The phototrophic metabolic behaviour of Candidatus accumulibacter.

The phototrophic capability of Candidatus Accumulibacter (Accumulibacter), a common polyphosphate accumulating organism (PAO) in enhanced biological phosphorus removal (EBPR) systems, was investigated in this study. Accumulibacter is phylogenetically related to the purple bacteria Rhodocyclus from the family Rhodocyclaceae, which belongs to the class Betaproteobacteria. Rhodocyclus typically exhibits both chemoheterotrophic and phototrophic growth, however, limited studies have evaluated the phototrophic potential of Accumulibacter. To address this gap, short and extended light cycle tests were conducted using a highly enriched Accumulibacter culture (95%) to evaluate its responses to illumination. Results showed that, after an initial period of adaptation to light conditions (approximately 4-5 h), Accumulibacter exhibited complete phosphorus (P) uptake by utilising polyhydroxyalkanoates (PHA), and additionally by consuming glycogen, which contrasted with its typical aerobic metabolism. Mass, energy, and redox balance analyses demonstrated that Accumulibacter needed to employ phototrophic metabolism to meet its energy requirements. Calculations revealed that the light reactions contributed to the generation of, at least more than 67% of the ATP necessary for P uptake and growth. Extended light tests, spanning 21 days with dark/light cycles, suggested that Accumulibacter generated ATP through light during initial operation, however, it likely reverted to conventional anaerobic/aerobic metabolism under dark/light conditions due to microalgal growth in the mixed culture, contributing to oxygen production. In contrast, extended light tests with an enriched Tetrasphaera culture, lacking phototrophic genes in its genome, clearly demonstrated that phototrophic P uptake did not occur. These findings highlight the adaptive metabolic capabilities of Accumulibacter, enabling it to utilise phototrophic pathways for energy generation during oxygen deprivation, which holds the potential to advance phototrophic-EBPR technology development.

Sensitive and selective phenol sensing in denitrifying Aromatoleum aromaticum EbN1T.

IMPORTANCE: Aromatic compounds are globally abundant organic molecules with a multitude of natural and anthropogenic sources, underpinning the relevance of their biodegradation. A. aromaticum EbN1T is a well-studied environmental betaproteobacterium specialized on the anaerobic degradation of aromatic compounds. The here studied responsiveness toward phenol in conjunction with the apparent high ligand selectivity (non-promiscuity) of its PheR sensor and those of the related p-cresol (PcrS) and p-ethylphenol (EtpR) sensors are in accord with the substrate-specificity and biochemical distinctiveness of the associated degradation pathways. Furthermore, the present findings advance our general understanding of the substrate-specific regulation of the strain's remarkable degradation network and of the concentration thresholds below which phenolic compounds become essentially undetectable and as a consequence should escape substantial biodegradation. Furthermore, the findings may inspire biomimetic sensor designs for detecting and quantifying phenolic contaminants in wastewater or environments.

Enhancing tellurite and selenite bioconversions by overexpressing a methyltransferase from Aromatoleum sp. CIB.

Pollution by metalloids, e.g., tellurite and selenite, is of serious environmental concern and, therefore, there is an increasing interest in searching for ecologically friendly solutions for their elimination. Some microorganisms are able to reduce toxic tellurite/selenite into less toxic elemental tellurium (Te) and selenium (Se). Here, we describe the use of the environmentally relevant ß-proteobacterium Aromatoleum sp. CIB as a platform for tellurite elimination. Aromatoleum sp. CIB was shown to tolerate 0.2 and 0.5 mM tellurite at aerobic and anaerobic conditions, respectively. Furthermore, the CIB strain was able to reduce tellurite into elemental Te producing rod-shaped Te nanoparticles (TeNPs) of around 200 nm length. A search in the genome of Aromatoleum sp. CIB revealed the presence of a gene, AzCIB_0135, which encodes a new methyltransferase that methylates tellurite and also selenite. AzCIB_0135 orthologs are widely distributed in bacterial genomes. The overexpression of the AzCIB_0135 gene both in Escherichia coli and Aromatoleum sp. CIB speeds up tellurite and selenite removal, and it enhances the production of rod-shaped TeNPs and spherical Se nanoparticles (SeNPs), respectively. Thus, the overexpression of a methylase becomes a new genetic strategy to optimize bacterial catalysts for tellurite/selenite bioremediation and for the programmed biosynthesis of metallic nanoparticles of biotechnological interest.

