Synergy effects of Methylomonas koyamae and Hyphomicrobium methylovorum under methanethiol stress.

Methanotrophs are able to metabolize volatile organic sulfur compounds (VOSCs), excrete organic carbon during CH4 oxidation, and influence microbial community structure and function of the ecosystem. In return, microbial community structure and environmental factors can affect the growth metabolism of methanotrophs. In this study, Methylomonas koyamae and Hyphomicrobium methylovorum were used for model organisms, and methanethiol (MT) was chosen for a typical VOSC to investigate the synergy effects under VOSC stress. The results showed that when Hyphomicrobium methylovorum was co-cultured with Methylomonas koyamae in the medium with CH4 used as the carbon source, the co-culture had better MT tolerance relative to Methylomonas koyamae and oxidized all CH4 within 120 h, even at the initial MT concentration of 2000 mg m-3. The optimal co-culture ratios of Methylomonas koyamae to Hyphomicrobium methylovorum were 4:1-12:1. Although MT could be converted spontaneously to dimethyl disulfide (DMDS), H2S, and CS2 in air, faster losses of MT, DMDS, H2S, and CS2 were observed in each strain mono-culture and the co-culture. Compared with Hyphomicrobium methylovorum, MT was degraded more quickly in the Methylomonas koyamae culture. During the co-culture, the CH4 oxidation process of Methylomonas koyamae could provide carbon and energy sources for the growth of Hyphomicrobium methylovorum, while Hyphomicrobium methylovorum oxidized MT to help Methylomonas koyamae detoxify. These findings are helpful to understand the synergy effects of Methylomonas koyamae and Hyphomicrobium methylovorum under MT stress and enrich the role of methanotrophs in the sulfur biogeochemical cycle. KEY POINTS: • The co-culture of Methylomonas and Hyphomicrobium has better tolerance to CH3SH. • Methylomonas can provide carbon sources for the growth of Hyphomicrobium. • The co-culture of Methylomonas and Hyphomicrobium enhances the removal of CH4 and CH3SH.

Detoxification Esterase StrH Initiates Strobilurin Fungicide Degradation in Hyphomicrobium sp. Strain DY-1.

Strobilurin fungicides are widely used in agricultural production due to their broad-spectrum and fungal mitochondrial inhibitory activities. However, their massive application has restrained the growth of eukaryotic algae and increased collateral damage in freshwater systems, notably harmful cyanobacterial blooms (HCBs). In this study, a strobilurin fungicide-degrading strain, Hyphomicrobium sp. strain DY-1, was isolated and characterized successfully. Moreover, a novel esterase gene, strH, responsible for the de-esterification of strobilurin fungicides, was cloned, and the enzymatic properties of StrH were studied. For trifloxystrobin, StrH displayed maximum activity at 50°C and pH 7.0. The catalytic efficiencies (kcat/Km ) of StrH for different strobilurin fungicides were 196.32 ± 2.30 µM-1 · s-1 (trifloxystrobin), 4.64 ± 0.05 µM-1 · s-1 (picoxystrobin), 2.94 ± 0.02 µM-1 · s-1 (pyraclostrobin), and (2.41 ± 0.19)×10-2 µM-1 · s-1 (azoxystrobin). StrH catalyzed the de-esterification of a variety of strobilurin fungicides, generating the corresponding parent acid to achieve the detoxification of strobilurin fungicides and relieve strobilurin fungicide growth inhibition of Chlorella This research will provide insight into the microbial remediation of strobilurin fungicide-contaminated environments.IMPORTANCE Strobilurin fungicides have been widely acknowledged as an essential group of pesticides worldwide. So far, their residues and toxic effects on aquatic organisms have been reported in different parts of the world. Microbial degradation can eliminate xenobiotics from the environment. Therefore, the degradation of strobilurin fungicides by microorganisms has also been reported. However, little is known about the involvement of enzymes or genes in strobilurin fungicide degradation. In this study, a novel esterase gene responsible for the detoxification of strobilurin fungicides, strH, was cloned in the newly isolated strain Hyphomicrobium sp. DY-1. This degradation process detoxifies the strobilurin fungicides and relieves their growth inhibition of Chlorella.

Two-stage oxygen supply strategy for enhancing fed-batch production of pyrroloquinoline quinone in Hyphomicrobium denitrificans FJNU-6.

