Changes of flavin-containing monooxygenases and trimethylamine-N-oxide may be involved in the promotion of non-alcoholic fatty liver disease by intestinal microbiota metabolite trimethylamine.

Evidence shows that trimethylamine (TMA)/trimethylamine-N-oxide (TMAO) is closely related to non-alcoholic fatty liver disease (NAFLD). The conversion of TMA to TMAO is mainly catalyzed by flavin-containing monooxygenases 3 (FMO3) and FMO1. In this study, we explored the role of TMA in the process of NAFLD. The human NAFLD liver puncture data set GSE89632 and rat TMAO gene chip GSE135856 was downloaded for gene differential expression analysis. Besides, oleic acid (OA) combined with palmitate were used to establish high-fat cell model. TMA, TMAO and FMO1-siRNA were used to stimulate L02 cells. Contents of free fatty acid (FFA), triglyceride (TG), TMAO, FMO1 and unfolded protein response (UPR) related proteins GRP78, XBP1, Derlin-1 were detected. Our results showed that FMO1 and PEG10 were important in the progression of NAFLD. Immunohistochemistry showed that FMO1 in NAFLD liver was increased. In addition, the contents of FFA, TG, FMO1 expression, and TMAO were significantly increased after OA + palmitate and TMA stimulation. However, after silencing FMO1 with siRNA, the expressions of these molecules were decreased. Besides, the protein levels of GRP78, XBP1, Derlin-1 were increased after TMAO treatment (all P < 0.05). In Conclusion, high fat and TMA could induce the expression of FMO1 and its metabolite TMAO. When FMO1 is silenced, the effects of high fat and TMA on TMAO are blocked. And the role of TMAO in NAFLD may be through the activation of UPR.

Constitutively solvent-tolerant Pseudomonas taiwanensis VLB120∆ C∆ ttgV supports particularly high-styrene epoxidation activities when grown under glucose excess conditions.

Solvent-tolerant bacteria represent an interesting option to deal with the substrate and product toxicity in bioprocesses. Recently, constitutive solvent tolerance was achieved for Pseudomonas taiwanensis VLB120 via knockout of the regulator TtgV, making tedious adaptation unnecessary. Remarkably, ttgV knockout increased styrene epoxidation activities of P. taiwanensis VLB120Δ C. With the aim to characterize and exploit the biocatalytic potential of P. taiwanensis VLB120Δ C and VLB120Δ CΔ ttgV, we investigated and correlated growth physiology, native styrene monooxygenase (StyAB) gene expression, whole-cell bioconversion kinetics, and epoxidation performance. Substrate inhibition kinetics was identified but was attenuated in two-liquid phase bioreactor setups. StyA fusion to the enhanced green fluorescent protein enabled precise enzyme level monitoring without affecting epoxidation activity. Glucose limitation compromised styAB expression and specific activities (30-40 U/g CDW for both strains), whereas unlimited batch cultivation enabled specific activities up to 180 U/g CDW for VLB120Δ CΔ ttgV strains, which is unrivaled for bioreactor-based whole-cell oxygenase biocatalysis. These extraordinarily high specific activities of constitutively solvent-tolerant P. taiwanensis VLB120∆ C∆ ttgV could be attributed to its high metabolic capacity, which also enabled high expression levels. This, together with the high product yields on glucose and biomass obtained qualifies the VLB120∆ ttgV strain as a highly attractive tool for the development of ecoefficient oxyfunctionalization processes and redox biocatalysis in general.

Functional expression and purification of CYP93C20, a plant membrane-associated cytochrome P450 from Medicago truncatula.

