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.

Differential responses of encoding-amoA nitrifiers and nir denitrifiers in activated sludge to anatase and rutile TiO2 nanoparticles: What is active functional guild in rate limiting step of nitrogen cycle?

The long-terms effects of different crystal-composition TiO2 nanoparticles (NPs) on nitrogen-cycle-related functional guilds in activated sludge remain unclear, especially under natural light irradiation. Accordingly, activated sludge was exposed to anatase TiO2-NPs (TiO2-A) and rutile TiO2-NPs (TiO2-R) for up to 45 days. With markedly (p < 0.05) reducing nitrification-/denitrification-enzymatic-activities and abundances of ammonia-oxidizing-microorganisms (AOMs) and nitrite-reducing-bacteria (NRB), TiO2-NPs triggered bacteria and archaea UPGMA clustering and a deep modification of N-cycling functional diversity guided by crystal structure. in situ13C-DNA-SIP confirmed ammonia-oxidizing-bacteria (AOB) (Nitrosomonas and Nitrosospira) in original sludge as main active AOMs with 75.4 times more abundance than ammonia-oxidizing-archaea (AOA), while AOA within Nitrosopumilus and Nitrososphaera genera were the main active AOMs and tended to aggregate inside sludge after 10-mg/L TiO2-NPs exposure. Encoding-nirK NRB were more sensitive, while encoding-nirS Zoogloea with a total share of 4.97% to 14.93%, etc. were the main active NRB. AOB was more sensitive to TiO2-A, while TiO2-R showed the stronger toxicity to AOA and NRB resulting from differences in water environmental behaviors and crystal characteristics of two TiO2-NPs. This work expands understanding of the ecological risks of titanium-dioxide-crystal-NPs in aquatic environment and may help devise better methods to alleviate environmental stress caused by NPs at wastewater treatment plants.

Long-chain flavodoxin FldX1 improves Paraburkholderia xenovorans LB400 tolerance to oxidative stress caused by paraquat and H2O2.

Flavodoxins are small electron transfer proteins containing flavin mononucleotide (FMN) as a prosthetic group, which play an important role during oxidative stress or iron limitation. The aims of this study were the identification and characterization of flavodoxins in the model aromatic-degrader Paraburkholderia xenovorans LB400 and the analyses of their protective effects during oxidative stress induced by paraquat and H2O2. Two genes (BxeA0278 and BxeB0391) encoding flavodoxins (hereafter referred to as fldX for flavodoxin from P. xenovorans), were identified at the LB400 major and minor chromosome. Genomic context of the flavodoxin-encoding genes showed genes encoding membrane proteins, transporters, and proteins involved in redox processes and biosynthesis of macromolecules. A secondary structure prediction of both LB400 flavodoxins showed the characteristic flavodoxin structure of five ß-sheets intercalated with five α-helices. FldX1 contains a loop intercalated in the fifth ß-strand, which indicates that it belongs to the long-chain flavodoxins, whereas FldX2 is a short-chain flavodoxin. A phylogenetic analysis of 73 flavodoxins from 43 bacterial genera revealed eight clusters (I-VIII), while FldX1 and FldX2 grouped separately within a long-chain and a short-chain flavodoxin clades. FldX1 and FldX2 were overexpressed in P. xenovorans. Interestingly, the strain overexpressing the long-chain flavodoxin FldX1 (p2-fldX1) showed a faster growth in glucose than the control strain. The recombinant strain overexpressing the long-chain flavodoxin FldX1 (p2-fldx1) exposed to paraquat (20 mM) possessed lower susceptibility to growth inhibition on plates and higher survival in liquid medium than the control strain. The strains overexpressing the flavodoxins FldX1 and FldX2 showed higher survival during exposure to 1 mM paraquat (>95%) than the control strain (68%). Compared to the control strain, strains overexpressing FldX1 and FldX2 showed lower lipid peroxidation (>20%) after exposure to 1 mM paraquat and a lower protein carbonylation (~30%) after exposure to 1 mM H2O2 was observed. During exposure to paraquat, strain p2-fldx1 downregulated the katG4, hpf, trxB1 and ohr genes (> 2-fold), whereas strain p2-fldx2 upregulated the oxyR and ahpC1 genes (> 2-fold). In conclusion, the flavodoxins FldX1 and FldX2 of P. xenovorans LB400 conferred protection to cells exposed to the oxidizing agents paraquat and H2O2.