Oxygen is a vital parameter for pyrroloquinoline quinone (PQQ) biosynthesis. In this study, the effects of oxygen supply on the biosynthesis of PQQ were first investigated systematically with Hyphomicrobium denitrificans FJNU-6. Following a kinetic analysis of the specific cell growth rate (µx) and specific PQQ formation rate (µp) in 5 L benchtop fermentation systems at various oxygen supply levels ranging from 0 to 60%, a novel, two-stage oxygen supply strategy was developed for enhancing PQQ production and productivity. Moreover, the transcription of genes involved in methanol oxidation and PQQ biosynthesis was analyzed throughout the process to outline the effect of oxygen supply on cell metabolism. Furthermore, with constant feeding of methanol at 0-1 g/L after the initial methanol was consumed completely, the PQQ concentration and productivity reached 1070 mg/L and 7.64 mg/L/h, respectively, after 140 h in a 5-L fermenter. The two-stage oxygen supply strategy developed in this study provides an effective and economical strategy for the industrial production of PQQ.Key Points• A novel, two-stage oxygen supply strategy was developed for enhancing PQQ production and productivity.•The transcription of genes involved in methanol oxidation and PQQ biosynthesis was regulated by changes in oxygen supply.• This study offers an effective and economical strategy for industrial or large-scale production of PQQ.

Development of a novel methanotrophic process with the helper micro-organism Hyphomicrobium sp. NM3.

AIMS: Microbial consortia can be more efficient at biological processes than single isolates. The purposes of this study were to design and evaluate a synthetic microbial consortium containing the methanotroph Methylocystis sp. M6 and the helper Hyphomicrobium sp. NM3, and develop a novel methanotrophic process for this consortium utilizing a dialysis membrane. METHODS AND RESULTS: Hyphomicrobium increased the methane-oxidation rate (MOR), biomass and stability at a dilution rate of 0·067 day-1 in fed-batch co-culture. qRT-PCR showed that Methylocystis population increased gradually with time, whereas Hyphomicrobium population remained stable despite cell washing, confirming synergistic population interaction. At 0·1 day-1 , spiking of Hyphomicrobium effectively increased the methanotrophic activity, after which Hyphomicrobium population decreased with time, indicating that the consortium is optimal at <0·1 day-1 . When Hyphomicrobium was grown in dialysis membrane within the bioreactor, MOR increased linearly up to 155·1 ± 1·0 mmol l-1  day-1 at 0·067, 0·1, 0·2 and 0·4 day-1 , which is the highest observed value for a methanotrophic reactor. CONCLUSIONS: Hyphomicrobium sp. NM3 is a promising helper micro-organism for methanotrophs. Hyphomicrobium-methanotroph consortia used concurrently with existing methods can produce an efficient and stable methane oxidation system. SIGNIFICANCE AND IMPACT OF THE STUDY: This novel methanotrophic process is superior to those previously reported in the literature, and can provide efficient and stable methane oxidation.

Lipoate-binding proteins and specific lipoate-protein ligases in microbial sulfur oxidation reveal an atpyical role for an old cofactor.

Many Bacteria and Archaea employ the heterodisulfide reductase (Hdr)-like sulfur oxidation pathway. The relevant genes are inevitably associated with genes encoding lipoate-binding proteins (LbpA). Here, deletion of the gene identified LbpA as an essential component of the Hdr-like sulfur-oxidizing system in the Alphaproteobacterium Hyphomicrobium denitrificans. Thus, a biological function was established for the universally conserved cofactor lipoate that is markedly different from its canonical roles in central metabolism. LbpAs likely function as sulfur-binding entities presenting substrate to different catalytic sites of the Hdr-like complex, similar to the substrate-channeling function of lipoate in carbon-metabolizing multienzyme complexes, for example pyruvate dehydrogenase. LbpAs serve a specific function in sulfur oxidation, cannot functionally replace the related GcvH protein in Bacillus subtilis and are not modified by the canonical E. coli and B. subtilis lipoyl attachment machineries. Instead, LplA-like lipoate-protein ligases encoded in or in immediate vicinity of hdr-lpbA gene clusters act specifically on these proteins.

A novel bacterial sulfur oxidation pathway provides a new link between the cycles of organic and inorganic sulfur compounds.