Plants possess very large numbers of biosynthetic cytochrome P450 enzymes. In spite of the importance of these enzymes for the synthesis of bioactive plant secondary metabolites, only two plant P450 structures has been obtained to date. Isoflavone synthase (IFS) is a membrane-associated cytochrome P450 enzyme catalyzing the entry-point reaction into isoflavonoid biosynthesis. IFS from the model legume Medicago truncatula (CYP93C20) was engineered by deleting the membrane-spanning domain and inserting a hydrophilic polypeptide in the N-terminus and a four histidine tag at the C-terminus. The truncated form exhibited dramatically enhanced expression and solubility. The engineered enzyme was expressed in Escherichia coli XL1-blue cells and was purified by Ni2+-NTA affinity chromatograph and size-exclusion chromatograph. The purified enzyme was characterized by enzyme assay, reduced carbon monoxide difference spectroscopy and peptide mass fingerprinting. The engineered soluble enzyme exhibited the same activity as the full length membrane-associated enzyme expressed in yeast. These studies suggest an approach for engineering plant membrane-associated P450s with enhanced expression and solubility for mechanistic and structural studies.

SFRP2/DPP4 and FMO1/LSP1 Define Major Fibroblast Populations in Human Skin.

Fibroblasts produce matrix, regulate inflammation, mediate reparative processes, and serve as pluripotent mesenchymal cells. Analyzing digested normal human skin by single-cell RNA sequencing, we explored different fibroblast populations. T-distributed stochastic neighbor embedding and clustering of single-cell RNA sequencing data from six biopsy samples showed two major fibroblast populations, defined by distinct genes, including SFRP2 and FMO1, expressed exclusively by these two major fibroblast populations. Further subpopulations were defined within each of the SFRP2 and FMO1 populations and five minor fibroblast populations, each expressing discrete genes: CRABP1, COL11A1, FMO2, PRG4, or C2ORF40. Immunofluorescent staining confirmed that SFRP2 and FMO1 define cell types of dramatically different morphology. SFRP2+ fibroblasts were small, elongated, and distributed between collagen bundles. FMO1+ fibroblasts were larger and distributed in both interstitial and perivascular locations. Differential gene expression by SFRP2+, FMO1+, and COL11A1+ fibroblasts suggests roles in matrix deposition, inflammatory cell retention, and connective tissue cell differentiation, respectively.

Copper and cerium-regulated gene expression in Methylosinus trichosporium OB3b.

In aerobic methanotrophs, copper and cerium control the expression and activity of different forms of methane monooxygenase and methanol dehydrogenase, respectively. To exploit methanotrophy for the valorization of methane, it is crucial to determine if these metals exert more global control on gene expression in methanotrophs. Using RNA-Seq analysis we compared the transcriptome of Methylosinus trichosporium OB3b grown in the presence of varying amounts of copper and cerium. When copper was added in the absence of cerium, expression of genes encoding for both soluble and particulate methane monooxygenases varied as expected. Genes encoding for copper uptake, storage, and efflux also increased, indicating that methanotrophs must carefully control copper homeostasis. When cerium was added in the absence of copper, expression of genes encoding for alternative methanol dehydrogenases varied as expected, but few other genes were found to have differential expression. When cerium concentrations were varied in the presence of copper, few genes were found to be either up- or downregulated, indicating that copper over rules any regulation by cerium. When copper was increased in the presence of cerium, however, many genes were upregulated, most notably multiple steps of the central methane oxidation pathway, the serine cycle, and the ethylmalonyl-CoA pathway. Many genes were also downregulated, including those encoding for nitrogenase and hydrogenase. Collectively, these data suggest that copper plays a larger role in regulating gene expression in methanotrophs, but that significant changes occur when both copper and cerium are present.

Transaldolase inhibition impairs mitochondrial respiration and induces a starvation-like longevity response in Caenorhabditis elegans.

Mitochondrial dysfunction can increase oxidative stress and extend lifespan in Caenorhabditis elegans. Homeostatic mechanisms exist to cope with disruptions to mitochondrial function that promote cellular health and organismal longevity. Previously, we determined that decreased expression of the cytosolic pentose phosphate pathway (PPP) enzyme transaldolase activates the mitochondrial unfolded protein response (UPRmt) and extends lifespan. Here we report that transaldolase (tald-1) deficiency impairs mitochondrial function in vivo, as evidenced by altered mitochondrial morphology, decreased respiration, and increased cellular H2O2 levels. Lifespan extension from knockdown of tald-1 is associated with an oxidative stress response involving p38 and c-Jun N-terminal kinase (JNK) MAPKs and a starvation-like response regulated by the transcription factor EB (TFEB) homolog HLH-30. The latter response promotes autophagy and increases expression of the flavin-containing monooxygenase 2 (fmo-2). We conclude that cytosolic redox established through the PPP is a key regulator of mitochondrial function and defines a new mechanism for mitochondrial regulation of longevity.

