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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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The long-terms effects of different crystal-composition TiO 2 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 TiO 2 -NPs (TiO 2 -A) and rutile TiO 2 -NPs (TiO 2 -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), TiO 2 -NPs triggered bacteria and archaea UPGMA clustering and a deep modification of N-cycling functional diversity guided by crystal structure. in situ 13 C-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 TiO 2 -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 TiO 2 -A, while TiO 2 -R showed the stronger toxicity to AOA and NRB resulting from differences in water environmental behaviors and crystal characteristics of two TiO 2 -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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

In this study, an expanded granular sludge blanket reactor (EGSB) was proposed to achieve stable mainstream deammonification process by adding hydrazine (N 2 H 4 ). Two N 2 H 4 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 ± 6%) and nitrogen removal rate (0.32 ± 0.02 kg N/(m 3 ·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 NH 4 + -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)., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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 β-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., Competing Interests: The authors have declared that no competing interests exist.

Aquatic animals are now recognized to be major hosts of potentially pathogenic Laribacter hongkongensis . A comparative study was carried out among extended-spectrum β-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, bla TEM-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.

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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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 (n gross ) rate and N 2 O 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 N 2 O emissions, n gross and net N nitrification (n net ) 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 n gross and n net rates and N 2 O production rate from the two pasture soils but to different extents. The inhibitory rates of NIs on n gross and n net reached 6.80-63.8 and 5.91-62.3%, respectively, whereas that on N 2 O production rate totaled 4.5-41.4% in the tested soils. NIs reduced nitrification and N 2 O 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 N 2 O emission by applying NIs in pasture soils.

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.

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., Competing Interests: The authors have declared that no competing interests exist.

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.

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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Grassland cultivation can mobilize large pools of N in the soil, with the potential for N leaching and N 2 O 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 N 2 O emissions were investigated. The soil NO 3 - pool was enriched with 15 N to trace sources of N 2 O. Ammonium was rapidly released from decomposing residues, and PAO was stimulated in soil near residues. DMPP effectively reduced NH 4 + 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 15 N enrichment of N 2 O showed that denitrification was responsible for 80 to 90% of emissions. With support from a control experiment without NO 3 - amendment, it was concluded that DMPP will generally reduce the potential for leaching of residue-derived N, whereas the effect of DMPP on N 2 O emissions will be significant only when soil NO 3 - 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 N 2 O 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 NO 3 - derived from residue decomposition, while the effect on reducing N 2 O emissions will be significant only when soil NO 3 - 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., (Copyright © 2016 American Society for Microbiology.)

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.

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., (Copyright © 2016 Gifford et al.)

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 β-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.

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., (© The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

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., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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.

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 β-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., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (Copyright © 2015 Durand et al.)

In the present study, we conducted a 2 week microcosm experiment with a natural freshwater bacterial community to assess the effects of titanium dioxide nanoparticles (TiO2-NPs) at various concentrations (0, 1, 10 and 100 mg/L) on planktonic and sessile bacteria under dark conditions. Results showed an increase of planktonic bacterial abundance at the highest TiO2-NP concentration, concomitant with a decrease from that of sessile bacteria. Bacterial assemblages were most affected by the 100 mg/L TiO2-NP exposure and overall diversity was found to be lower for planktonic bacteria and higher for sessile bacteria at this concentration. In both compartments, a 100 mg/L TiO2-NPs exposure induced a decrease in the ratio between the Betaproteobacteria and Bacteroidetes. For planktonic communities, a decrease of Comamonadaceae was observed concomitant with an increase of Oxalobacteraceae and Cytophagaceae (especially Emticicia). For sessile communities, results showed a strong decrease of Betaproteobacteria and particularly of Comamonadaceae., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

A bacterium was isolated from activated sewage sludge that has the ability to use ibuprofen as its sole carbon and energy source. Phylogenetic analysis of the 16S rRNA gene sequence placed the strain in the Variovorax genus within the β-proteobacteria. When grown on ibuprofen it accumulated a transient yellow intermediate that disappeared upon acidification, a trait consistent with meta ring-fission metabolites. GC/MS analysis of derivatized culture supernatant yielded two spectra consistent with trihydroxyibuprofen bearing all three hydroxyl groups on the aromatic ring. These metabolites were only detected when 3-fluorocatechol, a meta ring-fission inhibitor, was added to Ibu-1 cultures and the supernatant was then derivatized with aqueous acetic anhydride and diazomethane. These findings suggest the possibility of ibuprofen metabolism proceeding via a trihydroxyibuprofen meta ring-fission pathway. Identical spectra, consistent with these putative ring-hydroxylated trihydroxyibuprofen metabolites, were also obtained from ibuprofen-spiked sewage sludge, but only when it was poisoned with 3-fluorocatechol. The presence of the same trihydroxylated metabolites in both spiked sewage sludge and culture supernatants suggests that this trihydroxyibuprofen extradiol ring-cleavage pathway for the degradation of ibuprofen may have environmental relevance.

