IncP-type plasmids carrying genes for antibiotic resistance or for aromatic compound degradation are prevalent in sequenced Aromatoleum and Thauera strains.

Self-transferable plasmids of the incompatibility group P-1 (IncP-1) are considered important carriers of genes for antibiotic resistance and other adaptive functions. In the laboratory, these plasmids have a broad host range; however, little is known about their in situ host profile. In this study, we discovered that Thauera aromatica K172T , a facultative denitrifying microorganism capable of degrading various aromatic compounds, contains a plasmid highly similar to the IncP-1 ε archetype pKJK5. The plasmid harbours multiple antibiotic resistance genes and is maintained in strain K172T for at least 1000 generations without selection pressure from antibiotics. In a subsequent search, we found additional nine IncP-type plasmids in a total of 40 sequenced genomes of the closely related genera Aromatoleum and Thauera. Six of these plasmids form a novel IncP-1 subgroup designated θ, four of which carry genes for anaerobic or aerobic degradation of aromatic compounds. Pentanucleotide sequence analyses (k-mer profiling) indicated that Aromatoleum spp. and Thauera spp. are among the most suitable hosts for the θ plasmids. Our results highlight the importance of IncP-1 plasmids for the genetic adaptation of these common facultative denitrifying bacteria and provide novel insights into the in situ host profile of these plasmids.

Complete Genomes of the Anaerobic Degradation Specialists Aromatoleum petrolei ToN1T and Aromatoleum bremense PbN1T.

The betaproteobacterial genus Aromatoleum comprises facultative denitrifiers specialized in the anaerobic degradation of recalcitrant organic compounds (aromatic and terpenoid). This study reports on the complete and manually annotated genomes of Ar. petrolei ToN1T (5.41 Mbp) and Ar. bremense PbN1T (4.38 Mbp), which cover the phylogenetic breadth of the genus Aromatoleum together with previously genome sequenced Ar. aromaticum EbN1T [Rabus et al., Arch Microbiol. 2005 Jan;183(1):27-36]. The gene clusters for the anaerobic degradation of aromatic and terpenoid (strain ToN1T only) compounds are scattered across the genomes of strains ToN1T and PbN1T. The richness in mobile genetic elements is shared with other Aromatoleum spp., substantiating that horizontal gene transfer should have been a major driver in shaping the genomes of this genus. The composite catabolic network of strains ToN1T and PbN1T comprises 88 proteins, the coding genes of which occupy 86.1 and 76.4 kbp (1.59 and 1.75%) of the respective genome. The strain-specific gene clusters for anaerobic degradation of ethyl-/propylbenzene (strain PbN1T) and toluene/monoterpenes (strain ToN1T) share high similarity with their counterparts in Ar. aromaticum strains EbN1T and pCyN1, respectively. Glucose is degraded via the ED-pathway in strain ToN1T, while gluconeogenesis proceeds via the reverse EMP-pathway in strains ToN1T, PbN1T, and EbN1T. The diazotrophic, endophytic lifestyle of closest related genus Azoarcus is known to be associated with nitrogenase and type-6 secretion system (T6SS). By contrast, strains ToN1T, PbN1T, and EbN1T lack nif genes for nitrogenase (including cofactor synthesis and enzyme maturation). Moreover, strains PbN1T and EbN1T do not possess tss genes for T6SS, while strain ToN1T does and facultative endophytic "Aromatoleum" sp. CIB is known to even have both. These findings underpin the functional heterogeneity among Aromatoleum members, correlating with the high plasticity of their genomes.

Dechloromonas and close relatives prevail during hydrogenotrophic denitrification in stimulated microcosms with oxic aquifer material.

Globally occurring nitrate pollution in groundwater is harming the environment and human health. In situ hydrogen addition to stimulate denitrification has been proposed as a remediation strategy. However, observed nitrite accumulation and incomplete denitrification are severe drawbacks that possibly stem from the specific microbial community composition. We set up a microcosm experiment comprising sediment and groundwater from a nitrate polluted oxic oligotrophic aquifer. After the microcosms were sparged with hydrogen gas, samples were taken regularly within 122 h for nitrate and nitrite measurements, community composition analysis via 16S rRNA gene amplicon sequencing and gene and transcript quantification via qPCR of reductase genes essential for complete denitrification. The highest nitrate reduction rates and greatest increase in bacterial abundance coincided with a 15.3-fold increase in relative abundance of Rhodocyclaceae, specifically six ASVs that are closely related to the genus Dechloromonas. The denitrification reductase genes napA, nirS and clade I nosZ also increased significantly over the observation period. We conclude that taxa of the genus Dechloromonas are the prevailing hydrogenotrophic denitrifiers in this nitrate polluted aquifer and the ability of hydrogenotrophic denitrification under the given conditions is species-specific.