Different cucumber CsYUC genes regulate response to abiotic stresses and flower development.

The phytohormone auxin is essential for plant growth and development, and YUCCA (YUC) proteins catalyze a rate-limiting step for endogenous auxin biosynthesis. Despite YUC family genes have been isolated from several species, systematic expression analyses of YUCs in response to abiotic stress are lacking, and little is known about the function of YUC homologs in agricultural crops. Cucumber (Cucumis sativus L.) is a world cultivated vegetable crop with great economical and nutritional value. In this study, we isolated 10 YUC family genes (CsYUCs) from cucumber and explored their expression pattern under four types of stress treatments. Our data showed that CsYUC8 and CsYUC9 were specifically upregulated to elevate the auxin level under high temperature. CsYUC10b was dramatically increased but CsYUC4 was repressed in response to low temperature. CsYUC10a and CsYUC11 act against the upregulation of CsYUC10b under salinity stress, suggesting that distinct YUC members participate in different stress response, and may even antagonize each other to maintain the proper auxin levels in cucumber. Further, CsYUC11 was specifically expressed in the male flower in cucumber, and enhanced tolerance to salinity stress and regulated pedicel and stamen development through auxin biosynthesis in Arabidopsis.

Cloning of Toluene 4-Monooxygenase Genes and Application of Two-Phase System to the Production of the Anticancer Agent, Indirubin.

Indirubin is a strong inhibitor of several eukaryotic cell signaling pathways and shows promise as a treatment for myelocytic leukemia and Alzheimer's disease. The tmoABCDEF operon, encoding the components of a novel toluene 4-monooxygenase from the paint factory soil isolate, Pseudomonas sp. M4, was cloned and expressed in Escherichia coli. E. coli::pKSR12 expressing the tmo genes was used to develop a two-phase [dioctyl phthalate (DOP)/aqueous medium] culture system that was optimized to obtain maximal yields of indirubin from the starting substrate, indole. DOP was used as the organic phase to solubilize and sequester the toxic indole substrate, making possible the use of high indole concentrations that would otherwise interfere with growth in aqueous media. A 50 % (v/v) DOP two-phase system using tryptophan medium containing 3 mM cysteine, 5 mM indole, and 1 mM isatin yielded 102.4 mg/L of indirubin with no conversion of indole to the less valuable alternate product, indigo.

Production of ketocarotenoids in tobacco alters the photosynthetic efficiency by reducing photosystem II supercomplex and LHCII trimer stability.

The consequences of ketocarotenoid production in transgenic tobacco (Nicotiana tabacum) plants expressing a Chlamydomonas reinhardtii gene encoding a ß-carotene ketolase were examined concerning the functionality of the photosynthetic apparatus. T1 plants produced less photosynthetic pigments per dry weight, but Chl a/Chl b ratios remained unchanged. Almost as much ketocarotenoids as accessory xanthophylls accumulated per Chl a molecule. These ketocarotenoids were found mainly in the thylakoid membranes, but were not functionally bound to light-harvesting complexes, although LHCII is known to be able to bind astaxanthin. On the contrary, high amounts of ketocarotenoids probably changed the properties of the lipid phase of the thylakoids, thereby reducing the stability of photosystem II supercomplexes and LHCII trimers and ultimately decreasing grana formation. In addition, photosystem II function in electron transport was impaired, and plants exhibited less non-photochemical quenching compared to wild-type plants. Thus, in order not to disturb vital functions of the plants, production of astaxanthin and other nutritionally valuable ketocarotenoids apparently requires ways to sequester the additional carotenoids to plastoglobuli.