GSK1322322 is a novel antibacterial agent under development, and it has known antibacterial activities against multidrug-resistant respiratory and skin pathogens through its inhibition of the bacterial peptide deformylase. Here, we used next-generation sequencing (NGS) of the bacterial 16S rRNA genes from stool samples collected from 61 healthy volunteers at the predosing and end-of-study time points to determine the effects of GSK1322322 on the gastrointestinal (GI) microbiota in a phase I, randomized, double-blind, and placebo-controlled study. GSK1322322 was administered either intravenously (i.v.) only or in an oral-i.v. combination in single- and repeat-dose-escalation infusions. Analysis of the 16S rRNA sequence data found no significant changes in the relative abundances of GI operational taxonomic units (OTUs) between the prestudy and end-of-study samples for either the placebo- or i.v.-only-treated subjects. However, oral-i.v. treatment resulted in significant decreases in some bacterial taxa, the Firmicutes and Bacteroidales, and increases in others, the Betaproteobacteria, Gammaproteobacteria, and Bifidobacteriaceae. Microbiome diversity plots clearly differentiated the end-of-study oral-i.v.-dosed samples from all others collected. The changes in genome function as inferred from species composition suggest an increase in bacterial transporter and xenobiotic metabolism pathways in these samples. A phylogenetic analysis of the peptide deformylase protein sequences collected from the published genomes of clinical isolates previously tested for GSK1322322 in vitro susceptibility and GI bacterial reference genomes suggests that antibiotic target homology is one of several factors that influences the response of GI microbiota to this antibiotic. Our study shows that dosing regimen and target class are important factors when considering the impact of antibiotic usage on GI microbiota. (This clinical trial was registered at the GlaxoSmithKline Clinical Study Register under study identifier PDF 113376.)., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Owing to their wide application in industry and manufacturing, understanding the environmental safety of gold (Au) and silver (Ag) nanoparticles entering aquatic environment is a global issue of concern. For this study, ammonia-oxidizing bacteria (AOB) enrichment cultures reproduced from surface sediments taken from the Jiulong River estuary wetlands (Fujian Province, China) were spiked with nano-Ag and nano-Au to determine their impact on ammoxidation and the mechanisms involved in the process. Results showed that nano-Ag significantly inhibited bacterial ammoxidation in aquatic environment, with the average ammoxidation rate decreasing with increasing nano-Ag concentration. The average ammoxidation rate was significantly correlated to the Shannon index, the Simpson index, and AOB abundance. This suggested that ammoxidation inhibition resulted primarily from AOB biodiversity and abundance reduction, caused by the antibacterial property of nano-Ag. However, AOB biodiversity and abundance as well as bacterial ammoxidation were not inhibited by nano-Au (with a maximum experimental concentration of 2 mg L(-1)). Moreover, an insignificant correlation was found between AOB biodiversity and abundance and the average ammoxidation rate under the nano-Au treatment. Given that ammoxidation is regarded as a rate-limiting procedure in nitrogen (N) circulation, nano-Ag would affect N cycling but nano-Au would not after entering aquatic environments. Identified nano-Ag and nano-Au impacts on ammonium nitrogen transformation could be generalized in aquatic environment according to their extensive representation in the phylogenetic tree., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Lead (Pb) is a prominent toxic metal in natural and engineered systems. Current knowledge on Pb toxicity to the activated sludge has been limited to short-term (≤24 h) toxicity. The effect of extended Pb exposure on process performance, bacterial viability, and community compositions remains unknown. We quantified the 24-h and 7-day Pb toxicity to chemical oxygen demand (COD) and NH3–N removal, bacterial viability, and community compositions using lab-scale experiments. Our results showed that 7-day toxicity was significantly higher than the short-term 24-h toxicity. Ammonia-oxidizing bacteria were more susceptible than the heterotrophs to Pb toxicity. The specific oxygen uptake rate responded quickly to Pb addition and could serve as a rapid indicator for detecting Pb pollutions. Microbial viability decreased linearly with the amount of added Pb at extended exposure. The bacterial community diversity was markedly reduced with elevated Pb concentrations. Surface analysis suggested that the adsorbed form of Pb could have contributed to its toxicity along with the dissolved form. Our study provides for the first time a systematic investigation of the effect of extended exposure of Pb on the performance and microbiology of aerobic treatment processes, and it indicates that long-term Pb toxicity has been underappreciated by previous studies.

The genetic organization of the aioBA operon, encoding the arsenite oxidase of the moderately acidophilic and facultative chemoautotrophic bacterium Thiomonas arsenitoxydans, is different from that of the aioBA operon in the other arsenite oxidizers, in that it encodes AioF, a metalloprotein belonging to the ArsR/SmtB family. AioF is stabilized by arsenite, arsenate, or antimonite but not molybdate. Arsenic is tightly attached to AioF, likely by cysteine residues. When loaded with arsenite or arsenate, AioF is able to bind specifically to the regulatory region of the aio operon at two distinct positions. In Thiomonas arsenitoxydans, the promoters of aioX and aioB are convergent, suggesting that transcriptional interference occurs. These results indicate that the regulation of the aioBA operon is more complex in Thiomonas arsenitoxydans than in the other aioBA containing arsenite oxidizers and that the arsenic binding protein AioF is involved in this regulation. On the basis of these data, a model to explain the tight control of aioBA expression by arsenic in Thiomonas arsenitoxydans is proposed., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Microcosms were setup to investigate the possible impact of copper exposure on bacterial community structure and function in sediments of Jiaozhou Bay, China, by culture-independent microbial ecological techniques and community-level physiological profiling. Bacterial 16S rDNA libraries indicated that proportion of the bacteria in phyla Chloroflexi and Acidobacteria decreased, but that of Gammaproteobacteria and Planctomycetes slightly increased in copper-treated sediment. Denaturing gradient gel profiles showed that bacterial communities in control and copper exposed sediments developed into different directions, while the copper exposure did not change the pattern of ammonia oxidizing bacterial community. Microbial community-level physiological profiling revealed an obvious response to copper dosage. The copper pollution caused an acute decrease of carbon utilizing ability as well as bacterial functional diversity; the number of culturable heterotrophic bacteria was reduced by 90%. This study demonstrated that high copper input would obviously reduce culturable bacterial counts and seriously impact bacterial community function in marine sediments.

