Genome and proteome analysis of Pseudomonas chloritidismutans†AW-1T that grows on n-decane with chlorate or oxygen as electron acceptor.

Growth of Pseudomonas chloritidismutans AW-1T on C7 to C12 n-alkanes with oxygen or chlorate as electron acceptor was studied by genome and proteome analysis. Whole genome shotgun sequencing resulted in a 5 Mbp assembled sequence with a G + C content of 62.5%. The automatic annotation identified 4767 protein-encoding genes and a putative function could be assigned to almost 80% of the predicted proteins. The distinct phylogenetic position of P. chloritidismutans AW-1T within the Pseudomonas stutzeri cluster became clear by comparison of average nucleotide identity values of sequenced genomes. Analysis of the proteome of P. chloritidismutans AW-1T showed the versatility of this bacterium to adapt to aerobic and anaerobic growth conditions with acetate or n-decane as substrates. All enzymes involved in the alkane oxidation pathway were identified. An alkane monooxygenase was detected in n-decane-grown cells, but not in acetate-grown cells. The enzyme was found when grown in the presence of oxygen or chlorate, indicating that under both conditions an oxygenase-mediated pathway is employed for alkane degradation. Proteomic and biochemical data also showed that both chlorate reductase and chlorite dismutase are constitutively present, but most abundant under chlorate-reducing conditions.

Genomic, proteomic, and biochemical analysis of the organohalide respiratory pathway in Desulfitobacterium dehalogenans.

Desulfitobacterium dehalogenans is able to grow by organohalide respiration using 3-chloro-4-hydroxyphenyl acetate (Cl-OHPA) as an electron acceptor. We used a combination of genome sequencing, biochemical analysis of redox active components, and shotgun proteomics to study elements of the organohalide respiratory electron transport chain. The genome of Desulfitobacterium dehalogenans JW/IU-DC1(T) consists of a single circular chromosome of 4,321,753 bp with a GC content of 44.97%. The genome contains 4,252 genes, including six rRNA operons and six predicted reductive dehalogenases. One of the reductive dehalogenases, CprA, is encoded by a well-characterized cprTKZEBACD gene cluster. Redox active components were identified in concentrated suspensions of cells grown on formate and Cl-OHPA or formate and fumarate, using electron paramagnetic resonance (EPR), visible spectroscopy, and high-performance liquid chromatography (HPLC) analysis of membrane extracts. In cell suspensions, these components were reduced upon addition of formate and oxidized after addition of Cl-OHPA, indicating involvement in organohalide respiration. Genome analysis revealed genes that likely encode the identified components of the electron transport chain from formate to fumarate or Cl-OHPA. Data presented here suggest that the first part of the electron transport chain from formate to fumarate or Cl-OHPA is shared. Electrons are channeled from an outward-facing formate dehydrogenase via menaquinones to a fumarate reductase located at the cytoplasmic face of the membrane. When Cl-OHPA is the terminal electron acceptor, electrons are transferred from menaquinones to outward-facing CprA, via an as-yet-unidentified membrane complex, and potentially an extracellular flavoprotein acting as an electron shuttle between the quinol dehydrogenase membrane complex and CprA.

Microbial removal of the pharmaceutical compounds Ibuprofen and diclofenac from wastewater.

Studies on the occurrence of pharmaceuticals show that the widely used pharmaceuticals ibuprofen and diclofenac are present in relevant concentrations in the environment. A pilot plant treating hospital wastewater with relevant concentrations of these pharmaceuticals was evaluated for its performance to reduce the concentration of the pharmaceuticals. Ibuprofen was completely removed, whereas diclofenac yielded a residual concentration, showing the necessity of posttreatment to remove diclofenac, for example, activated carbon. Successively, detailed laboratory experiments with activated sludge from the same wastewater treatment plant showed bioremediation potential in the treatment plant. The biological degradation pathway was studied and showed a mineralisation of ibuprofen and degradation of diclofenac. The present microbes were further studied in laboratory experiments, and DGGE analyses showed the enrichment and isolation of highly purified cultures that degraded either ibuprofen or diclofenac. This research illuminates the importance of the involved bacteria for the effectiveness of the removal of pharmaceuticals in a wastewater treatment plant. A complete removal of pharmaceuticals from wastewater will stimulate water reuse, addressing the worldwide increasing demand for clean and safe fresh water.