Competition between metals for binding to methanobactin enables expression of soluble methane monooxygenase in the presence of copper.

It is well known that copper is a key factor regulating expression of the two forms of methane monooxygenase found in proteobacterial methanotrophs. Of these forms, the cytoplasmic, or soluble, methane monooxygenase (sMMO) is expressed only at low copper concentrations. The membrane-bound, or particulate, methane monooxygenase (pMMO) is constitutively expressed with respect to copper, and such expression increases with increasing copper. Recent findings have shown that copper uptake is mediated by a modified polypeptide, or chalkophore, termed methanobactin. Although methanobactin has high specificity for copper, it can bind other metals, e.g., gold. Here we show that in Methylosinus trichosporium OB3b, sMMO is expressed and active in the presence of copper if gold is also simultaneously present. Such expression appears to be due to gold binding to methanobactin produced by M. trichosporium OB3b, thereby limiting copper uptake. Such expression and activity, however, was significantly reduced if methanobactin preloaded with copper was also added. Further, quantitative reverse transcriptase PCR (RT-qPCR) of transcripts of genes encoding polypeptides of both forms of MMO and SDS-PAGE results indicate that both sMMO and pMMO can be expressed when copper and gold are present, as gold effectively competes with copper for binding to methanobactin. Such findings suggest that under certain geochemical conditions, both forms of MMO may be expressed and active in situ. Finally, these findings also suggest strategies whereby field sites can be manipulated to enhance sMMO expression, i.e., through the addition of a metal that can compete with copper for binding to methanobactin.

Temporomandibular Disorders in a Sample Population of the Brazilian Northeast

Temporomandibular disorder (TMD) is a common condition. This study is part of a research group and it investigated the prevalence of TMD and myofascial pain and its association with gender, age and socioeconomic class. The sample comprised 100 subjects, aged 15 to 70, users of the Family Health Units' services, in the city of Recife, PE, Brazil. The TMD degree was evaluated using the Research Diagnostic Criteria for TMD and socioeconomic class by the Economic Classification Criteria Brazil. Categorical variables were analyzed by chi-square test for proportions and Fisher's exact test for 2x2 tables, and binary logistic analysis to track the relationship between the independent and dependent variables. According to the results, 42% of the subjects had TMD and 14% myofascial pain. No statistically significant association could be found between TMD and gender or socioeconomic class, but it was found to have statistically significant association with age, and myofascial pain was associated with socioeconomic class. Considering that the results of the present study should be confirmed by further studies and the fact that this was a pilot study, the prevalence must be analyzed with caution.

Disfunção temporomandibular (DTM) é uma condição comum. Este estudo é parte de um grupo de pesquisa e investigou a prevalência de DTM e dor miofascial e suas associações com sexo, idade e classe socioeconômica. A amostra foi composta por 100 indivíduos, com idades entre 15 e 70 anos, usuários das Unidades de Saúde da Família, na cidade de Recife, PE. O grau de DTM foi avaliado usando os Critérios de Diagnósticos Científicos em DTM, e classe socioeconômica com o Critério de Classificação Econômica Brasil. As variáveis categóricas foram analisadas pelo teste do qui-quadrado para proporções e teste exato de Fisher para tabelas 2x2, e a análise logística binária para traçar a relação entre as variáveis independentes e dependentes. De acordo com os resultados, 42% dos indivíduos tinham DTM e 14% dor miofascial. Não houve associação estatisticamente significativa entre DTM e sexo ou classe socioeconômica, mas houve associação estatisticamente significativa com a idade e a dor miofascial foi associada com a classe socioeconômica. Considerando-se que os resultados do presente estudo devam ser confirmados em outros estudos e por causa de sua natureza piloto, a prevalência deve ser analisada com cautela.

Tolerance to acetaminophen hepatotoxicity in the mouse model of autoprotection is associated with induction of flavin-containing monooxygenase-3 (FMO3) in hepatocytes.