Comparative Genomics Provides Insights into the Taxonomy of Azoarcus and Reveals Separate Origins of Nif Genes in the Proposed Azoarcus and Aromatoleum Genera.

Among other attributes, the Betaproteobacterial genus Azoarcus has biotechnological importance for plant growth-promotion and remediation of petroleum waste-polluted water and soils. It comprises at least two phylogenetically distinct groups. The "plant-associated" group includes strains that are isolated from the rhizosphere or root interior of the C4 plant Kallar Grass, but also strains from soil and/or water; all are considered to be obligate aerobes and all are diazotrophic. The other group (now partly incorporated into the new genus Aromatoleum) comprises a diverse range of species and strains that live in water or soil that is contaminated with petroleum and/or aromatic compounds; all are facultative or obligate anaerobes. Some are diazotrophs. A comparative genome analysis of 32 genomes from 30 Azoarcus-Aromatoleum strains was performed in order to delineate generic boundaries more precisely than the single gene, 16S rRNA, that has been commonly used in bacterial taxonomy. The origin of diazotrophy in Azoarcus-Aromatoleum was also investigated by comparing full-length sequences of nif genes, and by physiological measurements of nitrogenase activity using the acetylene reduction assay. Based on average nucleotide identity (ANI) and whole genome analyses, three major groups could be discerned: (i) Azoarcus comprising Az. communis, Az. indigens and Az. olearius, and two unnamed species complexes, (ii) Aromatoleum Group 1 comprising Ar. anaerobium, Ar. aromaticum, Ar. bremense, and Ar. buckelii, and (iii) Aromatoleum Group 2 comprising Ar. diolicum, Ar. evansii, Ar. petrolei, Ar. toluclasticum, Ar. tolulyticum, Ar. toluolicum, and Ar. toluvorans. Single strain lineages such as Azoarcus sp. KH32C, Az. pumilus, and Az. taiwanensis were also revealed. Full length sequences of nif-cluster genes revealed two groups of diazotrophs in Azoarcus-Aromatoleum with nif being derived from Dechloromonas in Azoarcus sensu stricto (and two Thauera strains) and from Azospira in Aromatoleum Group 2. Diazotrophy was confirmed in several strains, and for the first time in Az. communis LMG5514, Azoarcus sp. TTM-91 and Ar. toluolicum TT. In terms of ecology, with the exception of a few plant-associated strains in Azoarcus (s.s.), across the group, most strains/species are found in soil and water (often contaminated with petroleum or related aromatic compounds), sewage sludge, and seawater. The possession of nar, nap, nir, nor, and nos genes by most Azoarcus-Aromatoleum strains suggests that they have the potential to derive energy through anaerobic nitrate respiration, so this ability cannot be usefully used as a phenotypic marker to distinguish genera. However, the possession of bzd genes indicating the ability to degrade benzoate anaerobically plus the type of diazotrophy (aerobic vs. anaerobic) could, after confirmation of their functionality, be considered as distinguishing phenotypes in any new generic delineations. The taxonomy of the Azoarcus-Aromatoleum group should be revisited; retaining the generic name Azoarcus for its entirety, or creating additional genera are both possible outcomes.

Characterization of nitrous oxide reduction by Azospira sp. HJ23 isolated from advanced wastewater treatment sludge.