Light excitation of humic matter generates reactive oxygen species (ROS) in surface waters of aquatic ecosystems. Abundant ROS generated in humic matter rich lakes include singlet oxygen ((1)O2) and hydrogen peroxide (H2O2). Because these ROS differ in half-life time and toxicity, we compared their effects on microbial activity ((14)C-Leucine incorporation) and bacterial community composition (BCC) in surface waters of humic Lake Grosse Fuchskuhle (North-eastern Germany). For this purpose, experiments with water samples collected from the lake were conducted in July 2006, September 2008 and August 2009. Artificially increased (1)O2 and H2O2 concentrations inhibited microbial activity in water samples to a similar extent, but the effect of the respective ROS on BCC varied strongly. BCC analysis by 16S rRNA gene clone libraries and RT-PCR DGGE revealed ROS specific changes in relative abundance and activity of major bacterial groups and composition of dominating phylotypes. These changes were consistent in the three experiments performed in different years. The relative abundance of Polynucleobacter necessarius, Limnohabitans-related phylotypes (Betaproteobacteria), and Novosphingobium acidiphilum (Alphaproteobacteria) increased or was not affected by photo-sensitized (1)O2 exposure, but decreased after H2O2 exposure. The opposite pattern was found for Actinobacteria of the freshwater AcI-B cluster which were highly sensitive to (1)O2 but not to H2O2 exposure. Furthermore, group-specific RT-PCR DGGE analysis revealed that particle-attached P. necessarius and Limnohabitans-related phylotypes exhibit higher resistance to (1)O2 exposure compared to free-living populations. These results imply that (1)O2 acts as a factor in niche separation of closely affiliated Polynucleobacter and Limnohabitans-related phylotypes. Consequently, oxidative stress caused by photochemical ROS generation should be regarded as an environmental variable determining abundance, activity, and phylotype composition of environmentally relevant bacterial groups, in particular in illuminated and humic matter rich waters.

So far, numerous genes have been found to associate with various strategies to resist and transform the toxic metalloid arsenic (here, we denote these genes as "arsenic-related genes"). However, our knowledge of the distribution, redundancies and organization of these genes in bacteria is still limited. In this study, we analyzed the 188 Burkholderiales genomes and found that 95% genomes harbored arsenic-related genes, with an average of 6.6 genes per genome. The results indicated: a) compared to a low frequency of distribution for aio (arsenite oxidase) (12 strains), arr (arsenate respiratory reductase) (1 strain) and arsM (arsenite methytransferase)-like genes (4 strains), the ars (arsenic resistance system)-like genes were identified in 174 strains including 1,051 genes; b) 2/3 ars-like genes were clustered as ars operon and displayed a high diversity of gene organizations (68 forms) which may suggest the rapid movement and evolution for ars-like genes in bacterial genomes; c) the arsenite efflux system was dominant with ACR3 form rather than ArsB in Burkholderiales; d) only a few numbers of arsM and arrAB are found indicating neither As III biomethylation nor AsV respiration is the primary mechanism in Burkholderiales members; (e) the aio-like gene is mostly flanked with ars-like genes and phosphate transport system, implying the close functional relatedness between arsenic and phosphorus metabolisms. On average, the number of arsenic-related genes per genome of strains isolated from arsenic-rich environments is more than four times higher than the strains from other environments. Compared with human, plant and animal pathogens, the environmental strains possess a larger average number of arsenic-related genes, which indicates that habitat is likely a key driver for bacterial arsenic resistance.

Background: Laribacter hongkongensis is a Gram-negative, urease-positive bacillus associated with invasive bacteremic infections in liver cirrhosis patients and fish-borne community-acquired gastroenteritis and traveler's diarrhea. Its mechanisms of adaptation to various environmental niches and host defense evasion are largely unknown. During the process of analyzing the L. hongkongensis genome, a complete urease cassette and two adjacent arc gene cassettes were found. We hypothesize that the urease cassette and/or the arc gene cassettes are important for L. hongkongensis to survive in acidic environment and macrophages. In this study, we tested this hypothesis by constructing single, double and triple non-polar deletion mutants of the urease and two arc gene cassettes of L. hongkongensis using the conjugation-mediated gene deletion system and examining their effects in acidic environment in vitro, in macrophages and in a mouse model., Results: HLHK9∆ureA, HLHK9∆ureC, HLHK9∆ureD and HLHK9∆ureE all exhibited no urease activity. HLHK9∆arcA1 and HLHK9∆arcA2 both exhibited arginine deiminase (ADI) activities, but HLHK9∆arcA1/arcA2 double deletion mutant exhibited no ADI activity. At pH 2 and 3, survival of HLHK9∆arcA1/arcA2 and HLHK9∆ureA/arcA1/arcA2 were markedly decreased (p < 0.001) but that of HLHK9∆ureA was slightly decreased (p < 0.05), compared to wild type L. hongkongensis HLHK9. Survival of HLHK9∆ureA/arcA1/arcA2 and HLHK9∆arcA1/arcA2 in macrophages were also markedly decreased (p < 0.001 and p < 0.01 respectively) but that of HLHK9∆ureA was slightly decreased (p < 0.05), compared to HLHK9, although expression of arcA1, arcA2 and ureA genes were all upregulated. Using a mouse model, HLHK9∆ureA exhibited similar survival compared to HLHK9 after passing through the murine stomach, but survival of HLHK9∆arcA1/arcA2 and HLHK9∆ureA/arcA1/arcA2 were markedly reduced (p < 0.01)., Conclusions: In contrast to other important gastrointestinal tract pathogens, ADI pathway is far more important than urease for acid resistance and intracellular survival in L. hongkongensis. The gene duplication of the arc gene cassettes could be a result of their functional importance in L. hongkongensis.