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.

Sulfuricystis multivorans gen. nov., sp. nov. and Sulfuricystis thermophila sp. nov., facultatively autotropic sulfur-oxidizing bacteria isolated from a hot spring, and emended description of the genus Rugosibacter.

Strains J5BT and M52T are facultatively autotrophic sulfur-oxidizing bacteria isolated from a microbial mat from a hot spring. They were isolated and partially characterized in previous studies, as facultative anaerobes which use nitrate as electron acceptor. In this study, additional characterizations were made to determine their taxonomic status. In both strains, major cellular fatty acids were C16:1 (C16:1ω7c and/or C16:1ω6c) and C16:0. Their chemolithoautotrophic growth was supported by thiosulfate and elemental sulfur. They used some organic acids as growth substrates. Their 16S rRNA gene sequences indicated the highest sequence identities to species in the family Sterolibacteriaceae, but the identities were 95% or lower. Phylogenetic analysis indicated that these strains do not belong to any existing genera. Values of average nucleotide identity and digital DNA-DNA hybridization between strains J5BT and M52T were 87.93% and 34.3%, respectively. On the basis of phenotypic and genomic characteristics, Sulfuricystis multivorans gen. nov. sp. nov., and Sulfuricystis thermophila sp. nov. are proposed, with type strains of J5BT and M52T, respectively. An emended description of the genus Rugosibacter is also proposed, for its reclassification to the family Sterolibacteriaceae.

Genetic characterization of the cyclohexane carboxylate degradation pathway in the denitrifying bacterium Aromatoleum sp. CIB.

The alicyclic compound cyclohexane carboxylate (CHC) is anaerobically degraded through a peripheral pathway that converges with the central benzoyl-CoA degradation pathway of aromatic compounds in Rhodopseudomonas palustris (bad pathway) and some strictly anaerobic bacteria. Here we show that in denitrifying bacteria, e.g. Aromatoleum sp. CIB strain, CHC is degraded through a bad-ali pathway similar to that reported in R. palustris but that does not share common intermediates with the benzoyl-CoA degradation pathway (bzd pathway) of this bacterium. The bad-ali genes are also involved in the aerobic degradation of CHC in strain CIB, and orthologous bad-ali clusters have been identified in the genomes of a wide variety of bacteria. Expression of bad-ali genes in strain CIB is under control of the BadR transcriptional repressor, which was shown to recognize CHC-CoA, the first intermediate of the pathway, as effector, and whose operator region (CAAN4 TTG) was conserved in bad-ali clusters from Gram-negative bacteria. The bad-ali and bzd pathways generate pimelyl-CoA and 3-hydroxypimelyl-CoA, respectively, that are metabolized through a common aab pathway whose genetic determinants form a supraoperonic clustering with the bad-ali genes. A synthetic bad-ali-aab catabolic module was engineered and it was shown to confer CHC degradation abilities to different bacterial hosts.

Complex and flexible catabolism in Aromatoleum aromaticum pCyN1.

Large quantities of organic matter are continuously deposited, and (a)biotic gradients intersect in the soil-rhizosphere, where biodegradation contributes to the global cycles of elements. The betaproteobacterial genus Aromatoleum comprises cosmopolitan, facultative denitrifying degradation specialists. Aromatoleum aromaticum. pCyN1 stands out for anaerobically decomposing plant-derived monoterpenes in addition to monoaromatic hydrocarbons, polar aromatics and aliphatics. The catabolic network's structure and flexibility in A. aromaticum pCyN1 were studied across 34 growth conditions by superimposing proteome profiles onto the manually annotated 4.37 Mbp genome. Strain pCyN1 employs three fundamentally different enzymes for C-H-bond cleavage at the methyl groups of p-cymene/4-ethyltoluene, toluene and p-cresol respectively. Regulation of degradation modules displayed substrate specificities ranging from narrow (toluene and cyclohexane carboxylate) via medium-wide (one module shared by p-cymene, 4-ethyltoluene, α-phellandrene, α-terpinene, γ-terpinene and limonene) to broad (central benzoyl-CoA pathway serving 16 aromatic substrates). Remarkably, three variants of ATP-dependent (class I) benzoyl-CoA reductase and four different ß-oxidation routes establish a degradation hub that accommodates the substrate diversity. The respiratory system displayed several conspicuous profiles, e.g. the presence of nitrous oxide reductase under oxic and of low-affinity oxidase under anoxic conditions. Overall, nutritional versatility in conjunction with network regulation endow A. aromaticum pCyN1 with broad adaptability.