A new nitrous oxide (N2O)-reducing bacterium was isolated from a consortium that was enriched using advanced wastewater treatment sludge as an inoculum and N2O as the sole nitrogen source. The isolated facultative anaerobe was identified as Azospira sp. HJ23. Azospira sp. HJ23 exhibited optimum N2O-reducing activity with a C/N ratio of 62 at pH 6 in the temperature range of 37 °C to 40 °C. The optimum carbon source for N2O reduction was a mixture of glucose and acetate. The maximum rate of N2O reduction by Azospira sp. HJ23 was 4.8 mmol·g-dry cell-1·h-1, and its N2O-reducing activity was higher than other known N2O reducers. Azospira sp. HJ23 possessed several functional genes for denitrification. These included narG (NO3- reductase), nirK (NO2- reductase), norB (NO reductase), and nosZ (N2O reductase) genes. These results suggest that Azospira sp. HJ23 can be applied in the denitrification process to minimalize N2O emission.

Nanomolar Responsiveness of an Anaerobic Degradation Specialist to Alkylphenol Pollutants.

Anaerobic degradation of p-cresol (4-methylphenol) by the denitrifying betaproteobacterium Aromatoleum aromaticum EbN1 is regulated with high substrate specificity, presumed to be mediated by the predicted σ54-dependent two-component system PcrSR. An unmarked, in-frame ΔpcrSR deletion mutant showed reduced expression of the genes cmh (21-fold) and hbd (8-fold) that encode the two enzymes for initial oxidation of p-cresol to p-hydroxybenzoate compared to their expression in the wild type. The expression of cmh and hbd was restored by in trans complementation with pcrSR in the ΔpcrSR background to even higher levels than in the wild type. This is likely due to ∼200-/∼30-fold more transcripts of pcrSR in the complemented mutant. The in vivo responsiveness of A. aromaticum EbN1 to p-cresol was studied in benzoate-limited anaerobic cultures by the addition of p-cresol at various concentrations (from 100 µM down to 0.1 nM). Time-resolved transcript profiling by quantitative reverse transcription-PCR (qRT-PCR) revealed that the lowest p-cresol concentrations just affording cmh and hbd expression (response threshold) ranged between 1 and 10 nM, which is even more sensitive than the respective odor receptors of insects. A similar response threshold was determined for another alkylphenol, p-ethylphenol, which strain EbN1 anaerobically degrades via a different route and senses by the σ54-dependent one-component system EtpR. Based on these data and theoretical considerations, p-cresol or p-ethylphenol added as a single pulse (10 nM) requires less than a fraction of a second to reach equilibrium between intra- and extracellular space (∼20 molecules per cell), with an estimated Kd (dissociation constant) of <100 nM alkylphenol (p-cresol or p-ethylphenol) for its respective sensory protein (PcrS or EtpR).IMPORTANCE Alkylphenols (like p-cresol and p-ethylphenol) represent bulk chemicals for industrial syntheses. Besides massive local damage events, large-scale micropollution is likewise of environmental and health concern. Next to understanding how such pollutants can be degraded by microorganisms, it is also relevant to determine the microorganisms' lower threshold of responsiveness. Aromatoleum aromaticum EbN1 is a specialist in anaerobic degradation of aromatic compounds, employing a complex and substrate-specifically regulated catabolic network. The present study aims at verifying the predicted role of the PcrSR system in sensing p-cresol and at determining the threshold of responsiveness for alkylphenols. The findings have implications for the enigmatic persistence of dissolved organic matter (escape from biodegradation) and for the lower limits of aromatic compounds required for bacterial growth.

Two Different Quinohemoprotein Amine Dehydrogenases Initiate Anaerobic Degradation of Aromatic Amines in Aromatoleum aromaticum EbN1.