The metagenomic approach was applied to characterize variations of microbial structure and functions in raw (RW) and treated water (TW) in a drinking water treatment plant (DWTP) at Pearl River Delta, China. Microbial structure was significantly influenced by the treatment processes, shifting from Gammaproteobacteria and Betaproteobacteria in RW to Alphaproteobacteria in TW. Further functional analysis indicated the basic metabolic functions of microorganisms in TW did not vary considerably. However, protective functions, i.e. glutathione synthesis genes in 'oxidative stress' and 'detoxification' subsystems, significantly increased, revealing the surviving bacteria may have higher chlorine resistance. Similar results were also found in glutathione metabolism pathway, which identified the major reaction for glutathione synthesis and supported more genes for glutathione metabolism existed in TW. This metagenomic study largely enhanced our knowledge about the influences of treatment processes, especially chlorination, on bacterial community structure and protective functions (e.g. glutathione metabolism) in ecosystems of DWTPs.

Ammonia (NH3)-oxidizing bacteria (AOB) and thaumarchaea (AOA) co-occupy most soils, yet no short-term growth-independent method exists to determine their relative contributions to nitrification in situ. Microbial monooxygenases differ in their vulnerability to inactivation by aliphatic n-alkynes, and we found that NH3 oxidation by the marine thaumarchaeon Nitrosopumilus maritimus was unaffected during a 24-h exposure to ≤ 20 μM concentrations of 1-alkynes C8 and C9. In contrast, NH3 oxidation by two AOB (Nitrosomonas europaea and Nitrosospira multiformis) was quickly and irreversibly inactivated by 1 μM C8 (octyne). Evidence that nitrification carried out by soilborne AOA was also insensitive to octyne was obtained. In incubations (21 or 28 days) of two different whole soils, both acetylene and octyne effectively prevented NH4(+)-stimulated increases in AOB population densities, but octyne did not prevent increases in AOA population densities that were prevented by acetylene. Furthermore, octyne-resistant, NH4(+)-stimulated net nitrification rates of 2 and 7 μg N/g soil/day persisted throughout the incubation of the two soils. Other evidence that octyne-resistant nitrification was due to AOA included (i) a positive correlation of octyne-resistant nitrification in soil slurries of cropped and noncropped soils with allylthiourea-resistant activity (100 μM) and (ii) the finding that the fraction of octyne-resistant nitrification in soil slurries correlated with the fraction of nitrification that recovered from irreversible acetylene inactivation in the presence of bacterial protein synthesis inhibitors and with the octyne-resistant fraction of NH4(+)-saturated net nitrification measured in whole soils. Octyne can be useful in short-term assays to discriminate AOA and AOB contributions to soil nitrification.

To study the influence of the clay minerals montmorillonite (M) and illite (I), the metal oxides ferrihydrite (F) and aluminum hydroxide (A), and charcoal (C) on soil bacterial communities, seven artificial soils with identical texture provided by quartz (Q) were mixed with sterilized manure as organic carbon source before adding a microbial inoculant derived from a Cambisol. Bacterial communities established in artificial soils after 90 days of incubation were compared by DGGE analysis of bacterial and taxon-specific 16S rRNA gene amplicons. The bacterial community structure of charcoal-containing soils highly differed from the other soils at all taxonomic levels studied. Effects of montmorillonite and illite were observed for Bacteria and Betaproteobacteria, but not for Actinobacteria or Alphaproteobacteria. A weak influence of metal oxides on Betaproteobacteria was found. Barcoded pyrosequencing of 16S rRNA gene amplicons done for QM, QI, QIF, and QMC revealed a high bacterial diversity in the artificial soils. The composition of the artificial soils was different from the inoculant, and the structure of the bacterial communities established in QMC soil was most different from the other soils, suggesting that charcoal provided distinct microenvironments and biogeochemical interfaces formed. Several populations with discriminative relative abundance between artificial soils were identified., (© 2013 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Background: In this study a large random collection (n=2188) of facultative oligotrophic bacteria, from 90 water samples gathered in three consecutive years (2007-2009) from three different sampling sites of River Mahananda in Siliguri, West Bengal, India, were investigated for the presence of class 1 integrons and sequences of the amplification products., Methodology/principal Findings: Replica plating method was employed for determining the antibiotic resistance profile of the randomly assorted facultative oligotrophic isolates. Genomic DNA from each isolate was analyzed by PCR for the presence of class 1 integron. Amplicons were cloned and sequenced. Numerical taxonomy and 16S rRNA gene sequence analyses were done to ascertain putative genera of the class 1 integron bearing isolates. Out of 2188 isolates, 1667 (76.19%) were antibiotic-resistant comprising of both single-antibiotic resistance (SAR) and multiple-antibiotic resistant (MAR), and 521 (23.81%) were sensitive to all twelve different antibiotics used in this study. Ninety out of 2188 isolates produced amplicon(s) of varying sizes from 0.15 to 3.45 KB. Chi-square (χ(2)) test revealed that the possession of class 1 integron in sensitive, SAR and MAR is not equally probable at the 1% level of significance. Diverse antibiotic-resistance gene cassettes, aadA1, aadA2, aadA4, aadA5, dfrA1, dfrA5, dfrA7, dfrA12, dfrA16, dfrA17, dfrA28, dfrA30, dfr-IIe, blaIMP-9, aacA4, Ac-6'-Ib, oxa1, oxa10 and arr2 were detected in 64 isolates. The novel cassettes encoding proteins unrelated to any known antibiotic resistance gene function were identified in 26 isolates. Antibiotic-sensitive isolates have a greater propensity to carry gene cassettes unrelated to known antibiotic-resistance genes. The integron-positive isolates under the class Betaproteobacteria comprised of only two genera, Comamonas and Acidovorax of family Comamonadaceae, while isolates under class Gammaproteobacteria fell under the families, Moraxellaceae, Pseudomonadaceae, Aeromonadaceae and Enterobacteriaceae., Conclusions: Oligotrophic bacteria are good sources of novel genes as well as potential reservoirs of antibiotic resistance gene casettes.