Dimethylsulfide (DMS) plays a globally significant role in carbon and sulfur cycling and impacts Earth's climate because its oxidation products serve as nuclei for cloud formation. While the initial steps of aerobic DMS degradation and the fate of its carbon atoms are reasonably well documented, oxidation of the contained sulfur is largely unexplored. Here, we identified a novel pathway of sulfur compound oxidation in the ubiquitously occurring DMS-degrader Hyphomicrobium denitrificans XT that links the oxidation of the volatile organosulfur compound with that of the inorganic sulfur compound thiosulfate. DMS is first transformed to methanethiol from which sulfide is released and fully oxidized to sulfate. Comparative proteomics indicated thiosulfate as an intermediate of this pathway and pointed at a heterodisulfide reductase (Hdr)-like system acting as a sulfur-oxidizing entity. Indeed, marker exchange mutagenesis of hdr-like genes disrupted the ability of H. denitrificans to metabolize DMS and also prevented formation of sulfate from thiosulfate provided as an additional electron source during chemoorganoheterotrophic growth. Complementation with the hdr-like genes under a constitutive promoter rescued the phenotype on thiosulfate as well as on DMS. The production of sulfate from an organosulfur precursor via the Hdr-like system is previously undocumented and provides a new shunt in the biogeochemical sulfur cycle. Furthermore, our findings fill a long-standing knowledge gap in microbial dissimilatory sulfur metabolism because the Hdr-like pathway is abundant not only in chemoheterotrophs, but also in a wide range of chemo- and photolithoautotrophic sulfur oxidizers acting as key players in global sulfur cycling.

Molecular processes underlying synergistic linuron mineralization in a triple-species bacterial consortium biofilm revealed by differential transcriptomics.

The proteobacteria Variovorax sp. WDL1, Comamonas testosteroni WDL7, and Hyphomicrobium sulfonivorans WDL6 compose a triple-species consortium that synergistically degrades and grows on the phenylurea herbicide linuron. To acquire a better insight into the interactions between the consortium members and the underlying molecular mechanisms, we compared the transcriptomes of the key biodegrading strains WDL7 and WDL1 grown as biofilms in either isolation or consortium conditions by differential RNAseq analysis. Differentially expressed pathways and cellular systems were inferred using the network-based algorithm PheNetic. Coculturing affected mainly metabolism in WDL1. Significantly enhanced expression of hylA encoding linuron hydrolase was observed. Moreover, differential expression of several pathways involved in carbohydrate, amino acid, nitrogen, and sulfur metabolism was observed indicating that WDL1 gains carbon and energy from linuron indirectly by consuming excretion products from WDL7 and/or WDL6. Moreover, in consortium conditions, WDL1 showed a pronounced stress response and overexpression of cell to cell interaction systems such as quorum sensing, contact-dependent inhibition, and Type VI secretion. Since the latter two systems can mediate interference competition, it prompts the question if synergistic linuron degradation is the result of true adaptive cooperation or rather a facultative interaction between bacteria that coincidentally occupy complementary metabolic niches.

On the Trails of the Proteasome Fold: Structural and Functional Analysis of the Ancestral ß-Subunit Protein Anbu.

The 20S proteasome is a key player in eukaryotic and archaeal protein degradation, but its progenitor in eubacteria is unknown. Recently, the ancestral ß-subunit protein (Anbu) was predicted to be the evolutionary precursor of the proteasome. We crystallized Anbu from Hyphomicrobium sp. strain MC1 in four different space groups and solved the structures by SAD-phasing and Patterson search calculation techniques. Our data reveal that Anbu adopts the classical fold of Ntn-hydrolases, but its oligomeric state differs from that of barrel-shaped proteases. In contrast to their typical architecture, the Anbu protomer is a tightly interacting dimer that can assemble into a helical superstructure. Although Anbu features a catalytic triad of Thr1Oγ, Asp17Oδ1 and Lys32Nε, it is unable to hydrolyze standard protease substrates. The lack of activity might be caused by the incapacity of Thr1NH2 to function as a Brønsted acid during substrate cleavage due to its missing activation via hydrogen bonding. Altogether, we demonstrate that the topology of the proteasomal fold is conserved in Anbu, but whether it acts as a protease still needs to be clarified.