Genome analysis and physiological comparison of Alicycliphilus denitrificans strains BC and K601(T.).

The genomes of the Betaproteobacteria Alicycliphilus denitrificans strains BC and K601(T) have been sequenced to get insight into the physiology of the two strains. Strain BC degrades benzene with chlorate as electron acceptor. The cyclohexanol-degrading denitrifying strain K601(T) is not able to use chlorate as electron acceptor, while strain BC cannot degrade cyclohexanol. The 16S rRNA sequences of strains BC and K601(T) are identical and the fatty acid methyl ester patterns of the strains are similar. Basic Local Alignment Search Tool (BLAST) analysis of predicted open reading frames of both strains showed most hits with Acidovorax sp. JS42, a bacterium that degrades nitro-aromatics. The genomes include strain-specific plasmids (pAlide201 in strain K601(T) and pAlide01 and pAlide02 in strain BC). Key genes of chlorate reduction in strain BC were located on a 120 kb megaplasmid (pAlide01), which was absent in strain K601(T). Genes involved in cyclohexanol degradation were only found in strain K601(T). Benzene and toluene are degraded via oxygenase-mediated pathways in both strains. Genes involved in the meta-cleavage pathway of catechol are present in the genomes of both strains. Strain BC also contains all genes of the ortho-cleavage pathway. The large number of mono- and dioxygenase genes in the genomes suggests that the two strains have a broader substrate range than known thus far.

Role of "Dehalococcoides" spp. in the anaerobic transformation of hexachlorobenzene in European rivers.

The diffuse pollution by chlorinated organic compounds in river basins is a concern, due to their potential adverse effects on human health and the environment. Organohalides, like hexachlorobenzene (HCB), are recalcitrant to aerobic microbial degradation, and "Dehalococcoides" spp. are the only known microorganisms capable of anaerobic transformation of these compounds coupled to their growth. In this study, sediments from four European rivers were studied in order to determine their HCB dechlorination capacities and the role of Dehalococcoides spp. in this process. Only a weak correlation was observed between Dehalococcoides species abundance and HCB transformation rates from different locations. In one of these locations, in the Ebro River sediment, HCB dechlorination could be linked to Dehalococcoides species growth and activity by 16S rRNA-based molecular methods. Furthermore, HCB dechlorination activity in this sediment was found over the full range of ambient temperatures that this sediment can be exposed to during different seasons throughout the year. The sediment contained several reductive dehalogenase (rdh) genes, and analysis of their transcription revealed the dominance of cbrA, previously shown to encode a trichlorobenzene reductive dehalogenase. This study investigated the role of Dehalococcoides spp. in HCB dechlorination in river sediments and evaluated if the current knowledge of rdh genes could be used to assess HCB bioremediation potential.

Nitrate and (per)chlorate reduction pathways in (per)chlorate-reducing bacteria.

The reduction of (per)chlorate and nitrate in (per)chlorate-reducing bacteria shows similarities and differences. (Per)chlorate reductase and nitrate reductase both belong to the type II DMSO family of enzymes and have a common bis(molybdopterin guanine dinucleotide)molybdenum cofactor. There are two types of dissimilatory nitrate reductases. With respect to their localization, (per)chlorate reductase is more similar to the dissimilatory periplasmic nitrate reductase. However, the periplasmic, unlike the membrane-bound, respiratory nitrate reductase, is not able to use chlorate. Structurally, (per)chlorate reductase is more similar to respiratory nitrate reductase, since these reductases have analogous subunits encoded by analogous genes. Both periplasmic (per)chlorate reductase and membrane-bound nitrate reductase activities are induced under anoxic conditions in the presence of (per)chlorate and nitrate respectively. During microbial (per)chlorate reduction, molecular oxygen is generated. This is not the case for nitrate reduction, although an atypical reaction in nitrite reduction linked to oxygen formation has been described recently. Microbial oxygen production during reduction of oxyanions may enhance biodegradation of pollutants under anoxic conditions.