Acetaminophen (APAP) pretreatment with a hepatotoxic dose (400 mg/kg) in mice results in resistance to a second, higher dose (600 mg/kg) of APAP (APAP autoprotection). Recent microarray work by our group showed a drastic induction of liver flavin containing monooxygenase-3 (Fmo3) mRNA expression in our mouse model of APAP autoprotection. The role of liver Fmo3, which detoxifies xenobiotics, in APAP autoprotection is unknown. The purpose of this study was to characterize the gene regulation and protein expression of liver Fmo3 during APAP hepatotoxicity. The functional consequences of Fmo3 induction were also investigated. Plasma and livers were collected from male C57BL/6J mice over a period of 72 h following a single dose of APAP (400 mg/kg) to measure Fmo3 mRNA and protein expression. Although Fmo3 mRNA levels increased significantly following APAP treatment, protein expression changed marginally. In contrast, both Fmo3 mRNA and protein expression were significantly higher in APAP autoprotected livers. Unlike male C57BL/6J mice, female mice have ∼80-times higher constitutive Fmo3 mRNA levels and are highly resistant to APAP hepatotoxicity. Coadministration of APAP with the FMO inhibitor methimazole rendered female mice susceptible to APAP hepatotoxicity, with no changes in susceptibility detected in male mice. Furthermore, a human hepatocyte cell line (HC-04) clone over-expressing human FMO3 showed enhanced resistance to APAP cytotoxicity. Taken together, these findings establish for the first time induction of Fmo3 protein expression and function by xenobiotic treatment. Our results also indicate that Fmo3 expression and function plays a role in protecting the liver from APAP-induced toxicity. Although the mechanism(s) of this protection remains to be elucidated, this work describes a novel protective function for this enzyme.

Biocatalysis with Escherichia coli-overexpressing cyclopentanone monooxygenase immobilized in polyvinyl alcohol gel.

UNLABELLED: This is the first reported study on the immobilization of living recombinant Escherichia coli cells that overexpress cyclopentanone monooxygenase in polyvinyl alcohol gel particles LentiKats®. Immobilized cells overexpressing cyclopentanone monooxygenase have been used as a model of biocatalyst for enantioselective Baeyer-Villiger biooxidation of rac-bicyclo[3.2.0]hept-2-en-6-one into regioisomeric lactones. This process is useful for the syntheses of cytostatic sarkomycin, several prostaglandins and other biologically active compounds. The original technique for qualitative analysis of enzyme expression within free cells and cells entrapped in LentiKats® using SDS-PAGE was developed and used for verification of optimal conditions for the induction of cyclopentanone monooxygenase. Here, we successfully performed six repeated batch Baeyer-Villiger biooxidations utilizing entrapped cells using 40% (w/v) polyvinyl alcohol gel particles in flasks with baffles. The latter conditions have been found to be the most appropriate achieving optimal oxygen transfer within LentiKats®. Moreover, immobilized cells retained their catalytic efficiency over six reaction cycles, while the catalytic efficiency of free cells decreased after three reaction cycles. SIGNIFICANCE AND IMPACT OF THE STUDY: Immobilization in polyvinylalcohol gel particles is desirable technique with presumptive impact on industrial applications of recombinant whole-cell Baeyer-Villiger monooxygenases as biocatalysts for production of bioactive compounds and precursors of potentially new drugs. An original immobilization of cells E. coli with overproduced Baeyer-Villiger monooxygenase improved their stability in repetitive batch biooxidations as compared to free cells. Detected autoinduction of recombinant enzyme in pET22b+ plays significant role in application of immobilized cells as it may increase specific activity of cells in repetitive use under growing reaction conditions. Original technique for qualitative analysis of enzyme expression within immobilized cells was developed.

Cloning, characterization and expression of OsFMO(t) in rice encoding a flavin monooxygenase.