Comparing two hydrazine addition strategies to stabilize mainstream deammonification: Performance and microbial community analysis.

In this study, an expanded granular sludge blanket reactor (EGSB) was proposed to achieve stable mainstream deammonification process by adding hydrazine (N2H4). Two N2H4 addition methods consisted of constant concentration (strategy A) and variable concentration (strategy B) both can inhibit nitrite oxidizing bacteria. A efficient performance was achieved with higher total nitrogen removal efficiency (82 ±â€¯6%) and nitrogen removal rate (0.32 ±â€¯0.02 kg N/(m3·d)) under strategy B. For strategy A, anaerobic ammonia oxidizing bacteria (AnAOB) in-situ activity was decreased from 2.76 to 0.68 mg N/(g VSS·h) at 42 mg/L NH4+-N. Candidatus Brocadia abundance increase from 14.62% to 20.07% under the strategy may indicated the self-regulate mechanism of AnAOB. Aerobic ammonia oxidizing bacteria (AOB, mainly Nitrosomonas) and AnAOB (mainly Candidatus Brocadia) were always dominated under two strategies. Strategy B provided better environment for most microorganisms (mainly Chloroflexri, Planctomycetes, Proteobacteria and Chlorobi).

Comparative Analysis of Antimicrobial Resistance, Integrons, and Virulence Genes Among Extended-Spectrum ß-Lactamase-Positive Laribacter hongkongensis from Edible Frogs and Freshwater Fish.

Aquatic animals are now recognized to be major hosts of potentially pathogenic Laribacter hongkongensis. A comparative study was carried out among extended-spectrum ß-lactamase (ESBL)-producing L. hongkongensis isolated from frogs (47 isolates) and fish (41 isolates) to examine phenotypic and genotypic antimicrobial resistance profiles, integrons, virulence factors, and genetic relatedness. Isolates from frogs showed a higher incidence of antibiotic resistance compared with those from fish for most of the antimicrobials tested, especially trimethoprim-sulfamethoxazole, tetracycline, ciprofloxacin, levofloxacin, and streptomycin. Multidrug-resistant strains were also found more frequently among frog isolates (5.44 traits on average) than among fish isolates (3.29 traits). In frog isolates, class 1 integrons and the resistance genes sul1, sul2, tetA, tetR, and aac(6')-Ib-cr showed a clearly higher incidence compared with isolates from fish. In contrast, blaTEM-1 was higher in fish isolates than in frog isolates. Correlation analysis showed that sul1, sul2, tetA, and tetR were significantly associated with class 1 integrons in frog isolates. The correlations indicated a potential co-selection risk of bacterial resistance to antibiotics. In addition, the distribution of three virulence-associated determinants for the type IV bundle-forming pili gene (bfpA), ferric aerobactin receptor gene (iucD), and iron-responsive element gene (ireA) was markedly higher in strains isolated from frogs than in those isolated from fish. No obvious genetic relatedness was observed between both populations. The large differences found in the incidence of antibiotic resistance, integrons along with the multiple resistance genes, virulence factors, and genetic fingerprints determined by pulsed-field gel electrophoresis suggest a high degree of antibiotic resistance and pathogenicity potential of ESBL-producing L. hongkongensis from isolates found in frogs.

Adaptive shifts of bacterioplankton communities in response to nitrogen enrichment in a highly polluted river.

Anthropogenic activity-mediated nutrient pollution, especially nitrogen enrichment, poses one of the major threats to river ecosystems. However, it remains unclear how and to which extent it affects aquatic microbial communities, especially in heavily polluted rivers. In this study, a significant environmental gradient, particularly nitrogen gradient, was observed along a wastewater receiving river, the North Canal River (NCR). The pollution level was highest, moderate, and lowest in the up-, middle, and down-streams, respectively. The community composition of bacterioplankton transitioned from being Betaproteobacteria-dominated upstream to Gammaproteobacteria-dominated downstream. Copiotrophic groups, such as Polynucleobacter (Betaproteobacteria) and Hydrogenophaga (Betaproteobacteria), were dominant in the upstream. Multiple statistical analyses indicated that total nitrogen (TN) was the most important factor driving the adaptive shifts of community structure. Analyses of co-occurrence networks showed that the complexity of networks was disrupted in the up- and middle streams, while enhanced in the downstream. Our findings here suggested that microbial interactions were reduced in response to the aggravation of nutrient pollution. Similar to these changes, we observed significant dissimilarity of composition of functional groups, with highest abundance of nitrogen metabolism members under the highest level of nitrogen enrichment. Further analyses indicated that most of these functional groups belonged to Betaproteobacteria, suggesting the potential coupling of community composition and function diversity. In summary, adaptive shifts of bacterioplankton community composition, as well as species interactions, occurred in response to nutrient pollution in highly polluted water bodies.