Concurrent hexachlorobenzene and chloroethene transformation by endogenous dechlorinating microorganisms in the Ebro River sediment.

The ability of Dehalococcoides spp. to reduce chlorinated compounds offers a great potential for bioremediation and/or bioaugmentation of contaminated environments. So far, however, our knowledge of the activity of Dehalococcoides spp. in situ is limited to only a few subsurface environments. The aim of this study was to broaden this knowledge to other environments, and we investigated the role of Dehalococcoides spp. in the transformation of chlorinated benzenes and chlorinated ethenes in the Ebro River (Spain) sediments. Lab-scale batch microcosms were used to follow the growth and abundance of Dehalococcoides spp. during the transformation of selected chlorinated compounds. We applied biomolecular tools targeting the 16S rRNA, the 16S rRNA gene and several functional genes involved in dechlorination in combination with chemical measurements. The growth of Dehalococcoides spp. and the differential expression of several reductive dehalogenase genes during the dechlorination process could be demonstrated. Furthermore, 16S rRNA gene-based clone libraries of dechlorinating river sediment showed a complex community structure and indicated the involvement of several additional bacterial genera in the transformation process, underlining the remarkable potential of this rivers' sediment to transform different halo-organic pollutants.

Chemical ecology of interactions between human skin microbiota and mosquitoes.

Microbiota on the human skin plays a major role in body odour production. The human microbial and chemical signature displays a qualitative and quantitative correlation. Genes may influence the chemical signature by shaping the composition of the microbiota. Recent studies on human skin microbiota, using 16S rRNA gene sequencing, found a high inter- and intrapersonal variation in bacterial species on the human skin, which is relatively stable over time. Human body odours mediate the attraction of mosquitoes to their blood hosts. Odours produced by skin microbiota are attractive to mosquitoes as shown by in vitro studies, and variation in bacterial species on the human skin may explain the variation in mosquito attraction between humans. Detailed knowledge of the ecology and genetics of human skin microbiota is needed in order to unravel the evolutionary mechanisms that underlie the interactions between mosquitoes and their hosts.

Differential attraction of malaria mosquitoes to volatile blends produced by human skin bacteria.

The malaria mosquito Anopheles gambiae sensu stricto is mainly guided by human odour components to find its blood host. Skin bacteria play an important role in the production of human body odour and when grown in vitro, skin bacteria produce volatiles that are attractive to A. gambiae. The role of single skin bacterial species in the production of volatiles that mediate the host-seeking behaviour of mosquitoes has remained largely unknown and is the subject of the present study. Headspace samples were taken to identify volatiles that mediate this behaviour. These volatiles could be used as mosquito attractants or repellents. Five commonly occurring species of skin bacteria were tested in an olfactometer for the production of volatiles that attract A. gambiae. Odour blends produced by some bacterial species were more attractive than blends produced by other species. In contrast to odours from the other bacterial species tested, odours produced by Pseudomonas aeruginosa were not attractive to A. gambiae. Headspace analysis of bacterial volatiles in combination with behavioural assays led to the identification of six compounds that elicited a behavioural effect in A. gambiae. Our results provide, to our knowledge, the first evidence for a role of selected bacterial species, common on the human skin, in determining the attractiveness of humans to malaria mosquitoes. This information will be used in the further development of a blend of semiochemicals for the manipulation of mosquito behaviour.

Cultured skin microbiota attracts malaria mosquitoes.