Aromatic amines like 2-phenylethylamine (2-PEA) and benzylamine (BAm) have been identified as novel growth substrates of the betaproteobacterium Aromatoleum aromaticum EbN1, which degrades a wide variety of aromatic compounds in the absence of oxygen under denitrifying growth conditions. The catabolic pathway of these amines was identified, starting with their oxidative deamination to the corresponding aldehydes, which are then further degraded via the enzymes of the phenylalanine or benzyl alcohol metabolic pathways. Two different periplasmic quinohemoprotein amine dehydrogenases involved in 2-PEA or BAm metabolism were identified and characterized. Both enzymes consist of three subunits, contain two heme c cofactors in their α-subunits, and exhibit extensive processing of their γ-subunits, generating four intramolecular thioether bonds and a cysteine tryptophylquinone (CTQ) cofactor. One of the enzymes was present in cells grown with 2-PEA or other substrates, showed an α2ß2γ2 composition, and had a rather broad substrate spectrum, which included 2-PEA, BAm, tyramine, and 1-butylamine. In contrast, the other enzyme was specifically induced in BAm-grown cells, showing an αßγ composition and activity only with BAm and 2-PEA. Since the former enzyme showed the highest catalytic efficiency with 2-PEA and the latter with BAm, they were designated 2-PEADH and benzylamine dehydrogenase (BAmDH). The catalytic properties and inhibition patterns of 2-PEADH and BAmDH showed considerable differences and were compared to previously characterized quinohemoproteins of the same enzyme family.IMPORTANCE The known substrate spectrum of A. aromaticum EbN1 is expanded toward aromatic amines, which are metabolized as sole substrates coupled to denitrification. The characterization of the two quinohemoprotein isoenzymes involved in degrading either 2-PEA or BAm expands the knowledge of this enzyme family and establishes for the first time that the necessary maturation of their quinoid CTQ cofactors does not require the presence of molecular oxygen. Moreover, the study revealed a highly interesting regulatory phenomenon, suggesting that growth with BAm leads to a complete replacement of 2-PEADH by BAmDH, which has considerably different catalytic and inhibition properties.

The 7α-hydroxysteroid dehydratase Hsh2 is essential for anaerobic degradation of the steroid skeleton of 7α-hydroxyl bile salts in the novel denitrifying bacterium Azoarcus sp. strain Aa7.

Bile salts are steroid compounds from the digestive tract of vertebrates and enter the environment via defecation. Many aerobic bile-salt degrading bacteria are known but no bacteria that completely degrade bile salts under anoxic conditions have been isolated so far. In this study, the facultatively anaerobic Betaproteobacterium Azoarcus sp. strain Aa7 was isolated that grew with bile salts as sole carbon source under anoxic conditions with nitrate as electron acceptor. Phenotypic and genomic characterization revealed that strain Aa7 used the 2,3-seco pathway for the degradation of bile salts as found in other denitrifying steroid-degrading bacteria such as Sterolibacterium denitrificans. Under oxic conditions strain Aa7 used the 9,10-seco pathway as found in, for example, Pseudomonas stutzeri Chol1. Metabolite analysis during anaerobic growth indicated a reductive dehydroxylation of 7α-hydroxyl bile salts. Deletion of the gene hsh2 Aa7 encoding a 7-hydroxysteroid dehydratase led to strongly impaired growth with cholate and chenodeoxycholate but not with deoxycholate lacking a hydroxyl group at C7. The hsh2 Aa7 deletion mutant degraded cholate and chenodeoxycholate to the corresponding C19 -androstadienediones only while no phenotype change was observed during aerobic degradation of cholate. These results showed that removal of the 7α-hydroxyl group was essential for cleavage of the steroid skeleton under anoxic conditions.

Genomic resolution of bacterial populations in saccharin and cyclamate degradation.

The benefits of extensive artificial sweeteners use come at a cost of their ubiquitous occurrence in the aquatic environment. Biodegradation is crucial for the removal of artificial sweeteners in the environment, yet comprehensive characterizations of the degradation consortia that degrade these compounds have not been initiated. Here, we performed metagenomic analysis of microbial communities fulfilling complete mineralization of two typical artificial sweeteners, i.e. saccharin and cyclamate. Genome-resolved metagenomics enabled the recovery and metabolic characterization of total 23 population genomes from 8 phyla in the two consortia, most of which represented novel species. The saccharin-degrading consortia was notably dominated by a betaproteobacterial genome from the family Rhodocyclaceae, accounting for 15.5% of total sequences. For the cyclamate enrichment, 28.1% of the total sequences were assigned to three similarly abundant Alphaproteobacteria population genomes belonging to the family Sphingomonadaceae and Methylobacteriaceae. The metabolic potential of these population genomes were examined to potentially identify the roles of these populations in biodegradation of artificial sweeteners, and focusing on the energy and nutrient metabolisms.

Type IV CRISPR RNA processing and effector complex formation in Aromatoleum aromaticum.