Flavin monooxygenases (FMO) play a key role in tryptophan (Trp)-dependent indole-acetic acid (IAA) biosynthesis in plants and regulate plant growth and development. In this study, the full-length genomic DNA and cDNA of OsFMO(t), a FMO gene that was originally identified from a rolled-leaf mutant in rice, was isolated and cloned from wild type of the rolled-leaf mutant. OsFMO(t) was found to have four exons and three introns, and encode a protein with 422 amino acid residues that contains two basic conserved motifs, with a 'GxGxxG' characteristic structure. OsFMO(t) showed high amino acid sequence identity with FMO proteins from other plants, in particular with YUCCA from Arabidopsis, FLOOZY from Petunia, and OsYUCCA1 from rice. Our phylogenetic analysis showed that OsFMO(t) and the homologous FMO proteins belong to the same clade in the evolutionary tree. Overexpression of OsFMO(t) in transformed rice calli produced IAA-excessive phenotypes that showed browning and lethal effects when exogenous auxins such as naphthylacetic acid (NAA) were added to the medium. These results suggested that the OsFMO(t) protein is involved in IAA biosynthesis in rice and its overexpression could lead to the malformation of calli. Spatio-temporal expression analysis using RT-PCR and histochemical analysis for GUS activity revealed that expression of OsFMO(t) was totally absent in the rolled-leaf mutant. However, in the wild type variety, this gene was expressed at different levels temporally and spatially, with the highest expression observed in tissues with fast growth and cell division such as shoot apexes, tender leaves and root tips. Our results demonstrated that IAA biosynthesis regulated by OsFMO(t) is likely localized and might play an essential role in shaping local IAA concentrations which, in turn, is critical for regulating normal growth and development in rice.

Methanobactin and MmoD work in concert to act as the 'copper-switch' in methanotrophs.

Biological oxidation of methane to methanol by aerobic bacteria is catalysed by two different enzymes, the cytoplasmic or soluble methane monooxygenase (sMMO) and the membrane-bound or particulate methane monooxygenase (pMMO). Expression of MMOs is controlled by a 'copper-switch', i.e. sMMO is only expressed at very low copper : biomass ratios, while pMMO expression increases as this ratio increases. Methanotrophs synthesize a chalkophore, methanobactin, for the binding and import of copper. Previous work suggested that methanobactin was formed from a polypeptide precursor. Here we report that deletion of the gene suspected to encode for this precursor, mbnA, in Methylosinus trichosporium OB3b, abolishes methanobactin production. Further, gene expression assays indicate that methanobactin, together with another polypeptide of previously unknown function, MmoD, play key roles in regulating expression of MMOs. Based on these data, we propose a general model explaining how expression of the MMO operons is regulated by copper, methanobactin and MmoD. The basis of the 'copper-switch' is MmoD, and methanobactin amplifies the magnitude of the switch. Bioinformatic analysis of bacterial genomes indicates that the production of methanobactin-like compounds is not confined to methanotrophs, suggesting that its use as a metal-binding agent and/or role in gene regulation may be widespread in nature.

Consumers of 4-chloro-2-methylphenoxyacetic acid from agricultural soil and drilosphere harbor cadA, r/sdpA, and tfdA-like gene encoding oxygenases.

Microbial degradation of 2-methyl-4-chlorophenoxyacetic acid (MCPA) in soil is enhanced by earthworms and initiated by tfdA-like, cadA and r/sdpA gene encoding oxygenases. Copy numbers of such genes increased during MCPA degradation in soil, and MCPA stimulated transcription of tfdA-like and r/sdpA genes up to 4×. Transcription of cadA was detected in the presence of MCPA only. DNA stable isotope probing after consumption of 0.6-0.8 mg 13C-MCPA gdw -1 in oxic microcosms indicated diverse labeled oxygenase genes in bulk soil, burrow walls, and cast. 9, 6, and 3 operational taxonomic units of tfdA-like, cadA, and r/sdpA genes, respectively, were labeled and affiliated with group 2 Alphaproteobacteria including Bradyrhizobia and group 1 class III Betaproteobacteria. New genes encoding putative MCPA degrading oxygenases were identified. Diversity of labeled OTUs tended to be lower for cast than for bulk soil. The collective data indicate (1) hitherto unknown active MCPA degraders and/or oxygenase genes in soil; (2) that multiple oxygenases are associated with MCPA degradation in soil at the same time; (3) that earthworms impact the capability of MCPA degraders in soil to respond to MCPA; and (4) the collective data enable a more in-depth analysis of MCPA degrader communities in soil by future structural gene-based experimental strategies.