Azospira inquinata sp. nov., a nitrate-reducing bacterium of the family Rhodocyclaceae isolated from contaminated groundwater.

Two novel Gram-stain-negative bacterial strains, Azo-3T and Azo-2, were isolated from a toluene-producing enrichment culture that originated from contaminated groundwater at a site in southeast Louisiana (USA). Cells are non-spore forming straight to curved rods with single polar flagella. Strains Azo-3T and Azo-2 are oxidase-positive, catalase-negative, use nitrate and nitrite as electron acceptors, and are able to fix nitrogen. Poly-ß-hydroxybutyrate storage granules are produced. Dominant fatty acids when grown in R2A medium at 37 °C are C16:0, summed feature 3 (C16:1 ω7c and/or C15:0 iso 2OH), C17:0 cyclo and C18:1 ω7c. 16S rRNA gene sequence based phylogenetic analysis indicated that the strains cluster within the family Rhodocyclaceae, class Betaproteobacteria, most closely related to but distinct from type strains of the species Azospira oryzae (96.94% similarity) and Azospira restricta (95.10% similarity). Complete genome sequences determined for strains Azo-3T and Azo-2 revealed DNA G+C content of 62.70 mol%. Genome-wide comparisons based on average nucleotide identity by orthology and estimated DNA-DNA hybridization values combined with phenotypic and chemotaxonomic traits and phylogenetic analysis indicate that strains Azo-3T and Azo-2 represent a novel species within the genus Azospira for which the name Azospira inquinata sp. nov. is proposed. The type strain of Azospira inquinata is Azo-3T (=NRRL B-65590T=DSM 112046T).

Dynamic interspecies interactions and robustness in a four-species model biofilm.

Interspecific interactions within biofilms determine relative species abundance, growth dynamics, community resilience, and success or failure of invasion by an extraneous organism. However, deciphering interspecific interactions and assessing their contribution to biofilm properties and function remain a challenge. Here, we describe the constitution of a model biofilm composed of four bacterial species belonging to four different genera (Rhodocyclus sp., Pseudomonas fluorescens, Kocuria varians, and Bacillus cereus), derived from a biofilm isolated from an industrial milk pasteurization unit. We demonstrate that the growth dynamics and equilibrium composition of this biofilm are highly reproducible. Based on its equilibrium composition, we show that the establishment of this four-species biofilm is highly robust against initial, transient perturbations but less so towards continuous perturbations. By comparing biofilms formed from different numbers and combinations of the constituent species and by fitting a growth model to the experimental data, we reveal a network of dynamic, positive, and negative interactions that determine the final composition of the biofilm. Furthermore, we reveal that the molecular determinant of one negative interaction is the thiocillin I synthesized by the B. cereus strain, and demonstrate its importance for species distribution and its impact on robustness by mutational analysis of the biofilm ecosystem.

Perchlorate Contamination: Sources, Effects, and Technologies for Remediation.

Perchlorate is a persistent pollutant, generated via natural and anthropogenic processes, that possesses a high potential for endocrine disruption in humans and biota. It inhibits iodine fixation, a major reason for eliminating this pollutant from ecosystems. Remediation of perchlorate can be achieved with various physicochemical treatments, especially at low concentrations. However, microbiological approaches using microorganisms, such as those from the genera Dechloromonas, Serratia, Propionivibrio, Wolinella, and Azospirillum, are promising when perchlorate pollution is extensive. Perchlorate-reducing bacteria, isolated from harsh environments, for example saline soils, mine sediments, thermal waters, wastewater treatment plants, underground gas storage facilities, and remote areas, including the Antarctica, can provide removal yields from 20 to 100%. Perchlorate reduction, carried out by a series of enzymes, such as perchlorate reductase and superoxide chlorite, depends on pH, temperature, salt concentration, metabolic inhibitors, nutritional conditions, time of contact, and cellular concentration. Microbial degradation is cost-effective, simple to implement, and environmentally friendly, rendering it a viable method for alleviating perchlorate pollution in the environment.