Two genes encoding structurally similar Copper P1B -type ATPases can be identified in several genomes. Notwithstanding the high sequence and structural similarities these ATPases held, it has been suggested that they fulfil distinct physiological roles. In deed, we have shown that the Cu(+) -ATPase CtpA is required only for the activity of cuproproteins in the purple bacterium Rubrivivax gelatinosus; herein, we show that CopA is not directly required for cytochrome c oxidase but is vital for copper tolerance. Interestingly, excess copper in the copA(-) mutant resulted in a substantial decrease of the cytochrome c oxidase and the photosystem under microaerobic and anaerobic conditions together with the extrusion of coproporphyrin III. The data indicated that copper targeted the tetrapyrrole biosynthesis pathway at the level of the coproporphyrinogen III oxidase HemN and thereby affects the oxidase and the photosystem. This is the first in vivo demonstration that copper, like oxygen, affects tetrapyrrole biosynthesis presumably at the level of the SAM and [4Fe-4S] containing HemN enzyme. In light of these results and similar findings in Escherichia coli, the potential role of copper ions in the evolution of [4Fe-4S] enzymes and the Cu(+) -ATPases is discussed., (© 2013 Blackwell Publishing Ltd.)

Methylmercury compounds are very toxic for most organisms. Here, we investigated the potential of earthworms to methylate inorganic-Hg. We hypothesized that the anaerobic and nutrient-rich conditions in the digestive tracts of earthworm's promote the methylation of Hg through the action of their gut bacteria. Earthworms were either grown in sterile soils treated with an inorganic (HgCl2) or organic (CH3HgCl) Hg source, or were left untreated. After 30 days of incubation, the total-Hg and methyl-Hg concentrations in the soils, earthworms, and their casts were analyzed. The impact of Hg on the bacterial community compositions in earthworms was also studied. Tissue concentrations of methyl-Hg in earthworms grown in soils treated with inorganic-Hg were about six times higher than in earthworms grown in soils without Hg. Concentrations of methyl-Hg in the soils and earthworm casts remained at significantly lower levels suggesting that Hg was mainly methylated in the earthworms. Bacterial communities in earthworms were mostly affected by methyl-Hg treatment. Terminal-restriction fragments (T-RFs) affiliated to Firmicutes were sensitive to inorganic and methyl-Hg, whereas T-RFs related to Betaproteobacteria were tolerant to the Hg treatments. Sulphate-reducing bacteria were detected in earthworms but not in soils.

Microbial solubilizing of metals in acid environments is successfully used in industrial bioleaching of ores or biomining to extract metals such as copper, gold, uranium and others. This is done mainly by acidophilic and other microorganisms that mobilize metals and generate acid mine drainage or AMD, causing serious environmental problems. However, bioremediation or removal of the toxic metals from contaminated soils can be achieved by using the specific properties of the acidophilic microorganisms interacting with these elements. These bacteria resist high levels of metals by using a few "canonical" systems such as active efflux or trapping of the metal ions by metal chaperones. Nonetheless, gene duplications, the presence of genomic islands, the existence of additional mechanisms such as passive instruments for pH and cation homeostasis in acidophiles and an inorganic polyphosphate-driven metal resistance mechanism have also been proposed. Horizontal gene transfer in environmental microorganisms present in natural ecosystems is considered to be an important mechanism in their adaptive evolution. This process is carried out by different mobile genetic elements, including genomic islands (GI), which increase the adaptability and versatility of the microorganism. This mini-review also describes the possible role of GIs in metal resistance of some environmental microorganisms of importance in biomining and bioremediation of metal polluted environments such as Thiomonas arsenitoxydans, a moderate acidophilic microorganism, Acidithiobacillus caldus and Acidithiobacillus ferrooxidans strains ATCC 23270 and ATCC 53993, all extreme acidophiles able to tolerate exceptionally high levels of heavy metals. Some of these bacteria contain variable numbers of GIs, most of which code for high numbers of genes related to metal resistance. In some cases there is an apparent correlation between the number of metal resistance genes and the metal tolerance of each of these microorganisms. It is expected that a detailed knowledge of the mechanisms that these environmental microorganisms use to adapt to their harsh niche will help to improve biomining and metal bioremediation in industrial processes.