Catabolic task division between two near-isogenic subpopulations co-existing in a herbicide-degrading bacterial consortium: consequences for the interspecies consortium metabolic model.

Variovorax sp. WDL1 mediates hydrolysis of the herbicide linuron into 3,4-dichloroaniline (DCA) and N,O-dimethylhydroxylamine in a tripartite bacterial consortium with Comamonas testosteroni WDL7 and Hyphomicrobium sulfonivorans WDL6. Although strain WDL1 contains the dcaQTA1A2B operon for DCA oxidation, this conversion is mainly performed by WDL7. Phenotypic diversification observed in WDL1 cultures and scrutiny of the WDL1 genome suggest that WDL1 cultures consist of two dedicated subpopulations, i.e., a linuron-hydrolysing subpopulation (Lin + DCA-) and a DCA-oxidizing subpopulation (Lin-DCA+). Whole genome analysis of strains representing the respective subpopulations revealed that they are identical, aside from the presence of hylA (in Lin + DCA- cells) and the dcaQTA1A2B gene cluster (in Lin-DCA+ cells), and that these catabolic gene modules replace each other at exactly the same locus on a 1380 kb extra-chromosomal element that shows plasmid gene functions including genes for transferability by conjugation. Both subpopulations proliferate in consortium biofilms fed with linuron, but Lin + DCA- cells compose the main WDL1 subpopulation. Our observations instigated revisiting the interactions within the consortium and suggest that the physical separation of two essential linuron catabolic gene clusters in WDL1 by mutually exclusive integration in the same mobile genetic element is key to the existence of WDL1 in a consortium mode.

Change in microbial community in landfill refuse contaminated with antibiotics facilitates denitrification more than the increase in ARG over long-term.

In this study, the addition of sulfamethazine (SMT) to landfill refuse decreased nitrogen intermediates (e.g. N2O and NO) and dinitrogen (N2) gas fluxes to <0.5 µg-N/kg-refuse·h-1, while the N2O and N2 flux were at ~1.5 and 5.0 µg-N/kg-refuse·h-1 respectively in samples to which oxytetracycline (OTC) had been added. The ARG (antibiotic resistance gene) levels in the refuse increased tenfold after long-term exposure to antibiotics, followed by a fourfold increase in the N2 flux, but SMT-amended samples with the largest resistome facilitated the denitrification (the nitrogen accumulated as NO gas at ~6 µg-N/kg-refuse·h-1) to a lesser extent than OTC-amended samples. Further, deep sequencing results show that long-term OTC exposure partially substituted Hyphomicrobium, Fulvivirga, and Caldilinea (>5%) for the dominant bacterial hosts (Rhodothermus, ~20%) harboring nosZ and norB genes that significantly correlated with nitrogen emission pattern, while sulfamethazine amendment completely reduced the relative abundance of the "original inhabitants" functioning to produce NOx gas reduction. The main ARG carriers (Pseudomonas) that were substantially enriched in the SMT group had lower levels of denitrifying functional genes, which could imply that denitrification is influenced more by bacterial dynamics than by abundance of ARGs under antibiotic pressures.

Metaproteomics Applied to Activated Sludge for Industrial Wastewater Treatment Revealed a Dominant Methylotrophic Metabolism of Hyphomicrobium zavarzinii.

In biological wastewater treatments, microbial populations of the so-called activated sludge work together in the abatement of pollutants. In this work, the metabolic behavior of the biomass of a lab-scale plant treating industrial pharmaceutical wastewater was investigated through a metaproteomic approach. The complete treatment process included a membrane biological reactor (MBR) coupled with an advanced oxidation process (AOP) for partial breakdown of non-biodegradable molecules. Proteins from biomass samples collected pre- and post-AOP application were investigated by two-dimensional gel electrophoresis (2DE), mass spectrometry (MS), and finally identified by database search. Results showed that most proteins remained constant between pre- and post-AOP. Methanol dehydrogenase (MDH) belonging to Hyphomicrobium zavarzinii appeared as the most constantly expressed protein in the studied consortium. Other identified proteins belonging to Hyphomicrobium spp. revealed a predominant methylotrophic metabolism, and H. zavarzinii appeared as a key actor in the studied microbial community.