Type IV CRISPR-Cas modules belong to class 1 prokaryotic adaptive immune systems, which are defined by the presence of multisubunit effector complexes. They usually lack the known Cas proteins involved in adaptation and target cleavage, and their function has not been experimentally addressed. To investigate RNA and protein components of this CRISPR-Cas type, we located a complete type IV cas gene locus and an adjacent CRISPR array on a megaplasmid of Aromatoleum aromaticum EbN1, which contains an additional type I-C system on its chromosome. RNA sequencing analyses verified CRISPR RNA (crRNA) production and maturation for both systems. Type IV crRNAs were shown to harbour unusually short 7 nucleotide 5'-repeat tags and stable 3' hairpin structures. A unique Cas6 variant (Csf5) was identified that generates crRNAs that are specifically incorporated into type IV CRISPR-ribonucleoprotein (crRNP) complexes. Structures of RNA-bound Csf5 were obtained. Recombinant production and purification of the type IV Cas proteins, together with electron microscopy, revealed that Csf2 acts as a helical backbone for type IV crRNPs that include Csf5, Csf3 and a large subunit (Csf1). Mass spectrometry analyses identified protein-protein and protein-RNA contact sites. These results highlight evolutionary connections between type IV and type I CRISPR-Cas systems and demonstrate that type IV CRISPR-Cas systems employ crRNA-guided effector complexes.

Towards the Response Threshold for p-Hydroxyacetophenone in the Denitrifying Bacterium "Aromatoleum aromaticum" EbN1.

The denitrifying betaproteobacterium "Aromatoleum aromaticum" EbN1 regulates the capacity to anaerobically degrade p-ethylphenol (via p-hydroxyacetophenone) with high substrate specificity. This process is mediated by the σ54-dependent transcriptional regulator EtpR, which apparently recognizes both aromatic compounds, yielding congruent expression profiles. The responsiveness of this regulatory system was studied with p-hydroxyacetophenone, which is more easily administered to cultures and traced analytically. Cultures of A. aromaticum EbN1 were initially cultivated under nitrate-reducing conditions with a growth-limiting supply of benzoate, upon the complete depletion of which p-hydroxyacetophenone was added at various concentrations (from 500 µM down to 0.1 nM). Depletion profiles of this aromatic substrate and presumptive effector were determined by highly sensitive micro-high-performance liquid chromatography (microHPLC). Irrespective of the added concentration of p-hydroxyacetophenone, depletion commenced after less than 5 min and suggested a response threshold of below 10 nM. This approximation was corroborated by time-resolved transcript profiles (quantitative reverse transcription-PCR) of selected degradation and efflux relevant genes (e.g., pchF, encoding a subunit of predicted p-ethylphenol methylenehydroxylase) and narrowed down to a range of 10 to 1 nM. The most pronounced transcriptional response was observed, as expected, for genes located at the beginning of the two operon-like structures, related to catabolism (i.e., acsA) and potential efflux (i.e., ebA335).IMPORTANCE Aromatic compounds are widespread microbial growth substrates with natural as well as anthropogenic sources, albeit with their in situ concentrations and their bioavailabilities varying over several orders of magnitude. Even though degradation pathways and underlying regulatory systems have long been studied with aerobic and, to a lesser extent, with anaerobic bacteria, comparatively little is known about the effector concentration-dependent responsiveness. A. aromaticum EbN1 is a model organism for the anaerobic degradation of aromatic compounds with the architecture of the catabolic network and its substrate-specific regulation having been intensively studied by means of differential proteogenomics. The present study aims at unraveling the minimal concentration of an aromatic growth substrate (p-hydroxyacetophenone here) required to initiate gene expression for its degradation pathway and to learn in principle about the lower limit of catabolic responsiveness of an anaerobic degradation specialist.

Stable isotope probing of hypoxic toluene degradation at the Siklós aquifer reveals prominent role of Rhodocyclaceae.

The availability of oxygen is often a limiting factor for the degradation of aromatic hydrocarbons in subsurface environments. However, while both aerobic and anaerobic degraders have been intensively studied, degradation betwixt, under micro- or hypoxic conditions has rarely been addressed. It is speculated that in environments with limited, but sustained oxygen supply, such as in the vicinity of groundwater monitoring wells, hypoxic degradation may take place. A large diversity of subfamily I.2.C extradiol dioxygenase genes has been previously detected in a BTEX-contaminated aquifer in Hungary. Older literature suggests that such catabolic potentials could be associated to hypoxic degradation. Bacterial communities dominated by members of the Rhodocyclaceae were found, but the majority of the detected C23O genotypes could not be affiliated to any known bacterial degrader lineages. To address this, a stable isotope probing (SIP) incubation of site sediments with 13C7-toluene was performed under microoxic conditions. A combination of 16S rRNA gene amplicon sequencing and T-RFLP fingerprinting of C23O genes from SIP gradient fractions revealed the central role of degraders within the Rhodocyclaceae in hypoxic toluene degradation. The main assimilators of 13C were identified as members of the genera Quatrionicoccus and Zoogloea, and a yet uncultured group of the Rhodocyclaceae.

