this inhibitory effect was greater for Type II. In addition, the affinity of Type II for phosphate was higher than that of Type I. Type IIA-B dominated at a phosphate concentration less than 5 mg P L-1, while Type IA was dominant at 50 and 500 mg P L-1. These patterns of enrichment may be explained by an inhibition kinetics model.

Influence of pH, EDTA/Fe(II) ratio, and microbial culture on Fe(II)-mediated autotrophic denitrification.

Fe(II)-mediated autotrophic denitrification with four different microbial cultures under different pH and EDTA/Fe(II) conditions was investigated in batch bioassays. Initially, the highest nitrate removal (72%) was achieved with an activated sludge inoculum. The use of pure cultures of Pseudogulbenkiania strain 2002 and Thiobacillus denitrificans resulted in a 55 and 52% nitrate removal, respectively. No denitrification was observed for a mixed culture dominated by Thiobacillus thioparus and T. denitrificans. A longer enrichment on Fe(II) and the supplementation of thiosulfate as additional electron donor were needed to stimulate the denitrifying activity of the Thiobacillus-mixed culture. A second subculture on Fe(II) as sole electron donor resulted in higher denitrification efficiencies for all microbial cultures. In particular, nitrate removal reached up to 84% with a specific nitrate removal rate of 1.160 mM·(g VSS·day)-1 in the bioassays seeded with the Thiobacillus-mixed culture. All cultures were favored by decreasing the EDTA/Fe(II) molar ratio from 2.0 to 0.5. The most significant denitrification enhancement was observed for the Pseudogulbenkiania species, indicating a lower tolerance to EDTA. The two pure cultures effectively maintained denitrification at pH 7.0 and were more sensitive to a pH decrease. Conversely, the optimal pH was 6.0 for the Thiobacillus-mixed and activated sludge cultures.

Bacterial Compatibility in Combined Inoculations Enhances the Growth of Potato Seedlings.

The compatibility of strains is crucial for formulating bioinoculants that promote plant growth. We herein assessed the compatibility of four potential bioinoculants isolated from potato roots and tubers (Sphingomonas sp. T168, Streptomyces sp. R170, Streptomyces sp. R181, and Methylibium sp. R182) that were co-inoculated in order to improve plant growth. We screened these strains using biochemical tests, and the results obtained showed that R170 had the highest potential as a bioinoculant, as indicated by its significant ability to produce plant growth-promoting substances, its higher tolerance against NaCl (2%) and AlCl3 (0.01%), and growth in a wider range of pH values (5.0-10.0) than the other three strains. Therefore, the compatibility of R170 with other strains was tested in combined inoculations, and the results showed that the co-inoculation of R170 with T168 or R182 synergistically increased plant weight over un-inoculated controls, indicating the compatibility of strains based on the increased production of plant growth promoters such as indole-3-acetic acid (IAA) and siderophores as well as co-localization on roots. However, a parallel test using strain R181, which is the same Streptomyces genus as R170, showed incompatibility with T168 and R182, as revealed by weaker plant growth promotion and a lack of co-localization. Collectively, our results suggest that compatibility among bacterial inoculants is important for efficient plant growth promotion, and that R170 has potential as a useful bioinoculant, particularly in combined inoculations that contain compatible bacteria.

Comparison of different two-pathway models for describing the combined effect of DO and nitrite on the nitrous oxide production by ammonia-oxidizing bacteria.

The aim of this work is to compare the capability of two recently proposed two-pathway models for predicting nitrous oxide (N2O) production by ammonia-oxidizing bacteria (AOB) for varying ranges of dissolved oxygen (DO) and nitrite. The first model includes the electron carriers whereas the second model is based on direct coupling of electron donors and acceptors. Simulations are confronted to extensive sets of experiments (43 batches) from different studies with three different microbial systems. Despite their different mathematical structures, both models could well and similarly describe the combined effect of DO and nitrite on N2O production rate and emission factor. The model-predicted contributions for nitrifier denitrification pathway and hydroxylamine pathway also matched well with the available isotopic measurements. Based on sensitivity analysis, calibration procedures are described and discussed for facilitating the future use of those models.