Bioaugmentation of nitrate-dependent anaerobic ferrous oxidation by heterotrophic denitrifying sludge addition: A promising way for promotion of chemoautotrophic denitrification.

Nitrate-dependent anaerobic ferrous oxidation (NAFO) is a new and valuable bio-process for the treatment of wastewaters with low C/N ratio, and the NAFO process is in state of the art. The heterotrophic denitrifying sludge (HDS), possessing NAFO activity, was used as bioaugmentation to enhance NAFO efficiency. At a dosage of 6% (V/V), the removal of nitrate and ferrous was 2.4 times and 2.3 times of as primary, and the volumetric removal rate (VRR) of nitrate and ferrous was 2.4 times and 2.2 times of as primary. Tracing experiments of HDS indicated that the bioaugmentation on NAFO reactor was resulted from the NAFO activity by HDS itself. The predominant bacteria in HDS were identified as Thauera (52.5%) and Hyphomicrobium (20.0%) which were typical denitrifying bacteria and had potential ability to oxidize ferrous. In conclusion, HDS could serve as bioaugmentation or a new seeding sludge for operating high-efficiency NAFO reactors.

Comparative Analysis of Denitrifying Activities of Hyphomicrobium nitrativorans, Hyphomicrobium denitrificans, and Hyphomicrobium zavarzinii.

Hyphomicrobium spp. are commonly identified as major players in denitrification systems supplied with methanol as a carbon source. However, denitrifying Hyphomicrobium species are poorly characterized, and very few studies have provided information on the genetic and physiological aspects of denitrification in pure cultures of these bacteria. This is a comparative study of three denitrifying Hyphomicrobium species, H. denitrificans ATCC 51888, H. zavarzinii ZV622, and a newly described species, H. nitrativorans NL23, which was isolated from a denitrification system treating seawater. Whole-genome sequence analyses revealed that although they share numerous orthologous genes, these three species differ greatly in their nitrate reductases, with gene clusters encoding a periplasmic nitrate reductase (Nap) in H. nitrativorans, a membrane-bound nitrate reductase (Nar) in H. denitrificans, and one Nap and two Nar enzymes in H. zavarzinii. Concurrently with these differences observed at the genetic level, important differences in the denitrification capacities of these Hyphomicrobium species were determined. H. nitrativorans grew and denitrified at higher nitrate and NaCl concentrations than did the two other species, without significant nitrite accumulation. Significant increases in the relative gene expression levels of the nitrate (napA) and nitrite (nirK) reductase genes were also noted for H. nitrativorans at higher nitrate and NaCl concentrations. Oxygen was also found to be a strong regulator of denitrification gene expression in both H. nitrativorans and H. zavarzinii, although individual genes responded differently in these two species. Taken together, the results presented in this study highlight the potential of H. nitrativorans as an efficient and adaptable bacterium that is able to perform complete denitrification under various conditions.

Potentialities of coupling biological processes (biotrickler/biofilter) for the degradation of a mixture of sulphur compounds.

This study deals with the potential of biological processes combining a biotrickler and a biofilter to treat a mixture of sulphur-reduced compounds including dimethyl sulphide (DMS), dimethyl disulphide (DMDS) and hydrogen sulphide (H2S). As a reference, duplicated biofilters were implemented, and operating conditions were similar for all bioprocesses. The first step of this work was to determine the efficiency removal level achieved for each compound of the mixture and in a second step, to assess the longitudinal distribution of biodegradation activities and evaluate the total bacteria, Hyphomicrobium sp. and Thiobacillus thioparus densities along the bed height. A complete removal of hydrogen sulphide is reached at the start of the experiment within the first stage (biotrickler) of the coupling. This study highlighted that the coupling of a biotrickling filter and a biofilter is an interesting way to improve both removal efficiency levels (15-20% more) and kinetics of recalcitrant sulphur compounds such as DMS and DMDS. The total cell densities remained similar (around 1 × 10(10) 16S recombinant DNA (rDNA) copies g dry packing material) for duplicated biofilters and the biofilter below the biotrickling filter. The relative abundances of Hyphomicrobium sp. and T. thioparus have been estimated to an average of 10 ± 7.0 and 0.23 ± 0.07%, respectively, for all biofilters. Further investigation should allow achieving complete removal of DMS by starting the organic sulphur compound degradation within the first stage and surveying microbial community structure colonizing this complex system.