Evolution of a xenobiotic degradation pathway: formation and capture of the labile phthaloyl-CoA intermediate during anaerobic phthalate degradation.

Xenobiotic phthalates are industrially produced on the annual million ton scale. The oxygen-independent enzymatic reactions involved in anaerobic phthalate degradation have only recently been elucidated. In vitro assays suggested that phthalate is first activated to phthaloyl-CoA followed by decarboxylation to benzoyl-CoA. Here, we report the heterologous production and characterization of the enzyme initiating anaerobic phthalate degradation from 'Aromatoleum aromaticum': a highly specific succinyl-CoA:phthalate CoA transferase (SPT, class III CoA transferase). Phthaloyl-CoA formed by SPT accumulated only to sub-micromolar concentrations due to the extreme lability of the product towards intramolecular substitution with a half-life of around 7 min. Upon addition of excess phthaloyl-CoA decarboxylase (PCD), the combined activity of both enzymes was drastically shifted towards physiologically relevant benzoyl-CoA formation. In conclusion, a massive overproduction of PCD in phthalate-grown cells to concentrations >140 µM was observed that allowed for efficient phthaloyl-CoA conversion at concentrations 250-fold below the apparent Km -value of PCD. The results obtained provide insights into an only recently evolved xenobiotic degradation pathway where a massive cellular overproduction of PCD compensates for the formation of the probably most unstable CoA ester intermediate in biology.

Uliginosibacterium sediminicola sp. nov., isolated from freshwater sediment.

Strain M1-21T is a Gram-stain-negative, strictly aerobic and short-rod-shaped bacterium, motile by means of a single polar flagellum; it was isolated from freshwater sediment in Korea. It grew at 10-40 °C (optimum 25 °C), pH 6.0-8.0 (optimum pH 7.0) and with 0-0.75 % (w/v) NaCl (optimal growth occurred in the absence of NaCl) on R2A agar, and it accumulated poly-ß-hydroxybutyrate granules inside the cells. According to 16S rRNA gene sequence analysis, strain M1-21T showed highest sequence similarity with Uliginosibacterium gangwonense (94.7 %) and Uliginosibacterium paludis (94.4 %). Phylogenetic analysis of the 16S rRNA gene sequences revealed that strain M1-21T belongs to the genus Uliginosibacterium. The DNA G+C content of strain M1-21T was 61.9 mol%. The predominant respiratory quinone was ubiquinone-8. The major fatty acids (>10 % of the total) were C16 : 0 and summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c), and the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. Strain M1-21T showed distinct phenotypic characteristics that differentiated it from species of the genus Uliginosibacterium. Based on these results, strain M1-21T represents a novel species of the genus Uliginosibacterium, for which the name Uliginosibacterium sediminicola sp. nov. is proposed. The type strain is M1-21T (=KACC 19271T=JCM 32000T).

Growth yield and selection of nosZ clade II types in a continuous enrichment culture of N2 O respiring bacteria.

Nitrous oxide (N2 O) reducing microorganisms may be key in the mitigation of N2 O emissions from managed ecosystems. However, there is still no clear understanding of the physiological and bioenergetic implications of microorganisms possessing either of the two N2 O reductase genes (nosZ), clade I and the more recently described clade II type nosZ. It has been suggested that organisms with nosZ clade II have higher growth yields and a lower affinity constant (Ks ) for N2 O. We compared N2 O reducing communities with different nosZI/nosZII ratios selected in chemostat enrichment cultures, inoculated with activated sludge, fed with N2 O as a sole electron acceptor and growth limiting factor and acetate as electron donor. From the sequencing of the 16S rRNA gene, FISH and quantitative PCR of nosZ and nir genes, we concluded that betaproteobacterial denitrifying organisms dominated the enrichments with members within the family Rhodocyclaceae being highly abundant. When comparing cultures with different nosZI/nosZII ratios, we did not find support for (i) a more energy conserving N2 O respiration pathway in nosZ clade II systems, as reflected in the growth yield per mole of substrate, or (ii) a higher affinity for N2 O, defined by µmax /Ks , in organisms with nosZ clade II.