Four species of bacteria deterministically assemble to form a stable biofilm in a millifluidic channel.

Multispecies microbial adherent communities are widespread in nature and organisms, although the principles of their assembly and development remain unclear. Here, we test the possibility of establishing a simplified but relevant model of multispecies biofilm in a non-invasive laboratory setup for the real-time monitoring of community development. We demonstrate that the four chosen species (Bacillus thuringiensis, Pseudomonas fluorescens, Kocuria varians, and Rhodocyclus sp.) form a dynamic community that deterministically reaches its equilibrium after ~30 h of growth. We reveal the emergence of complexity in this simplified community as reported by an increase in spatial heterogeneity and non-monotonic developmental kinetics. Importantly, we find interspecies interactions consisting of competition for resources-particularly oxygen-and both direct and indirect physical interactions. The simplified experimental model opens new avenues to the study of adherent bacterial communities and their behavior in the context of rapid global change.

Niveibacterium microcysteis sp. nov., isolated from a cyanobacterial bloom sample.

A novel bacterial strain, named HC41T, was isolated from a cyanobacterial bloom sample and was characterized as Gram-stain-negative, rod-shaped and non-motile. According to 16S rRNA phylogenetic analyses, this strain HC41T belongs to the family Rhodocyclaceae and is most closely related to Niveibacterium umoris KACC 17062T (=MIC 2059T; 98.63 %) and Uliginosibacterium gangwonense 5YN10-9 T (=KACC 11603T; 93.64 %). The genome size and DNA G+C content of strain HC41T were 4.8 Mbp and 64.17 mol%, respectively. Moreover, the average nucleotide identity, digital DNA-DNA hybridization and amino acid identity values between strain HC41T and N. umoris KACC 17062T were 81.8, 43.1 and 90.89 %, respectively. Additionally, strain HC41T exhibited weak catalase and oxidase activities and had no motility (swimming and swarming motilities). The cells grew at 11-40 °C (optimum, 30 °C), pH 5.5-8.0 (optimum, pH 7) and with 0-1.0 % (w/v) NaCl (optimum, 0 % NaCl) in Reasoner's 2A medium. Its major respiratory quinone was ubiquinone-8 and its major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. Furthermore, C16 : 0 and summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c; C16 : 1 ω6c and/or C16 : 1 ω7c) were the predominant cellular fatty acids in strain HC41T according to fatty acid methyl ester analysis. Based on its genotypic and phenotypic characteristics, strain HC41T was identified as representing a novel Niveibacterium species, for which the name Niveibacterium microcysteis sp. nov. is proposed (=KACC 22091T=DSM 111425T).

Benzylmalonyl-CoA dehydrogenase, an enzyme involved in bacterial auxin degradation.

A novel acyl-CoA dehydrogenase involved in degradation of the auxin indoleacetate by Aromatoleum aromaticum was identified as a decarboxylating benzylmalonyl-CoA dehydrogenase (IaaF). It is encoded within the iaa operon coding for enzymes of indoleacetate catabolism. Using enzymatically produced benzylmalonyl-CoA, the reaction was characterized as simultaneous oxidation and decarboxylation of benzylmalonyl-CoA to cinnamoyl-CoA and CO2. Oxygen served as electron acceptor and was reduced to H2O2, whereas electron transfer flavoprotein or artificial dyes serving as electron acceptors for other acyl-CoA dehydrogenases were not used. The enzyme is homotetrameric, contains an FAD cofactor and is enantiospecific in benzylmalonyl-CoA turnover. It shows high catalytic efficiency and strong substrate inhibition with benzylmalonyl-CoA, but otherwise accepts only a few medium-chain alkylmalonyl-CoA compounds as alternative substrates with low activities. Its reactivity of oxidizing 2-carboxyacyl-CoA with simultaneous decarboxylation is unprecedented and indicates a modified reaction mechanism for acyl-CoA dehydrogenases, where elimination of the 2-carboxy group replaces proton abstraction from C2.