Litter soil cover constitutes an important micro-ecosystem in sustainable viticulture having a key role in nutrient cycling and serving as a habitat of complex microbial communities. Ammonia-oxidizing bacteria (AOB) and archaea (AOA) are known to regulate nitrification in soil while little is known regarding their function and diversity in litter. We investigated the effects of two fungicides, penconazole and cyprodinil, commonly used in vineyards, on the function and diversity of total and active AOB and AOA in a microcosm study. Functional changes measured via potential nitrification and structural changes assessed via denaturating gradient gel electrophoresis (DGGE) at the DNA and RNA levels were contrasted with pesticide dissipation in the litter layer. The latter was inversely correlated with potential nitrification, which was temporarily inhibited at the initial sampling dates (0 to 21 days) when nearly 100 % of the applied pesticide amounts was still present in the litter. Fungicides induced changes in AOB and AOA communities with RNA-DGGE analysis showing a higher sensitivity. AOA were more responsive to pesticide application compared to AOB. Potential nitrification was less sensitive to the fungicides and was restored faster than structural changes, which persisted. These results support the theory of microbial redundancy for nitrification in a stressed litter environment.

The ability of microorganisms to perform dissimilatory (per)chlorate reduction is, for most species, known to be oxygen sensitive. Consequently, bioremediation processes for the removal of oxochlorates will be disturbed if oxygen is present. We measured the expression of chlorite dismutase and chlorate reductase in the presence of different terminal electron acceptors in the chlorate reducer Ideonella dechloratans. Enzyme activity assays and mRNA analyses by real-time quantitative reverse transcription (qRT)-PCR were performed on cell extracts from cells grown aerobically with and without chlorate and on cells grown anaerobically in the presence of chlorate. Our results showed that both chlorite dismutase and chlorate reductase are expressed during aerobic growth. However, transfer to anaerobic conditions with chlorate resulted in significantly enhanced enzyme activities and mRNA levels for both enzymes. Absence of oxygen was necessary for the induction to occur, since chlorate addition under aerobic conditions produced neither increased enzyme activities nor higher relative levels of mRNA. For chlorite dismutase, the observed increase in activity was on the same order of magnitude as the increase in the relative mRNA level, indicating gene regulation at the transcriptional level. However, chlorate reductase showed about 200 times higher enzyme activity in anaerobically induced cells, whereas the increase in mRNA was only about 10-fold, suggesting additional mechanisms influence the enzyme activity.

The microbial composition in a pulp and paper wastewater aerated lagoon system was analysed using fluorescence in situ hybridisation (FISH) to gain further understanding of the effect of substrate composition on microbial diversity for improved management of wastewater treatment systems. Few experiments have been conducted to tease apart the factors influencing the composition and abundance of certain groups within these wastewaters. Specific probes were used to investigate and enumerate the different bacterial groups present at particular stages through the treatment system over an extended period. Community composition and abundance of specific groups differed through the system however temporal stability was retained despite significant variability in the wastewater. Middle stream wastewater samples were enriched to explore the impact of different carbon/nitrogen/phosphorus (C:N:P) ratios on community composition and provide functionality to groups of micro-organisms within the microbial consortia. Nitrogen and phosphorus conditions did not impact community composition of methanol-fed cultures, which exhibited a dominance of Betaproteobacteria (>75%), namely Methylotrophic bacteria. This was confirmed through 16S rRNA gene sequencing and specific FISH probing, reflecting population observations at the beginning of the treatment system. We conclude that the nutrient and carbon combinations used in the enrichments created an interactive effect, altering the community composition and mimicking the main substrate load in the different stages of the treatment system. Finally, pulp and paper wastewater microbial composition was highly variable across the treatment system but was stable within the time sampled, with the enrichments emulating the substrate loads in the full scale system., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Biofilms represent the most common microbial lifestyle, allowing the survival of microbial populations exposed to harsh environmental conditions. Here, we show that the biofilm development of a bacterial species belonging to the Thiomonas genus, frequently found in arsenic polluted sites and playing a key role in arsenic natural remediation, is markedly modified when exposed to subinhibitory doses of this toxic element. Indeed, arsenite [As(III)] exposure led to a considerable impact on biofilm maturation by strongly increasing the extracellular matrix synthesis and by promoting significant cell death and lysis within microcolonies. These events were followed by the development of complex 3D-biofilm structures and subsequently by the dispersal of remobilized cells observed inside the previously formed hollow voids. Our results demonstrate that this biofilm community responds to arsenite stress in a multimodal way, enhancing both survival and dispersal. Addressing this complex bacterial response to As(III) stress, which might be used by other microorganisms under various adverse conditions, may be essential to understand how Thiomonas strains persist in extreme environments.

The antiviral drug Tamiflu has received particular attention because of its recommended use against the influenza A H5N1 and H1N1 viruses. Given its resistance to degradation and its hydrophilicity the active metabolite, Oseltamivir Carboxylate (OC), is expected to enter the aquatic ecosystem from sewage treatment plants. In the present paper the bacterial community of surface water samples, treated with OC (1.5 mg L⁻¹), was characterized by Fluorescence In Situ Hybridization (FISH) in microcosm experiments. The α-, β- and γ-Proteobacteria increased in OC-treated versus non-treated water samples during the incubation period, suggesting these bacterial groups had an active role in OC degradation.