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.

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.

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.

Screening and optimizing of inhibitors for ammonia-oxidizing bacteria in sediments of malodorous river.

The proper use of selective ammonia-oxidizing archaea (AOA) and/or ammonia-oxidizing bacteria (AOB) inhibitors is critical to distinguish AOA and AOB contribution. In this research, three inhibitors including ampicillin, dicyandiamide (DCD), and allylthiourea (ATU) were examined mainly focusing on inhibiting dosage, adaptability, and effects. The results showed that the optimized inhibitory dosage of ampicillin, DCD, and ATU was separately 1.5 g L-1, 1 mM, and 25 µM. Among the three inhibitors, ATU exhibited the strongest and persistent inhibition effects and resulted in up to 90% inhibition in the AOB-enriched culture. The seemingly weakening inhibiting effects of ATU in the simulated river systems can be attributed to the involved role of AOA, the uneven spatial distribution of ATU, and protection by sediment structure in complex malodorous rivers. The high-throughput pyrosequencing analysis showed the AOB-related genus Nitrosomonas and Nitrosococcus were mostly affected by ATU in the enrichments and the river systems, respectively. The inhibition of ATU was realized mainly by reducing the abundance and activity of AOB. The decrease of the ratio of AOB/AOA amoA gene copy numbers after addition of ATU further confirmed the inhibiting effectiveness of ATU in complex microbial community of malodorous rivers.

Effects of different agricultural wastes on the dissipation of PAHs and the PAH-degrading genes in a PAH-contaminated soil.

Land application of agricultural wastes is considered as a promising bioremediation approach for cleaning up soils contaminated by aged polycyclic aromatic hydrocarbons (PAHs). However, it remains largely unknown about how microbial PAH-degraders, which play a key role in the biodegradation of soil PAHs, respond to the amendments of agricultural wastes. Here, a 90-day soil microcosm study was conducted to compare the effects of three agricultural wastes (i.e. WS, wheat stalk; MCSW, mushroom cultivation substrate waste; and CM, cow manure) on the dissipation of aged PAHs and the abundance and community structure of PAH-degrading microorganisms. The results showed that all the three agricultural wastes accelerated the dissipation of aged PAHs and significantly increased abundances of the bacterial 16S rRNA and PAH-degrading genes (i.e. pdo1 and nah). CM and MCSW with lower ratios of C:N eliminated soil PAHs more efficiently than WS with a high ratio of C:N. Low molecular weight PAHs were dissipated more quickly than those with high molecular weight. Phylogenetic analysis revealed that all of the nah and C12O clones were affiliated within Betaproteobacteria and Gammaproteobacteria, and application of agricultural wastes significantly changed the community structure of the microorganisms harboring nah and C12O genes, particularly in the CM treatment. Taken together, our findings suggest that the three tested agricultural wastes could accelerate the degradation of aged PAHs most likely through changing the abundances and community structure of microbial PAH degraders.

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.

Development of a toolbox to dissect host-endosymbiont interactions and protein trafficking in the trypanosomatid Angomonas deanei.