Responsiveness of Aromatoleum aromaticum EbN1T to Lignin-Derived Phenylpropanoids.

The betaproteobacterial degradation specialist Aromatoleum aromaticum EbN1T utilizes several plant-derived 3-phenylpropanoids coupled to denitrification. In vivo responsiveness of A. aromaticum EbN1T was studied by exposing nonadapted cells to distinct pulses (spanning 100 µM to 0.1 nM) of 3-phenylpropanoate, cinnamate, 3-(4-hydroxyphenyl)propanoate, or p-coumarate. Time-resolved, targeted transcript analyses via quantitative reverse transcription-PCR of four selected 3-phenylpropanoid genes revealed a response threshold of 30 to 50 nM for p-coumarate and 1 to 10 nM for the other three tested 3-phenylpropanoids. At these concentrations, transmembrane effector equilibration is attained by passive diffusion rather than active uptake via the ABC transporter, presumably serving the studied 3-phenylpropanoids as well as benzoate. Highly substrate-specific enzyme formation (EbA5316 to EbA5321 [EbA5316-21]) for the shared peripheral degradation pathway putatively involves the predicted TetR-type transcriptional repressor PprR. Accordingly, relative transcript abundances of ebA5316-21 are lower in succinate- and benzoate-grown wild-type cells than in an unmarked in-frame ΔpprR mutant. In trans-complementation of pprR into the ΔpprR background restored wild-type-like transcript levels. When adapted to p-coumarate, the three genotypes had relative transcript abundances similar to those of ebA5316-21 despite a significantly longer lag phase of the pprR-complemented mutant (∼100-fold higher pprR transcript level than the wild type). Notably, transcript levels of ebA5316-21 were ∼10- to 100-fold higher in p-coumarate- than succinate- or benzoate-adapted cells across all three genotypes. This indicates the additional involvement of an unknown transcriptional regulator. Furthermore, physiological, transcriptional, and (aromatic) acyl-coenzyme A ester intermediate analyses of the wild type and ΔpprR mutant grown with binary substrate mixtures suggest a mode of catabolite repression of superior order to PprR.IMPORTANCE Lignin is a ubiquitous heterobiopolymer built from a suite of 3-phenylpropanoid subunits. It accounts for more than 30% of the global plant dry material, and lignin-related compounds are increasingly released into the environment from anthropogenic sources, i.e., by wastewater effluents from the paper and pulp industry. Hence, following biological or industrial decomplexation of lignin, vast amounts of structurally diverse 3-phenylpropanoids enter terrestrial and aquatic habitats, where they serve as substrates for microbial degradation. This raises the question of what signaling systems environmental bacteria employ to detect these nutritionally attractive compounds and to adjust their catabolism accordingly. Moreover, determining in vivo response thresholds of an anaerobic degradation specialist such as A. aromaticum EbN1T for these aromatic compounds provides insights into the environmental fate of the latter, i.e., when they could escape biodegradation due to too low ambient concentrations.

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.

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.

Temperature and oxygen level determine N2 O respiration activities of heterotrophic N2 O-reducing bacteria: Biokinetic study.

Nitrous oxide (N2 O), a potent greenhouse gas, is reduced to N2 gas by N2 O-reducing bacteria (N2 ORB), a process which represents an N2 O sink in natural and engineered ecosystems. The N2 O sink activity by N2 ORB depends on temperature and O2 exposure, yet the specifics are not yet understood. This study explores the effects of temperature and oxygen exposure on biokinetics of pure culture N2 ORB. Four N2 ORB, representing either clade I type nosZ (Pseudomonas stutzeri JCM5965 and Paracoccus denitrificans NBRC102528) or clade II type nosZ (Azospira sp. strains I09 and I13), were individually tested. The higher activation energy for N2 O by Azospira sp. strain I13 (114.0 ± 22.6 kJ mol-1 ) compared with the other tested N2 ORB (38.3-60.1 kJ mol-1 ) indicates that N2 ORB can adapt to different temperatures. The O2 inhibition constants (KI ) of Azospira sp. strain I09 and Ps. stutzeri JCM5965 increased from 0.06 ± 0.05 and 0.05 ± 0.02 µmol L-1 to 0.92 ± 0.24 and 0.84 ± 0.31 µmol L-1 , respectively, as the temperature increased from 15°C to 35°C, while that of Azospira sp. strain I13 was temperature-independent (p = 0.106). Within the range of temperatures examined, Azospira sp. strain I13 had a faster recovery after O2 exposure compared with Azospira sp. strain I09 and Ps. stutzeri JCM5965 (p < 0.05). These results suggest that temperature and O2 exposure result in the growth of ecophysiologically distinct N2 ORB as N2 O sinks. This knowledge can help develop a suitable N2 O mitigation strategy according to the physiologies of the predominant N2 ORB.