The temporal activity, abundance and diversity of microbial communities were evaluated across a metal-contamination gradient around a Superfund site in Montana. In order to analyze short-term variability, samples were collected from six sites on four occasions over 12 months. Measurements of community activity, diversity and richness, quantified by dehydrogenase activity and through denaturant gradient gel electrophoresis (DGGE), respectively, were higher at contaminated sites adjacent to the smelter, relative to reference sites. 16S rRNA gene copy numbers, measured by quantitative PCR, showed seasonal variability, yet were generally higher within polluted sediments. Jaccard similarity coefficients of DGGE profiles, found sites to cluster based primarily on geographical proximity rather than geochemical similarities. Intra-site clustering of the most polluted sites also suggests a stable metal-tolerant community. Sequences from DGGE-extracted bands were predominantly Beta and Gammaproteobacteria, although the communities at all sites generally maintained a diverse phylogeny changing in composition throughout the sampling period. Spearman's rank correlations analysis found statistically significant relationships between community composition and organic carbon (r-value = 0.786) and metals (r-values As = 0.65; Cu = 0.63; Zn = 0.62). A diverse and abundant community at the most polluted site indicates that historical contamination selects for a metal-resistant microbial community, a finding that must be accounted for when using the microbial community within ecosystem monitoring studies. This study highlights the importance of using multiple time-points to draw conclusions on the affect of metal contamination.

The ability of some microbial species to oxidize monomethylamine via glutamate-mediated pathways was proposed in the 1960s; however, genetic determinants of the pathways have never been described. In the present study we describe a gene cluster essential for operation of the N-methylglutamate pathway in the methylotrophic beta-proteobacterium Methyloversatilis universalis FAM5. Four major polypeptides from protein fractions displaying high activities of N-methylglutamate synthetase, N-methylglutamate dehydrogenase and gamma-glutamylmethylamide synthetase were selected for mass spectrometry-based identification. The activities of enzymes were associated with the presence of peptides identified as ferredoxin-dependent glutamate synthase (GltB2), large subunit of putative heterotetrameric sarcosine oxidase (SoxA) and glutamine synthetase type III (GSIII) respectively. A gene cluster (8.3 kb) harbouring gltB2, soxA and gsIII-like genes was amplified from M. universalis FAM5, sequenced and assembled. Two partial and six complete open reading frames arranged in the order soxBDAG-gsIII-gltB132 were identified and subjected to mutational analysis, functional and metabolic profiling. We demonstrated that gltB-like and sox-like genes play a key role in methylamine utilization and encode N-methylglutamate synthetase and N-methylglutamate dehydrogenase respectively. Metabolic, enzymatic and mutational analyses showed that the gsIII-like gene encodes gamma-glutamylmethylamide synthetase; however, this enzyme is not essential for oxidation of methylamine.

As disinfection strategies could support a shift of some bacterial populations, the biodiversity of drinking water biofilms depending on the disinfectant concentrations was explored. The effect of different chlorine sequences applied for several weeks (0.1-0.4-0.1 mg Cl(2)L(-1) or vice versa) was tested on the abundance of the alpha-, beta- and gamma-proteobacteria populations, used as indicators of changes in bacterial populations within drinking water biofilms. Using dynamic (industrial pilot) and batch (bench scale) conditions, our work demonstrated the ability of the 3 proteobacteria subclasses to re-organize following discontinuous chlorinations. The beta- and gamma-proteobacteria subclasses were favoured by high free residual chlorine concentrations (0.4 mg Cl(2)L(-1)) while alpha-proteobacteria population was sensitive to this oxidant level. The proteobacteria population shifts within the biofilm exposed to discontinuous chlorination were reversible. The resilience of the biofilm proteobacteria populations exposed to oxidant stress questioned the emergence of bacterial population less sensitive to chlorine.

Cupriavidus metallidurans strain CH34 is a beta-Proteobacterium that thrives in low concentrations of heavy metals. The genetic determinants of resistance to heavy metals are located on its two chromosomes, and are particularly abundant in the two megaplasmids, pMOL28 and pMOL30. We explored the involvement of mobile genetic elements in acquiring these and others traits that might be advantageous in this strain using genome comparison of Cupriavidus/Ralstonia strains and related beta-Proteobacteria. At least eleven genomic islands were identified on the main replicon, three on pMOL28 and two on pMOL30. Multiple islands contained genes for heavy metal resistance or other genetic determinants putatively responding to harsh environmental conditions. However, cryptic elements also were noted. New mobile genetic elements (or variations of known ones) were identified through synteny analysis, allowing the detection of mobile genetic elements outside the bias of a selectable marker. Tn4371-like conjugative transposons involved in chemolithotrophy and degradation of aromatic compounds were identified in strain CH34, while similar elements involved in heavy metal resistance were found in Delftia acidovorans SPH-1 and Bordetella petrii DSM12804. We defined new transposons, viz., Tn6048 putatively involved in the response to heavy metals and Tn6050 carrying accessory genes not classically associated with transposons. Syntenic analysis also revealed new transposons carrying metal response genes in Burkholderia xenovorans LB400, and other bacteria. Finally, other putative mobile elements, which were previously unnoticed but apparently common in several bacteria, were also revealed. This was the case for triads of tyrosine-based site-specific recombinases and for an int gene paired with a putative repressor and associated with chromate resistance.