BACKGROUND: Bacterial endosymbionts are found across the eukaryotic kingdom and profoundly impacted eukaryote evolution. In many endosymbiotic associations with vertically inherited symbionts, highly complementary metabolic functions encoded by host and endosymbiont genomes indicate integration of metabolic processes between the partner organisms. While endosymbionts were initially expected to exchange only metabolites with their hosts, recent evidence has demonstrated that also host-encoded proteins can be targeted to the bacterial symbionts in various endosymbiotic systems. These proteins seem to participate in regulating symbiont growth and physiology. However, mechanisms required for protein targeting and the specific endosymbiont targets of these trafficked proteins are currently unexplored owing to a lack of molecular tools that enable functional studies of endosymbiotic systems. RESULTS: Here we show that the trypanosomatid Angomonas deanei, which harbors a ß-proteobacterial endosymbiont, is readily amenable to genetic manipulation. Its rapid growth, availability of full genome and transcriptome sequences, ease of transfection, and high frequency of homologous recombination have allowed us to stably integrate transgenes into the A. deanei nuclear genome, efficiently generate null mutants, and elucidate protein localization by heterologous expression of a fluorescent protein fused to various putative targeting signals. Combining these novel tools with proteomic analysis was key for demonstrating the routing of a host-encoded protein to the endosymbiont, suggesting the existence of a specific endosymbiont-sorting machinery in A. deanei. CONCLUSIONS: After previous reports from plants, insects, and a cercozoan amoeba we found here that also in A. deanei, i.e. a member of a fourth eukaryotic supergroup, host-encoded proteins can be routed to the bacterial endosymbiont. This finding adds further evidence to our view that the targeting of host proteins is a general strategy of eukaryotes to gain control over and interact with a bacterial endosymbiont. The molecular resources reported here establish A. deanei as a time and cost efficient reference system that allows for a rigorous dissection of host-symbiont interactions that have been, and are still being shaped over evolutionary time. We expect this system to greatly enhance our understanding of the biology of endosymbiosis.

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.

Tetracycline Selective Pressure and Homologous Recombination Shape the Evolution of Chlamydia suis: A Recently Identified Zoonotic Pathogen.

Species closely related to the human pathogen Chlamydia trachomatis (Ct) have recently been found to cause zoonotic infections, posing a public health threat especially in the case of tetracycline resistant Chlamydia suis (Cs) strains. These strains acquired a tet(C)-containing cassette via horizontal gene transfer (HGT). Genomes of 11 Cs strains from various tissues were sequenced to reconstruct evolutionary pathway(s) for tet(C) HGT. Cs had the highest recombination rate of Chlamydia species studied to date. Admixture occurred among Cs strains and with Chlamydia muridarum but not with Ct Although in vitro tet(C) cassette exchange with Ct has been documented, in vivo evidence may require examining human samples from Ct and Cs co-infected sites. Molecular-clock dating indicated that ancestral clades of resistant Cs strains predated the 1947 discovery of tetracycline, which was subsequently used in animal feed. The cassette likely spread throughout Cs strains by homologous recombination after acquisition from an external source, and our analysis suggests Betaproteobacteria as the origin. Selective pressure from tetracycline may be responsible for recent bottlenecks in Cs populations. Since tetracycline is an important antibiotic for treating Ct, zoonotic infections at mutual sites of infection indicate the possibility for cassette transfer and major public health repercussions.

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.

Nitric oxide scavengers differentially inhibit ammonia oxidation in ammonia-oxidizing archaea and bacteria.

Differential inhibitors are important for measuring the relative contributions of microbial groups, such as ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA), to biogeochemical processes in environmental samples. In particular, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO) represents a nitric oxide scavenger used for the specific inhibition of AOA, implicating nitric oxide as an intermediate of thaumarchaeotal ammonia oxidation. This study investigated four alternative nitric oxide scavengers for their ability to differentially inhibit AOA and AOB in comparison to PTIO. Caffeic acid, curcumin, methylene blue hydrate and trolox were tested onNitrosopumilus maritimus, two unpublished AOA representatives (AOA-6f and AOA-G6) as well as the AOB representative Nitrosomonas europaea All four scavengers inhibited ammonia oxidation by AOA at lower concentrations than for AOB. In particular, differential inhibition of AOA and AOB by caffeic acid (100 µM) and methylene blue hydrate (3 µM) was comparable to carboxy-PTIO (100 µM) in pure and enrichment culture incubations. However, when added to aquarium sponge biofilm microcosms, both scavengers were unable to inhibit ammonia oxidation consistently, likely due to degradation of the inhibitors themselves. This study provides evidence that a variety of nitric oxide scavengers result in differential inhibition of ammonia oxidation in AOA and AOB, and provides support to the proposed role of nitric oxide as a key intermediate in the thaumarchaeotal ammonia oxidation pathway.

Functional and structural responses of soil N-cycling microbial communities to the herbicide mesotrione: a dose-effect microcosm approach.