BACKGROUND: Host-seeking of the African malaria mosquito, Anopheles gambiae sensu stricto, is guided by human odours. The precise nature of the odours, and the composition of attractive blends of volatiles, remains largely unknown. Skin microbiota plays an important role in the production of human body odours. It is hypothesized that host attractiveness and selection of An. gambiae is affected by the species composition, density, and metabolic activity of the skin microbiota. A study is presented in which the production and constituency of volatile organic compounds (VOCs) by human skin microbiota is examined and the behavioural responses of An. gambiae to VOCs from skin microbiota are investigated. METHODS: Blood agar plates incubated with skin microbiota from human feet or with a reference strain of Staphylococcus epidermidis were tested for their attractiveness to An. gambiae in olfactometer bioassays and indoor trapping experiments. Entrained air collected from blood agar plates incubated with natural skin microbiota or with S. epidermidis were analysed using GC-MS. A synthetic blend of the compounds identified was tested for its attractiveness to An. gambiae. Behavioural data were analysed by a chi(2)-test and GLM. GC-MS results were analysed by fitting an exponential regression line to test the effect of the concentration of bacteria. RESULTS: More An. gambiae were caught with blood agar plates incubated with skin bacteria than with sterile blood agar plates, with a significant effect of incubation time and dilution of the skin microbiota. When bacteria from the feet of four other volunteers were tested, similar effects were found. Fourteen putative attractants were found in the headspace of the skin bacteria. A synthetic blend of 10 of these was attractive to An. gambiae. CONCLUSIONS: The discovery that volatiles produced by human skin microorganisms in vitro mediate An. gambiae host-seeking behaviour creates new opportunities for the development of odour-baited trapping systems. Additionally, identification of bacterial volatiles provides a new method to develop synthetic blends, attractive to An. gambiae and possibly other anthropophilic disease vectors.

Growth of Pseudomonas chloritidismutans AW-1(T) on n-alkanes with chlorate as electron acceptor.

Microbial (per)chlorate reduction is a unique process in which molecular oxygen is formed during the dismutation of chlorite. The oxygen thus formed may be used to degrade hydrocarbons by means of oxygenases under seemingly anoxic conditions. Up to now, no bacterium has been described that grows on aliphatic hydrocarbons with chlorate. Here, we report that Pseudomonas chloritidismutans AW-1(T) grows on n-alkanes (ranging from C7 until C12) with chlorate as electron acceptor. Strain AW-1(T) also grows on the intermediates of the presumed n-alkane degradation pathway. The specific growth rates on n-decane and chlorate and n-decane and oxygen were 0.5 +/- 0.1 and 0.4 +/- 0.02 day(-1), respectively. The key enzymes chlorate reductase and chlorite dismutase were assayed and found to be present. The oxygen-dependent alkane oxidation was demonstrated in whole-cell suspensions. The strain degrades n-alkanes with oxygen and chlorate but not with nitrate, thus suggesting that the strain employs oxygenase-dependent pathways for the breakdown of n-alkanes.

Tracking functional guilds: "Dehalococcoides" spp. in European river basins contaminated with hexachlorobenzene.

Hexachlorobenzene (HCB) has been widely used in chemical manufacturing processes and as a pesticide. Due to its resistance to biological degradation, HCB has mainly accumulated in freshwater bodies and agricultural soils. "Dehalococcoides" spp., anaerobic dechlorinating bacteria that are capable of degrading HCB, were previously isolated from river sediments. Yet there is limited knowledge about the abundance, diversity, and activity of this genus in the environment. This study focused on the molecular analysis of the composition and abundance of active Dehalococcoides spp. in HCB-contaminated European river basins. 16S rRNA-based real-time quantitative PCR and denaturing gradient gel electrophoresis in combination with multivariate statistics were applied. Moreover, a functional gene array was used to determine reductive dehalogenase (rdh) gene diversity. Spatial and temporal fluctuations were observed not only in the abundance of Dehalococcoides spp. but also in the composition of the populations and rdh gene diversity. Multivariate statistics revealed that Dehalococcoides sp. abundance is primarily affected by spatial differences, whereas species composition is under the influence of several environmental parameters, such as seasonal changes, total organic carbon and/or nitrogen content, and HCB contamination. This study provides new insight into the natural occurrence and dynamics of active Dehalococcoides spp. in HCB-contaminated river basins.