Hydrogen and carbon isotope fractionation during degradation of chloromethane by methylotrophic bacteria.

Chloromethane (CH3 Cl) is a widely studied volatile halocarbon involved in the destruction of ozone in the stratosphere. Nevertheless, its global budget still remains debated. Stable isotope analysis is a powerful tool to constrain fluxes of chloromethane between various environmental compartments which involve a multiplicity of sources and sinks, and both biotic and abiotic processes. In this study, we measured hydrogen and carbon isotope fractionation of the remaining untransformed chloromethane following its degradation by methylotrophic bacterial strains Methylobacterium extorquens CM4 and Hyphomicrobium sp. MC1, which belong to different genera but both use the cmu pathway, the only pathway for bacterial degradation of chloromethane characterized so far. Hydrogen isotope fractionation for degradation of chloromethane was determined for the first time, and yielded enrichment factors (ε) of -29‰ and -27‰ for strains CM4 and MC1, respectively. In agreement with previous studies, enrichment in (13) C of untransformed CH3 Cl was also observed, and similar isotope enrichment factors (ε) of -41‰ and -38‰ were obtained for degradation of chloromethane by strains CM4 and MC1, respectively. These combined hydrogen and carbon isotopic data for bacterial degradation of chloromethane will contribute to refine models of the global atmospheric budget of chloromethane.

Hyphomicrobium nitrativorans sp. nov., isolated from the biofilm of a methanol-fed denitrification system treating seawater at the Montreal Biodome.

A budding prosthecate bacterial strain, designated NL23(T), was isolated from a methanol-fed denitrification system treating seawater at the Montreal Biodome, Canada. Phylogenetic analysis based on 16S rRNA (rRNA) gene sequences showed that the strain was affiliated with the genus Hyphomicrobium of the Alphaproteobacteria and was most closely related to Hyphomicrobium zavarzinii with 99.4 % sequence similarity. Despite this high level of 16S rRNA gene sequence similarity, DNA-DNA hybridization assays showed that strain NL23(T) was only distantly related to H. zavarzinii ZV-622(T) (12 %). Strain NL23(T) grew aerobically, but also had the capacity to grow under denitrifying conditions in the presence of nitrate without nitrite accumulation. Growth occurred at pH 7.0-9.5, with 0-1 % NaCl and at temperatures of 15-35 °C. Major fatty acids were C18 : 1ω7c or ω6c (84.6 %) and C18 : 0 (8.5 %), and major quinones were Q8 (5 %) and Q9 (95 %). The complete genome of the strain was sequenced and showed a DNA G+C content of 63.8 mol%. Genome analysis predicted open reading frames (ORF) encoding the key enzymes of the serine pathway as well as enzymes involved in methylotrophy. Also, ORF encoding a periplasmic nitrate reductase (Nap), a nitrite reductase (Nir), a nitric oxide reductase (Nor) and a nitrous oxide reductase (Nos) were identified. Our results support that strain NL23(T) represents a novel species within the genus Hyphomicrobium, for which the name Hyphomicrobium nitrativorans sp. nov. is proposed. The type strain is NL23(T) ( = ATCC BAA-2476(T) = LMG 27277(T)).

Dimethyl sulfide biofiltration using immobilized Hyphomicrobium VS and Thiobacillus thioparus TK-m in sugarcane bagasse.

Sugarcane bagasse was used as a carrier material of microorganisms in two different biofilters used to remove dimethyl sulfide (DMS) from a gas stream. The first biofilter was inoculated with Hyphomicrobium VS and the second with Thiobacillus thioparus Tk-m. During the operation of the biofilters the empty bed residence time (EBRT) was varied from 90 to 180 seconds and the inlet concentration of DMS from 12 to 50 ppmv. The inlet load was varied in the range of 0.62 to 5.2 (g DMS/m3 h). The maximum elimination capacity (EC) of the biofilter inoculated with Hyphomicrobium VS was 5 g DMS/m3 h; however, for the biofilter inoculated with T. thioparus Tk-m the maximum EC was 3.9 g DMS/m3 h. For T. thioparus TK-m the maximum removal efficiency (RE) obtained was 85.1 +/- 5.2% at 12 ppmv DMS inlet concentration, inlet load of 0.62 g DMS/m3 h and 180 s EBRT. The highest removal efficiency for Hyphomicrobium VS was 97.6 + 4.8% at 12 ppmv DMS inlet concentration, load of 0.62 g DMS/m3 h and 180 s EBRT.