Rethinking growth and decay kinetics in activated sludge - towards a new adaptive kinetics approach.

Growth kinetics in activated sludge modelling (ASM) are typically assumed to be the result of intrinsic growth and decay properties and thus process parameters are deemed to be constant. The activity change in a microbial population is expressed in terms of variance of the active biomass fraction and not actual shifts in bacterial cellular activities. This approach is limited, in that it does not recognise the reality that active biomass is highly physiologically adaptive. Here, a strong correlation between maximum specific growth rate (µmax) and decay rate (be) of ordinary heterotrophic organisms was revealed in both low solids retention times (SRT) and high SRT activated sludge systems. This relationship is indicative of physiological adaptation either for growth (high µmax and be) or survival optimization (low µmax and be). Further, the nitrifier decay process was investigated using molecular techniques to measure decay rates of ammonia oxidizing bacteria and nitrite oxidizing bacteria over a range of temperatures. This approach revealed decay rates 10-12% lower than values previously accepted and used in ASM. These findings highlight potential benefits of incorporating physiological adaptation of heterotrophic and nitrifying populations in future ASM.

Predicting N2O emissions from nitrifying and denitrifying biofilms: a modeling study.

Wastewater treatment plants can be significant sources of nitrous oxide (N2O), a potent greenhouse gas. While our understanding of N2O emissions from suspended-growth processes has advanced significantly, less is known about emissions from biofilm processes. Biofilms may behave differently due to their substrate gradients and microbial stratification. In this study, we used mathematical modeling to explore the mechanisms of N2O emissions from nitrifying and denitrifying biofilms. Our ammonia-oxidizing bacteria biofilm model suggests that N2O emissions from biofilm can be significantly greater than from suspended-growth systems. The driving factor is the diffusion of hydroxylamine, a nitrification intermediate, from the aerobic to the anoxic regions of the biofilm. The presence of nitrite-oxidizing bacteria further increased emissions. For denitrifying biofilms, our results suggest that emissions are generally greater than for suspended-growth systems. However, the magnitude of the difference depends on the bulk dissolved oxygen, chemical oxygen demand, and nitrate concentrations, as well as the biofilm thickness. Overall, the accumulation and diffusion of key intermediates, i.e. hydroxylamine and nitrite, distinguish biofilms from suspended-growth systems. Our research suggests that the mechanisms of N2O emissions from biofilms are much more complex than suspended-growth systems, and that emissions may be higher in many cases.

Mechanism of electron transport during thiosulfate oxidation in an obligately mixotrophic bacterium Thiomonas bhubaneswarensis strain S10 (DSM 18181T).

This study describes the thiosulfate-supported respiratory electron transport activity of Thiomonas bhubaneswarensis strain S10 (DSM 18181T). Whole-genome sequence analysis revealed the presence of complete sox (sulfur oxidation) gene cluster (soxCDYZAXB) including the sulfur oxygenase reductase (SOR), sulfide quinone reductase (SQR), sulfide dehydrogenase (flavocytochrome c (fcc)), thiosulfate dehydrogenase (Tsd), sulfite dehydrogenase (SorAB), and intracellular sulfur oxidation protein (DsrE/DsrF). In addition, genes encoding respiratory electron transport chain components viz. complex I (NADH dehydrogenase), complex II (succinate dehydrogenase), complex III (ubiquinone-cytochrome c reductase), and various types of terminal oxidases (cytochrome c and quinol oxidase) were identified in the genome. Using site-specific electron donors and inhibitors and by analyzing the cytochrome spectra, we identified the shortest thiosulfate-dependent electron transport chain in T. bhubaneswarensis DSM 18181T. Our results showed that thiosulfate supports the electron transport activity in a bifurcated manner, donating electrons to quinol (bd) and cytochrome c (Caa 3 ) oxidase; these two sites (quinol oxidase and cytochrome c oxidase) also showed differences in their phosphate esterification potential (oxidative phosphorylation efficiency (P/O)). Further, it was evidenced that the substrate-level phosphorylation is the major contributor to the total energy budget in this bacterium.