Exocellular electron transfer in anaerobic microbial communities.

Exocellular electron transfer plays an important role in anaerobic microbial communities that degrade organic matter. Interspecies hydrogen transfer between microorganisms is the driving force for complete biodegradation in methanogenic environments. Many organic compounds are degraded by obligatory syntrophic consortia of proton-reducing acetogenic bacteria and hydrogen-consuming methanogenic archaea. Anaerobic microorganisms that use insoluble electron acceptors for growth, such as iron- and manganese-oxide as well as inert graphite electrodes in microbial fuel cells, also transfer electrons exocellularly. Soluble compounds, like humic substances, quinones, phenazines and riboflavin, can function as exocellular electron mediators enhancing this type of anaerobic respiration. However, direct electron transfer by cell-cell contact is important as well. This review addresses the mechanisms of exocellular electron transfer in anaerobic microbial communities. There are fundamental differences but also similarities between electron transfer to another microorganism or to an insoluble electron acceptor. The physical separation of the electron donor and electron acceptor metabolism allows energy conservation in compounds as methane and hydrogen or as electricity. Furthermore, this separation is essential in the donation or acceptance of electrons in some environmental technological processes, e.g. soil remediation, wastewater purification and corrosion.

Reductive dechlorination of beta-hexachlorocyclohexane (beta-HCH) by a Dehalobacter species in coculture with a Sedimentibacter sp.

An anaerobic coculture was enriched from a hexachlorocyclohexane (HCH) polluted soil. The coculture reductively dechlorinates the beta-HCH isomer to benzene and chlorobenzene in a ratio of 0.5-2 depending on the amount of beta-HCH degraded. The culture grows with H(2) as electron donor and beta-HCH as electron acceptor, indicating that dechlorination is a respiratory process. Phylogenetic analysis indicated that the coculture consists of two bacteria that are both related to gram-positive bacteria with a low G + C content of the DNA. One bacterium was identified as a Dehalobacter sp. This bacterium is responsible for the dechlorination. The other bacterium was isolated and characterized as being a Sedimentibacter sp. This strain is not able to dechlorinate beta-HCH. The Dehalobacter sp. requires the presence of Sedimentibacter for growth and dechlorination, but the function of the latter bacterium is not clear. This is the first report on the metabolic dechlorination of beta-HCH by a defined anaerobic bacterial culture.

Reductive dechlorination of hexachlorocyclohexane (HCH) isomers in soil under anaerobic conditions.

The biological anaerobic reductive dechlorination of beta-hexachlorocyclohexane under methanogenic conditions was tested in a number of contaminated soil samples from two locations in the Netherlands. Soils from a heavily polluted location showed rapid dechlorination of beta-hexachlorocyclohexane to benzene and chlorobenzene with lactate as electron donor. Soils from an adjacent slightly polluted location did not show substantial dechlorination of beta-hexachlorocyclohexane within 4 months. A heavily polluted sample was selected to optimise the dechlorination. All tested hexachlorocyclohexane isomers (alpha-, beta-, gamma-, and delta-), either added separately or simultaneously, were dechlorinated in this soil sample. The most rapid dechlorination was observed at a temperature of 30 degrees C. Dechlorination of beta-hexachlorocyclohexane was observed with acetate, propionate, lactate, methanol, H2, yeast extract and landfill leachate as electron donors. In a soil percolation column, packed with a selected heavily polluted soil sample, the presence of 10 mM sulphate in the influent led to simultaneous dechlorination of beta-hexachlorocyclohexane and sulphate reduction. When the column was fed with 10 mM nitrate instead of sulphate, dechlorination ceased immediately. After omitting nitrate from the influent, dechlorination activity recovered in about 1 month. Also in a separate column, the addition of nitrate from the start of the experiment did not result in dechlorination of beta-HCH. The significance of these experiments for in situ bioremediation of polluted soils is discussed.

Degradation pathway of 2-chloroethanol in Pseudomonas stutzeri strain JJ under denitrifying conditions.