One-year monitoring of meta-cleavage dioxygenase gene expression and microbial community dynamics reveals the relevance of subfamily I.2.C extradiol dioxygenases in hypoxic, BTEX-contaminated groundwater.

Aromatic hydrocarbons including benzene, toluene, ethyl-benzene, and xylene (BTEX) are frequent contaminants of groundwater, the major drinking water resource. Bioremediation is the only sustainable process to clean up these environments. Microbial degradation of BTEX compounds occurs rapidly under aerobic conditions but, in subsurface environments, the availability of oxygen is commonly restricted. Even so, the microaerobic degradation of aromatic compounds is still poorly understood. Hence, the dynamics of a bacterial community and the expression of meta-cleavage dioxygenase genes, with particular emphasis on subfamily I.2.C extradiol dioxygenase genes, were assessed over a 13-month period in a hypoxic, aromatic hydrocarbon-contaminated shallow groundwater by using sequence-aided terminal-restriction fragment length polymorphism (T-RFLP) and single-nucleotide primer extension (SNuPE), respectively. The bacterial 16S rRNA fingerprinting revealed the predominance of members of Rhodoferax, Azoarcus, Pseudomonas, and unknown bacteria related to Rhodocyclaceae. It was observed that mRNA transcripts of subfamily I.2.C extradiol dioxygenase genes were detected constantly over the monitoring period, and the detected sequences clustered into six distinct clusters. In order to reveal changes in the expression of these clusters over the monitoring period a SNuPE assay was developed. This quasi fingerprinting of functional gene expression provided the opportunity to link the investigated function to specific microbial populations. The results obtained can improve our understanding of aromatic hydrocarbon degradation under oxygen limitation and may benefit bioremediation research by demonstrating the usefulness of SNuPE for the monitoring of microbial populations involved in degradation process.

Potential immunogenic polypeptides of Burkholderia pseudomallei identified by shotgun expression library and evaluation of their efficacy for serodiagnosis of melioidosis.

The search for novel immunogenic polypeptides to improve the accuracy and reliability of serologic diagnostic methods for Burkholderia pseudomallei infection is ongoing. We employed a rapid and efficient approach to identify such polypeptides with sera from melioidosis patients using a small insert genomic expression library created from clinically confirmed local virulent isolates of B. pseudomallei. After 2 rounds of immunoscreening, 6 sero-positive clones expressing immunogenic peptides were sequenced and their identities were: benzoate 1,2-dioxygenase beta subunit, a putative 200 kDa antigen p200, phosphotransferase enzyme family protein, short chain dehydrogenase and 2 hypothetical proteins. These immunogens were then transferred to an ELISA platform for further large scale screening. By combining shotgun expression library and ELISA assays, we identified 2 polypeptides BPSS1904 (benzoate 1,2-dioxygenase beta subunit) and BPSL3130 (hypothetical protein), which had sensitivities of 78.9% and 79.4% and specificities of 88.1% and 94.8%, respectively in ELISA test, thus suggesting that both are potential candidate antigens for the serodiagnosis of infections caused by B. pseudomallei.

Environmental transcription of mmoX by methane-oxidizing Proteobacteria in a subarctic Palsa Peatland.

Methane-oxidizing bacteria (MOB) that possess the soluble form of methane monooxygenase (sMMO) are present in various environments, but unlike the prevalent particulate methane monooxygenase (pMMO), the in situ activity of sMMO has not been documented. Here we report on the environmental transcription of a gene (mmoX) for this enzyme, which was attributed mainly to MOB lacking a pMMO. Our study indicates that the sMMO is an active enzyme in acidic peat ecosystems, but its importance for the mitigation of methane releases remains unknown.