Microbial N Transformations and N2O Emission after Simulated Grassland Cultivation: Effects of the Nitrification Inhibitor 3,4-Dimethylpyrazole Phosphate (DMPP).

Grassland cultivation can mobilize large pools of N in the soil, with the potential for N leaching and N2O emissions. Spraying with the nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP) before cultivation was simulated by use of soil columns in which the residue distribution corresponded to plowing or rotovation to study the effects of soil-residue contact on N transformations. DMPP was sprayed on aboveground parts of ryegrass and white clover plants before incorporation. During a 42-day incubation, soil mineral N dynamics, potential ammonia oxidation (PAO), denitrifying enzyme activity (DEA), nitrifier and denitrifier populations, and N2O emissions were investigated. The soil NO3- pool was enriched with 15N to trace sources of N2O. Ammonium was rapidly released from decomposing residues, and PAO was stimulated in soil near residues. DMPP effectively reduced NH4+ transformation irrespective of residue distribution. Ammonia-oxidizing archaea (AOA) and bacteria (AOB) were both present, but only the AOB amoA transcript abundance correlated with PAO. DMPP inhibited the transcription of AOB amoA genes. Denitrifier genes and transcripts (nirK, nirS, and clades I and II of nosZ) were recovered, and a correlation was found between nirS mRNA and DEA. DMPP showed no adverse effects on the abundance or activity of denitrifiers. The 15N enrichment of N2O showed that denitrification was responsible for 80 to 90% of emissions. With support from a control experiment without NO3- amendment, it was concluded that DMPP will generally reduce the potential for leaching of residue-derived N, whereas the effect of DMPP on N2O emissions will be significant only when soil NO3- availability is limiting. IMPORTANCE: Residue incorporation following grassland cultivation can lead to mobilization of large pools of N and potentially to significant N losses via leaching and N2O emissions. This study proposed a mitigation strategy of applying 3,4-dimethylpyrazole phosphate (DMPP) prior to grassland cultivation and investigated its efficacy in a laboratory incubation study. DMPP inhibited the growth and activity of ammonia-oxidizing bacteria but had no adverse effects on ammonia-oxidizing archaea and denitrifiers. DMPP can effectively reduce the potential for leaching of NO3- derived from residue decomposition, while the effect on reducing N2O emissions will be significant only when soil NO3- availability is limiting. Our findings provide insight into how DMPP affects soil nitrifier and denitrifier populations and have direct implications for improving N use efficiency and reducing environmental impacts during grassland cultivation.

Quantitative Transcriptomics Reveals the Growth- and Nutrient-Dependent Response of a Streamlined Marine Methylotroph to Methanol and Naturally Occurring Dissolved Organic Matter.