The pathway of 2-chloroethanol degradation in the denitrifying Pseudomonas stutzeri strain JJ was investigated. In cell-free extracts, activities of a phenazine methosulfate (PMS)-dependent chloroethanol dehydrogenase, an NAD-dependent chloroacetaldehyde dehydrogenase, and a chloroacetate dehalogenase were detected. This suggested that the 2-chloroethanol degradation pathway in this denitrifying strain is the same as found in aerobic bacteria that degrade chloroethanol. Activity towards primary alcohols, secondary alcohols, diols, and other chlorinated alcohols could be measured in cell-free extracts with chloroethanol dehydrogenase (CE-DH) activity. PMS and phenazine ethosulfate (PES) were used as primary electron acceptors, but not NAD, NADP or ferricyanide. Cells of strain JJ cultured in a continuous culture under nitrate limitation exhibited chloroethanol dehydrogenase activity that was a 12 times higher than in cells grown in batch culture. However, under chloroethanol-limiting conditions, CE-DH activity was in the same range as in batch culture. Cells grown on ethanol did not exhibit CE-DH activity. Instead, NAD-dependent ethanol dehydrogenase (E-DH) activity and PMS-dependent E-DH activity were detected.

Hydrogen threshold concentrations in pure cultures of halorespiring bacteria and at a site polluted with chlorinated ethenes.

Halorespiring microorganisms are not only able to oxidize organic electron donors such as formate, acetate, pyruvate and lactate, but also H(2). Because these microorganisms have a high affinity for H(2), this may be the most important electron donor for halorespiration in the environment. We have studied the role of H(2)-threshold concentrations in pure halorespiring cultures and compared them with mixed cultures and field data. We have found H(2)-threshold values between 0.05 and 0.08 nM for Sulfurospirillum halorespirans, S. multivorans and Dehalobacter restrictus under PCE-reducing and nitrate-reducing conditions. The reduction of PCE and TCE can proceed at H(2) concentrations of below 1 nM at a polluted site. However, for the reduction of lower chlorinated ethenes a higher H(2) concentration is required. This indicates that the measured H(2) concentration in situ can be an indicator of the extent of anaerobic reductive dechlorination.

Anaerobic reduction and oxidation of quinone moieties and the reduction of oxidized metals by halorespiring and related organisms.

Halorespiring microorganisms have been detected in soils that were not polluted with chlorinated compounds. In this study, we describe alternative electron acceptor utilization by some halorespiring bacteria and phylogenetically related bacteria. It appears that oxidized metals like selenate, arsenate and manganese are rather common electron acceptors for halorespiring species of Desulfitobacterium and Sulfurospirillum and related bacteria. All tested microorganisms are able to reduce anthraquinone-2,6-disulfonate (AQDS) and four tested organisms (Desulfitobacterium hafniense DP7, Sulfurospirillum barnesii, Sulfurospirillum deleyianum and Sulfurospirillum arsenophilum) are able to oxidize reduced anthrahydroquinone-2,6,-disulfonate (AH(2)QDS) as well. The characteristic to reduce oxidized metals, and to reduce and oxidize quinone moieties coupled to energy conservation is a likely explanation for the presence of halorespiring microorganisms in unpolluted soils.

Description of Sulfurospirillum halorespirans sp. nov., an anaerobic, tetrachloroethene-respiring bacterium, and transfer of Dehalospirillum multivorans to the genus Sulfurospirillum as Sulfurospirillum multivorans comb. nov.

An anaerobic, halorespiring bacterium (strain PCE-M2(T) = DSM 13726(T) = ATCC BAA-583(T)) able to reduce tetrachloroethene to cis-dichloroethene was isolated from an anaerobic soil polluted with chlorinated aliphatic compounds. The isolate is assigned to the genus Sulfurospirillum as a novel species, Sulfurospirillum halorespirans sp. nov. Furthermore, on the basis of all available data, a related organism, Dehalospirillum multivorans DSM 12446(T), is reclassified to the genus Sulfurospirillum as Sulfurospirillum multivorans comb. nov.