Functional Characterization of Three Specific Acyl-Coenzyme A Synthetases Involved in Anaerobic Cholesterol Degradation in Sterolibacterium denitrificans Chol1S.

The denitrifying betaproteobacterium Sterolibacterium denitrificans Chol1S catabolizes steroids such as cholesterol via an oxygen-independent pathway. It involves enzyme reaction sequences described for aerobic cholesterol and bile acid degradation as well as enzymes uniquely found in anaerobic steroid-degrading bacteria. Recent studies provided evidence that in S. denitrificans, the cholest-4-en-3-one intermediate is oxygen-independently oxidized to Δ4-dafachronic acid (C26-oic acid), which is subsequently activated by a substrate-specific acyl-coenzyme A (acyl-CoA) synthetase (ACS). Further degradation was suggested to proceed via unconventional ß-oxidation, where aldolases, aldehyde dehydrogenases, and additional ACSs substitute for classical ß-hydroxyacyl-CoA dehydrogenases and thiolases. Here, we heterologously expressed three cholesterol-induced genes that putatively code for AMP-forming ACSs and characterized two of the products as specific 3ß-hydroxy-Δ5-cholenoyl-CoA (C24-oic acid)- and pregn-4-en-3-one-22-oyl-CoA (C22-oic acid)-forming ACSs, respectively. A third heterologously produced ATP-dependent ACS was inactive with C26-, C24-, or C22-oic-acids but activated 3aα-H-4α-(3'propanoate)-7aß-methylhexahydro-1,5-indanedione (HIP) to HIP-CoA, a rather late intermediate of aerobic cholesterol degradation that still contains the CD rings of the sterane skeleton. This work provides experimental evidence that anaerobic steroid degradation proceeds via numerous alternate CoA-ester-dependent or -independent enzymatic reaction sequences as a result of aldolytic side chain and hydrolytic sterane ring C-C bond cleavages. The aldolytic side chain degradation pathway comprising highly exergonic ACSs and aldehyde dehydrogenases is considered to be essential for driving the unfavorable oxygen-independent C26 hydroxylation forward.IMPORTANCE The biological degradation of ubiquitously abundant steroids is hampered by their low solubility and the presence of two quaternary carbon atoms. The degradation of cholesterol by aerobic Actinobacteria has been studied in detail for more than 30 years and involves a number of oxygenase-dependent reactions. In contrast, much less is known about the oxygen-independent degradation of steroids in denitrifying bacteria. In the cholesterol-degrading anaerobic model organism Sterolibacterium denitrificans Chol1S, initial evidence has been obtained that steroid degradation proceeds via numerous alternate coenzyme A (CoA)-ester-dependent/independent reaction sequences. Here, we describe the heterologous expression of three highly specific and characteristic acyl-CoA synthetases, two of which play key roles in the degradation of the side chain, whereas a third one is specifically involved in the B ring degradation. The results obtained shed light into oxygen-independent steroid degradation comprising more than 40 enzymatic reactions.

Identification of active denitrifiers by DNA-stable isotope probing and amplicon sequencing reveals Betaproteobacteria as responsible for attenuation of nitrate contamination in a low impacted aquifer.

Groundwater reservoirs constitute important freshwater resources. However, these ecosystems are highly vulnerable to contamination and have to rely on the resident microbiota to attenuate the impact of this contamination. Nitrate is one of the main contaminants found in groundwater, and denitrification is the main process that removes the compound. In this study, the response to nutrient load on indigenous microbial communities in groundwater from a low impacted aquifer in Uruguay was evaluated. Denitrification rates were measured in groundwater samples from three different sites with nitrate, acetate and pyrite amendments. Results showed that denitrification is feasible under in situ nitrate and electron donor concentrations, although the lack of readily available organic energy source would limit the attenuation of higher nitrate concentrations. DNA-stable isotope probing, combined with amplicon sequencing of 16S rRNA, nirS and nirK genes, was used to identify the active denitrifiers. Members of the phylum Betaproteobacteria were the dominant denitrifiers in two of three sites, with different families being observed; members of the genus Vogesella (Neisseriaceae) were key denitrifiers at one site, while the genera Dechloromonas (Rhodocyclaceae) and Comamonas (Comamonadaceae) were the main denitrifiers detected at the other sites.