Complete genome sequence of the chloromethane-degrading Hyphomicrobium sp. strain MC1.

Hyphomicrobium sp. strain MC1 is an aerobic methylotroph originally isolated from industrial sewage. This prosthecate bacterium was the first strain reported to grow with chloromethane as the sole carbon and energy source. Its genome, consisting of a single 4.76-Mb chromosome, is the first for a chloromethane-degrading bacterium to be formally reported.

Detection and isolation of chloromethane-degrading bacteria from the Arabidopsis thaliana phyllosphere, and characterization of chloromethane utilization genes.

Chloromethane gas is produced naturally in the phyllosphere, the compartment defined as the aboveground parts of vegetation, which hosts a rich bacterial flora. Chloromethane may serve as a growth substrate for specialized aerobic methylotrophic bacteria, which have been isolated from soil and water environments, and use cmu genes for chloromethane utilization. Evidence for the presence of chloromethane-degrading bacteria on the leaf surfaces of Arabidopsis thaliana was obtained by specific quantitative PCR of the cmuA gene encoding the two-domain methyltransferase corrinoid protein of chloromethane dehalogenase. Bacterial strains were isolated on a solid mineral medium with chloromethane as the sole carbon source from liquid mineral medium enrichment cultures inoculated with leaves of A. thaliana. Restriction analysis-based genotyping of cmuA PCR products was used to evaluate the diversity of chloromethane-degrading bacteria during enrichment and after strain isolation. The isolates obtained, affiliated to the genus Hyphomicrobium based on their 16S rRNA gene sequence and the presence of characteristic hyphae, dehalogenate chloromethane, and grow in a liquid culture with chloromethane as the sole carbon and energy source. The cmu genes of these isolates were analysed using new PCR primers, and their sequences were compared with those of previously reported aerobic chloromethane-degrading strains. The three isolates featured a colinear cmuBCA gene arrangement similar to that of all previously characterized strains, except Methylobacterium extorquens CM4 of known genome sequence.

Purification and characterization of dimethylsulfide monooxygenase from Hyphomicrobium sulfonivorans.

Dimethylsulfide (DMS) is a volatile organosulfur compound which has been implicated in the biogeochemical cycling of sulfur and in climate control. Microbial degradation is a major sink for DMS. DMS metabolism in some bacteria involves its oxidation by a DMS monooxygenase in the first step of the degradation pathway; however, this enzyme has remained uncharacterized until now. We have purified a DMS monooxygenase from Hyphomicrobium sulfonivorans, which was previously isolated from garden soil. The enzyme is a member of the flavin-linked monooxygenases of the luciferase family and is most closely related to nitrilotriacetate monooxygenases. It consists of two subunits: DmoA, a 53-kDa FMNH2-dependent monooxygenase, and DmoB, a 19-kDa NAD(P)H-dependent flavin oxidoreductase. Enzyme kinetics were investigated with a range of substrates and inhibitors. The enzyme had a K(m) of 17.2 (± 0.48) µM for DMS (k(cat) = 5.45 s⁻¹) and a V(max) of 1.25 (± 0.01) µmol NADH oxidized min⁻¹ (mg protein⁻¹). It was inhibited by umbelliferone, 8-anilinonaphthalenesulfonate, a range of metal-chelating agents, and Hg²(+), Cd²(+), and Pb²(+) ions. The purified enzyme had no activity with the substrates of related enzymes, including alkanesulfonates, aldehydes, nitrilotriacetate, or dibenzothiophenesulfone. The gene encoding the 53-kDa enzyme subunit has been cloned and matched to the enzyme subunit by mass spectrometry. DMS monooxygenase represents a new class of FMNH2-dependent monooxygenases, based on its specificity for dimethylsulfide and the molecular phylogeny of its predicted amino acid sequence. The gene encoding the large subunit of DMS monooxygenase is colocated with genes encoding putative flavin reductases, homologues of enzymes of inorganic and organic sulfur compound metabolism, and enzymes involved in riboflavin synthesis.