Cells of Methylibium petroleiphilum PM1 were immobilized in gel beads to degrade methyl tert-butyl ether (MTBE). Calcium alginate, agar, polyacrylamide and polyvinvyl alcohol were screened as suitable immobilization matrices, with calcium alginate demonstrating the fastest MTBE-degradation rate. The rate was accelerated by 1.8-fold when the beads had been treated in physiological saline for 24h at 28 degrees C. MTBE degradation in mineral salts medium (MSM) was accompanied by the increase of biomass. The half-life of MTBE-degradation activity for the encapsulated cells stored at 28 degrees C was about 120 h, which was obviously longer than that of free cells (approximately 36 h). Efficient reusability of the beads up to 30 batches was achieved in poor nutrition solution as compared to only 6 batches in MSM. The immobilized cells could be operated in a packed-bed reactor for degradation of 10 mg L(-1) MTBE in groundwater with more than 99% removal efficiency at hydraulic retention time of 20 min. These results suggested that immobilized cells of PM1 in bioreactor might be applicable to a groundwater treatment system for the removal of MTBE.

Chlorhexidine is a common-use antibacterial agent found in a range of personal-care products. We used rotating annular reactors to cultivate river biofilms under the influence of chlorhexidine or its molar equivalent in nutrients. Studies of the degradation of [(14)C]chlorhexidine demonstrated that no mineralization of the compound occurred. During studies with 100 microg liter(-1) chlorhexidine, significant changes were observed in the protozoan and micrometazoan populations, the algal and cyanobacterial biomass, the bacterial biomass, and carbon utilization. Denaturing gradient gel electrophoresis (DGGE) in combination with statistical analyses showed that the communities developing under control and 100 microg liter(-1) chlorhexidine were significantly different. At 10 microg liter(-1) chlorhexidine, there was significantly increased algal and cyanobacterial biomass while the bacterial biomass was not significantly affected (P < 0.05). No significant effects on protozoan or metazoan grazing were detected at the 10-microg liter(-1) chlorhexidine level. Fluorescent in situ hybridization indicated a significant reduction in the abundance of betaproteobacteria and gammaproteobacteria (P < 0.05). Archaeal cell counts were significantly reduced by both chlorhexidine and nutrient treatments. DGGE and statistical analyses indicated that 10 microg liter(-1) chlorhexidine and molar equivalent nutrient treatments were significantly different from control communities. In contrast to community level observations, toxicological testing with a panel of cyanobacteria, algae, and protozoa indicated no detectable effects at 10, 50, and 100 microg liter(-1) chlorhexidine. Thus, community level assessment indicated a risk of low levels of chlorhexidine in aquatic habitats while conventional approaches did not.

Pharmaceuticals are abundant at low concentrations (i.e. ng L(-1); microg L(-1)) in natural aquatic systems. However, very little is known about their effects on microorganisms. This study investigated the influence of the pharmaceuticals phenazone, amoxicillin and erythromycin, at low, non-toxic concentrations (i.e. 0.5-50 microg L(-1)), on the initial adhesion of bacteria to uncoated and iron-coated polystyrene. The influence of the pharmaceuticals on the initial adhesion of bacterial pure cultures (Escherichia coli, Aquabacterium commune, Bacillus subtilis) isolated from natural aquatic systems, was investigated with a plate assay. Initial adhesion of the pure cultures depended on the selected pharmaceutical, its concentration, the bacterial strain and the adhesion surface. Different combinations of these parameters resulted in inhibition, enhancement or had no effect on initial adhesion. In addition, a continuous flow system was used to investigate the influence of the pharmaceuticals on the initial adhesion of a drinking water microbial community. The drinking water community showed decreased adhesion in the presence of the pharmaceuticals regardless of adhesion surface. The results show that pharmaceuticals at environmentally relevant concentrations can influence the initial adhesion of bacteria. Thus, pharmaceutical compounds that are introduced to natural aquatic systems are able to exert subtle effects on bacteria.

The denitrifying betaproteobacterium "Aromatoleum aromaticum" strain EbN1 degrades several aromatic compounds, including ethylbenzene, toluene, p-cresol, and phenol, under anoxic conditions. The hydrophobicity of these aromatic solvents determines their toxic properties. Here, we investigated the response of strain EbN1 to aromatic substrates at semi-inhibitory (about 50% growth inhibition) concentrations under two different conditions: first, during anaerobic growth with ethylbenzene (0.32 mM) or toluene (0.74 mM); and second, when anaerobic succinate-utilizing cultures were shocked with ethylbenzene (0.5 mM), toluene (1.2 mM), p-cresol (3.0 mM), and phenol (6.5 mM) as single stressors or as a mixture (total solvent concentration, 2.7 mM). Under all tested conditions impaired growth was paralleled by decelerated nitrate-nitrite consumption. Additionally, alkylbenzene-utilizing cultures accumulated poly(3-hydroxybutyrate) (PHB) up to 10% of the cell dry weight. These physiological responses were also reflected on the proteomic level (as determined by two-dimensional difference gel electrophoresis), e.g., up-regulation of PHB granule-associated phasins, cytochrome cd(1) nitrite reductase of denitrification, and several proteins involved in oxidative (e.g., SodB) and general (e.g., ClpB) stress responses.