Interaction of "Candidatus Accumulibacter" and nitrifying bacteria to achieve energy-efficient denitrifying phosphorus removal via nitrite pathway from sewage.

To achieve energy-efficient denitrifying phosphorus removal via nitrite pathway from sewage, interaction of "Candidatus Accumulibacter" and nitrifying bacteria was investigated in a continuous-flow process. When nitrite in returned sludge of secondary settler was above 13mg/L, nitrite inhibition on anaerobic P-release of poly-phosphate organisms (PAOs) occurred. Clades IIC and IID were dominant, reaching 3.1%-11.9% of total bacteria. Clade IIC was sensitive to nitrite. Under low concentration of nitrite (<8mg/L), clade IIC primarily contributed to anoxic P-uptake. Clade IID had a strong tolerance to nitrite exposure. At high nitrite level (above 16mg/L), anoxic P-uptake was mainly performed by clade IID due to its strong tolerance to nitrite exposure. Ammonia oxidizing bacteria (AOB), nitrite oxidizing bacteria (NOB) and Accumulibacter interacted through variations of nitrite accumulation. High AOB abundance coupled with inhibition of NOB favored denitrifying phosphorus removal by clade IID. All Accumulibacter lineages were sorted into four clades of Type II. The most dominant ppk1 gene homologs were affiliated with clade IID, accounting for 69% of ppk1 clone library, and thus played an important role in denitrifying phosphorus removal via nitrite pathway.

Viridibacterium curvum gen. nov., sp. nov., isolated from freshwater.

A Gram stain-negative, yellowish green-pigmented, facultatively anaerobic, motile, curved rod-shaped bacterium designated as strain JJ016T was isolated from an artificial lake in South Korea, and characterized using a polyphasic approach. The 16S rRNA gene sequence of strain JJ016T indicated that the isolate belonged to the family Rhodocyclaceae and exhibited 95.0% identity to Uliginosibacterium gangwonense 5YN10-9T. The major cellular fatty acids of the novel strain were summed feature 3 (C16:1 ω6c and/or C16:1 ω7c), C16:0, C14:0, and summed feature 8 (C18:1 ω7c and/or C18:1 ω6c). The DNA G+C content of strain JJ016T was 61.9 mol%. The major respiratory quinone and major polar lipid of strain JJ016T were ubiquinone-8 and phosphatidylethanolamine, respectively. Based on the morphological and physiological properties and the biochemical evidence presented, we concluded that strain JJ016T represents a novel species of a new genus in the family Rhodocyclaceae, for which the name Viridibacterium curvum gen. nov., sp. nov. is proposed. The type strain is JJ016T (=KACC 16899T =JCM 18715T).

Spatio-Temporal Variations in the Abundance and Structure of Denitrifier Communities in Sediments Differing in Nitrate Content.

Spatial and temporal variations related to hydric seasonality in abundance and diversity of denitrifier communities were examined in sediments taken from two sites differing in nitrate concentration along a stream Doñana National Park during a 3-year study. We found a positive relationship between the relative abundance of denitrifiers, determined as narG, napA, nirK, nirS and nosZ denitrification genes, and sediment nitrate content, with similar spatial and seasonal variations. However, we did not find association between denitrification activity and the community structure of denitrifiers. Because nosZ showed the strongest correlation with the content of nitrate in sediments, we used this gene as a molecular marker to construct eight genomic libraries. Analysis of these genomic libraries revealed that diversity of the nosZ-bearing communities was higher in the site with higher nitrate content. Regardless of nitrate concentration in the sediments, the Bradyrhizobiaceae and Rhodocyclaceae were the most abundant families. On the contrary, Rhizobiaceae was exclusively present in sediments with higher nitrate content. Results showed that differences in sediment nitrate concentration affect the composition and diversityof nosZ-bearing communities.