Unexpected photosensitivity of the well-characterized heme enzyme chlorite dismutase.

Chlorite dismutase is a heme enzyme that catalyzes the conversion of the toxic compound ClO2- (chlorite) to innocuous Cl- and O2. The reaction is a very rare case of enzymatic O-O bond formation, which has sparked the interest to elucidate the reaction mechanism using pre-steady-state kinetics. During stopped-flow experiments, spectroscopic and structural changes of the enzyme were observed in the absence of a substrate in the time range from milliseconds to minutes. These effects are a consequence of illumination with UV-visible light during the stopped-flow experiment. The changes in the UV-visible spectrum in the initial 200 s of the reaction indicate a possible involvement of a ferric superoxide/ferrous oxo or ferric hydroxide intermediate during the photochemical inactivation. Observed EPR spectral changes after 30 min reaction time indicate the loss of the heme and release of iron during the process. During prolonged illumination, the oligomeric state of the enzyme changes from homo-pentameric to monomeric with subsequent protein precipitation. Understanding the effects of UV-visible light illumination induced changes of chlorite dismutase will help us to understand the nature and mechanism of photosensitivity of heme enzymes in general. Furthermore, previously reported stopped-flow data of chlorite dismutase and potentially other heme enzymes will need to be re-evaluated in the context of the photosensitivity. Illumination of recombinantly expressed Azospira oryzae Chlorite dismutase (AoCld) with a high-intensity light source, common in stopped-flow equipment, results in disruption of the bond between FeIII and the axial histidine. This leads to the enzyme losing its heme cofactor and changing its oligomeric state as shown by spectroscopic changes and loss of activity.

Bacterial catabolism of indole-3-acetic acid.

Indole-3-acetic acid (IAA) is a molecule with the chemical formula C10H9NO2, with a demonstrated presence in various environments and organisms, and with a biological function in several of these organisms, most notably in plants where it acts as a growth hormone. The existence of microorganisms with the ability to catabolize or assimilate IAA has long been recognized. To date, two sets of gene clusters underlying this property in bacteria have been identified and characterized: one (iac) is responsible for the aerobic degradation of IAA into catechol, and another (iaa) for the anaerobic conversion of IAA to 2-aminobenzoyl-CoA. Here, we summarize the literature on the products, reactions, and pathways that these gene clusters encode. We explore two hypotheses about the benefit that iac/iaa gene clusters confer upon their bacterial hosts: (1) exploitation of IAA as a source of carbon, nitrogen, and energy; and (2) interference with IAA-dependent processes and functions in other organisms, including plants. The evidence for both hypotheses will be reviewed for iac/iaa-carrying model strains of Pseudomonas putida, Enterobacter soli, Acinetobacter baumannii, Paraburkholderia phytofirmans, Caballeronia glathei, Aromatoleum evansii, and Aromatoleum aromaticum, more specifically in the context of access to IAA in the environments from which these bacteria were originally isolated, which include not only plants, but also soils and sediment, as well as patients in hospital environments. We end the mini-review with an outlook for iac/iaa-inspired research that addresses current gaps in knowledge, biotechnological applications of iac/iaa-encoded enzymology, and the use of IAA-destroying bacteria to treat pathologies related to IAA excess in plants and humans. KEY POINTS: • The iac/iaa gene clusters encode bacterial catabolism of the plant growth hormone IAA. • Plants are not the only environment where IAA or IAA-degrading bacteria can be found. • The iac/iaa genes allow growth at the expense of IAA; other benefits remain unknown.