Microbial communities driving the nitrogen cycle contribute to ecosystem services such as crop production and air, soil, and water quality. The responses to herbicide stress of ammonia-oxidizing and ammonia-denitrifying microbial communities were investigated by an analysis of changes in structure-function relationships. Their potential activities, abundances (quantitative PCR), and genetic structure (denaturing gradient gel electrophoresis) were assessed in a microcosm experiment. The application rate (1 × FR, 0.45 µg g(-1) soil) of the mesotrione herbicide did not strongly affect soil N-nutrient dynamics or microbial community structure and abundances. Doses of the commercial product Callisto® (10 × FR and 100 × FR) or pure mesotrione (100 × FR) exceeding field rates induced short-term inhibition of nitrification and a lasting stimulation of denitrification. These effects could play a part in the increase in soil ammonium content and decrease in nitrate contents observed in treated soils. These functional impacts were mainly correlated with abundance shifts of ammonia-oxidizing Bacteria (AOB) and Archaea (AOA) or denitrifying bacteria. The sustained restoration of nitrification activity, from day 42 in the 100 × FR-treated soils, was likely promoted by changes in the community size and composition of AOB, which suggests a leading role, rather than AOA, for soil nitrification restoration after herbicide stress. This ecotoxicological community approach provides a nonesuch multiparameter assessment of responses of N-cycling microbial guilds to pesticide stress.

c-Type Cytochrome Assembly Is a Key Target of Copper Toxicity within the Bacterial Periplasm.

UNLABELLED: In the absence of a tight control of copper entrance into cells, bacteria have evolved different systems to control copper concentration within the cytoplasm and the periplasm. Central to these systems, the Cu(+) ATPase CopA plays a major role in copper tolerance and translocates copper from the cytoplasm to the periplasm. The fate of copper in the periplasm varies among species. Copper can be sequestered, oxidized, or released outside the cells. Here we describe the identification of CopI, a periplasmic protein present in many proteobacteria, and show its requirement for copper tolerance in Rubrivivax gelatinosus. The ΔcopI mutant is more susceptible to copper than the Cu(+) ATPase copA mutant. CopI is induced by copper, localized in the periplasm and could bind copper. Interestingly, copper affects cytochrome c membrane complexes (cbb3 oxidase and photosystem) in both ΔcopI and copA-null mutants, but the causes are different. In the copA mutant, heme and chlorophyll synthesis are affected, whereas in ΔcopI mutant, the decrease is a consequence of impaired cytochrome c assembly. This impact on c-type cytochromes would contribute also to the copper toxicity in the periplasm of the wild-type cells when they are exposed to high copper concentrations. IMPORTANCE: Copper is an essential cation required as a cofactor in enzymes involved in vital processes such as respiration, photosynthesis, free radical scavenging, and pathogenesis. However, copper is highly toxic and has been implicated in disorders in all organisms, including humans, because it can catalyze the production of toxic reactive oxygen species and targets various biosynthesis pathways. Identifying copper targets, provides insights into copper toxicity and homeostatic mechanisms for copper tolerance. In this work, we describe for the first time a direct effect of excess copper on cytochrome c assembly. We show that excess copper specifically affects periplasmic and membrane cytochromes c, thus suggesting that the copper toxicity targets c-type cytochrome biogenesis.

Assessment of microbial communities in mung bean (Vigna radiata) rhizosphere upon exposure to phytotoxic levels of Copper.

Pollution of agricultural soils by Cu is of concern as it could bring about alterations in microbial communities, ultimately eliminating certain plant beneficial bacteria thus disturbing soil fertility and plant growth. To understand the response of rhizobacterial communities upon Cu perturbation, mung bean (Vigna radiata) plants were grown in agricultural soil amended with CuSO4 (0-1000 mg kg(-1) ) under laboratory conditions. Culture-independent and -dependent Denaturing Gradient Gel Electrophoresis (CI-DGGE and CD-DGGE) fingerprinting techniques were employed to monitor rhizobacterial community shifts upon Cu amendment. In group specific PCR-DGGE, a negative impact was seen on α-Proteobacteria followed by ß-Proteobacteria resulting in a concomitant decrease in diversity indices with increased Cu concentration. No significant changes were observed in Firmicutes and Actinomycetes populations. In CD-DGGE rhizobacterial community shift was observed above 500 mg kg(-1) (CuSO4 ), however certain bands were predominantly present in all treatments. Plants showed toxic effects by reduction in growth and elevated Cu accumulation, with root system being affected prominently. From this study it is evident that above 250 mg kg(-1) , rhizobacterial communities are adversely affected. α-Proteobacteria was found to be a sensitive bio-indicator for Cu toxicity and is of particular significance since this group includes majority of plant growth promoting rhizobacteria.