The members of the OM43 clade of Betaproteobacteria are abundant coastal methylotrophs with a range of carbon-utilizing capabilities. However, their underlying transcriptional and metabolic responses to shifting conditions or different carbon substrates remain poorly understood. We examined the transcriptional dynamics of OM43 isolate NB0046 subjected to various inorganic nutrient, vitamin, and carbon substrate regimes over different growth phases to (i) develop a quantitative model of its mRNA content; (ii) identify transcriptional markers of physiological activity, nutritional state, and carbon and energy utilization; and (iii) identify pathways involved in methanol or naturally occurring dissolved organic matter (DOM) metabolism. Quantitative transcriptomics, achieved through addition of internal RNA standards, allowed for analyses on a transcripts-per-cell scale. This streamlined bacterium exhibited substantial shifts in total mRNA content (ranging from 1,800 to 17 transcripts cell-1 in the exponential and deep stationary phases, respectively) and gene-specific transcript abundances (>1,000-fold increases in some cases), depending on the growth phase and nutrient conditions. Carbon metabolism genes exhibited substantial dynamics, including those for ribulose monophosphate, tricarboxylic acid (TCA), and proteorhodopsin, as well as methanol dehydrogenase (xoxF), which, while always the most abundant transcript, increased from 5 to 120 transcripts cell-1 when cultures were nutrient and vitamin amended. In the DOM treatment, upregulation of TCA cycle, methylcitrate cycle, vitamin, and organic phosphorus genes suggested a metabolic route for this complex mixture of carbon substrates. The genome-wide inventory of transcript abundances produced here provides insight into a streamlined marine bacterium's regulation of carbon metabolism and energy flow, providing benchmarks for evaluating the activity of OM43 populations in situ IMPORTANCE: Bacteria exert a substantial influence on marine organic matter flux, yet the carbon components targeted by specific bacterial groups, as well as how those groups' metabolic activities change under different conditions, are not well understood. Gene expression studies of model organisms can identify these responses under defined conditions, which can then be compared to environmental transcriptomes to elucidate in situ activities. This integration, however, is limited by the data's relative nature. Here, we report the fully quantitative transcriptome of a marine bacterium, providing a genome-wide survey of cellular transcript abundances and how they change with different states of growth, nutrient conditions, and carbon substrates. The results revealed the dynamic metabolic strategies this methylotroph has for processing both simple one-carbon compounds and the complex multicarbon substrates of naturally derived marine organic matter and provide baseline quantitative data for identifying their in situ activities and impact on the marine carbon cycle.

Sublethal concentrations of silver nanoparticles affect the mechanical stability of biofilms.

Bacterial biofilms are most likely confronted with silver nanoparticles (Ag NPs) as a pollutant stressor in aquatic systems. In this study, biofilms of Aquabacterium citratiphilum were exposed for 20 h to 30 and 70 nm citrate stabilized Ag NPs in low-dose concentrations ranging from 600 to 2400 µg l-1, and the Ag NP-mediated effects on descriptive, structural, and functional biofilm characteristics, including viability, protein content, architecture, and mechanical stability, were investigated. Viability, based on the bacterial cell membrane integrity of A. citratiphilum, as determined by epifluorescence microscopy, remained unaffected after Ag NP exposure. Moreover, in contrast to information in the current literature, protein contents of cells and extracellular polymeric substances (EPS) and biofilm architecture, including dry mass, thickness, and density, were not significantly impacted by exposure to Ag NPs. However, the biofilms themselves served as effective sinks for Ag NPs, exhibiting enrichment factors from 5 to 8. Biofilms showed a greater capacity to accumulate 30 nm sized Ag NPs than 70 nm Ag NPs. Furthermore, Ag NPs significantly threatened the mechanical stability of biofilms, as determined by a newly developed assay. For 30 nm Ag NPs, the mechanical stability of biofilms decreased as the Ag NP concentrations applied to them increased. In contrast, 70 nm Ag NPs produced a similar decrease in mechanical stability for each applied concentration. Overall, this finding demonstrates that exposure to Ag NPs triggers remarkable changes in biofilm adhesion and/or cohesiveness. Because of biofilm-mediated ecological services, this response raises environmental concerns regarding Ag NP release into freshwater systems, even in sublethal concentrations.

Repeated replacement of an intrabacterial symbiont in the tripartite nested mealybug symbiosis.

Stable endosymbiosis of a bacterium into a host cell promotes cellular and genomic complexity. The mealybug Planococcus citri has two bacterial endosymbionts with an unusual nested arrangement: the γ-proteobacterium Moranella endobia lives in the cytoplasm of the ß-proteobacterium Tremblaya princeps These two bacteria, along with genes horizontally transferred from other bacteria to the P. citri genome, encode gene sets that form an interdependent metabolic patchwork. Here, we test the stability of this three-way symbiosis by sequencing host and symbiont genomes for five diverse mealybug species and find marked fluidity over evolutionary time. Although Tremblaya is the result of a single infection in the ancestor of mealybugs, the γ-proteobacterial symbionts result from multiple replacements of inferred different ages from related but distinct bacterial lineages. Our data show that symbiont replacement can happen even in the most intricate symbiotic arrangements and that preexisting horizontally transferred genes can remain stable on genomes in the face of extensive symbiont turnover.